Research ArticleIn Vitro Antioxidant and Antidiabetic Potentials ofSyzygium caryophyllatum L Alston

Herath Pathiranage Thathmi Wathsara1 Hasitha Dhananjaya Weeratunge2

Mohamed Naeem Ahammadu Mubarak3 Pahan Indika Godakumbura1

and Pathmasiri Ranasinghe 2

1Department of Chemistry Faculty of Applied Sciences University of Sri Jayewardenepura Nugegoda Sri Lanka2Herbal Technology Section (HTS) Modern Research amp Development Complex (MRDC) Industrial Technology Institute (ITI)503A Halbarawa Gardens Malabe Sri Lanka3Residue Analysis Laboratory Industrial Technology Institute (ITI) 363 Bauddhaloka Mawatha Colombo 07 Sri Lanka

Correspondence should be addressed to Pathmasiri Ranasinghe pathmasiriitilk

Received 10 April 2020 Revised 19 June 2020 Accepted 3 July 2020 Published 28 July 2020

Guest Editor Saheed Sabiu

Copyright copy 2020 Herath Pathiranage (athmi Wathsara et al (is is an open access article distributed under the CreativeCommons Attribution License which permits unrestricted use distribution and reproduction in any medium provided theoriginal work is properly cited

Syzygium caryophyllatum L Alston (Family Myrtaceae Sinhala Heendan) is a red-listed plant that has been used in traditionalmedicine in Sri Lanka for the treatment of diabetes but it is yet to be exploited for its potential uses as a functional food or a source ofsupplements (e present study focused on the evaluation of antidiabetic property of S caryophyllatum fruits and leaves assessingantioxidant antiglycation and antiamylase activities and functional mineral element composition (e crude extracts (CR) of leavesand fruits were fractionated into hexane (Hex) ethyl acetate (EA) and aqueous (AQ) and evaluated for bioactivities along with thecrude extracts (e isolated fraction (C3) of Hex fraction of fruit showed significantly high (plt 005) antiamylase activity with IC50value 227plusmn 181μgmLwhere the Hex fraction of fruits exhibited the IC50 value as 4720plusmn 03 μgmLwhich was higher than acarbose(IC50 8796plusmn 143 μgmL) (e EA fraction of leaves showed highest values for DPPH radical scavenging activity ferric reducingantioxidant power and oxygen radical absorbance capacity Significantly high (plt 005) ABTS radical scavenging activity and ironchelating activity were observed in Hex fraction of fruit (e composition of volatiles in leaf oil was studied with GC-MS and 58compounds were identified Inductively coupled plasma-mass spectrometry data revealed the presence of biologically significanttrace elements such as Fe Zn Mg Cu Se and Sr in leaves and fruits It is concluded that the Hex fraction of S caryophyllatum fruitswill be a good source for the formulation of supplements for diabetic management with further evaluation of potency and efficacy

1 Introduction

(e prevalence of diabetes mellitus has shown a rapid in-crease over the years and according to the reports of theWHO individuals suffering from diabetes mellitus world-wide will show a marked increase of up to 592 million in2035 from 422 million in 2014 Further in 2015 16 milliondeaths were estimated to be directly or indirectly caused bydiabetes At present developing countries are much affectedby the more predominant form of diabetesmdashType 2 diabetesthat accounts for increased morbidity and disability causinga threat to the economic growth in most developingcountries However reports revealed that more than 80 of

deaths resulting from diabetes in developing countries aredue to rapid urbanization and poor healthcare facilities [1]

Diabetes Mellitus (DM) is a metabolic disorder ofmultiple etiology characterized by chronic hyperglycemiawhich is mainly caused by one or combination of factorsincluding the ineffectiveness of insulin deficiency of insulinsecretion and insulin resistance of the cells in particularskeletal muscle tissues [2] Changes in food patterns andlifestyle such as high intake of calories less exercise andphysiological stress conditions are also closely associatedwith the development and progression of DM [3]

Prolonged hyperglycemic conditions in diabetes patientsinduce nonenzymatic glycation which is a reaction between

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2020 Article ID 9529042 15 pageshttpsdoiorg10115520209529042

the amino group of proteins and the carbonyl group ofreducing sugar to form a fluorescent Advanced GlycationEnd-products (AGEs) [4] Apart from elevated sugar levelsin the blood exposure to exogenous free radicals andconsumption of processed foods with a high content offructose may also accelerate the accumulation of AGEs [5]Studies have proven that hyperglycemia and signal-trans-ducing receptor interactions of AGEs formation induceincreased production of free radicals leading to oxidativestress [6 7] (is in turn leads to various pathologicalconditions such as cancer neurological disorders athero-sclerosis hypertension type 2 diabetes acute respiratorydistress syndrome idiopathic pulmonary fibrosis chronicobstructive pulmonary disease and asthma [6]

Studies have further revealed that the imbalance of severalessential trace elements leads to the development and pro-gression of DM Further clinical studies have suggested thatpatients with diabetes have an increased risk of trace elementdeficiency Hence it is recommended to increase the dietaryintake of some trace elements or as a dietary supplement tomanage this condition during DM [8] (e mineral elementsplay a pivotal role in numerous basic physiological functionsof the human body and the deficiency thus leads to a variety ofextensive diseases and disorders [9] Several mineral elementshave been identified as the cofactors of antioxidative enzymesand play an important role in protecting the insulin-secretingpancreatic β-cells [8] (us management of type 2 DM in-cludes reduction of postprandial glucose concentrations byinhibition of α-amylase and α-glucosidase enzymes whichdelays the digestion of carbohydrates thus lowering thehyperglycemic condition

In recent years the usage and the vitality of naturalproducts have created great curiosity among the scientificcommunity (is is mainly due to the benefits of naturalproducts such as high effectiveness low risk of side effectslow cost and abundant availability

Ethnobotanical information reveals that there is a widearray of plants that possessed potential antidiabetic prop-erties and antioxidant properties [10 11] In this regard over200 pure compounds from herbal plants are scientificallyproven to have hypoglycemic activity [12]

Syzygium is a genus from the Myrtaceae family pos-sessing 1200ndash1800 species Syzygium caryophyllatum (Sin-hala Heendan) is one of the species that has been classifiedas endangered species under the International Union forConservation of Nature (IUCN) and is native to Sri Lankaand India [13] In India S caryophyllatum plant has beenstudied for its antibacterial and antioxidant activities usingsome in vitro antioxidant assays [14] However this is the 1stSri Lankan study to report the antioxidant activities andantidiabetic activities of S caryophyllatum In traditionalfolklore medical practices the decoction of S car-yophyllatum is used in the treatment for ailments of diabetesmellitus [15] In Sri Lanka S caryophyllatum is consideredas an underutilized wild fruit amidst all its beneficialproperties due to the lack of reported data on its nutritionalvalue poor awareness on the beneficial roles of the fruitupcoming modern agricultural practices and over importedfruits [16]

(erefore the present study aimed to evaluate in vitrobioactivities such as antioxidant antiamylase and anti-glycation and quantify mineral elements and volatile oilcompositions of Syzygium caryophyllatum in Sri LankaFurthermore the outcome of this study will be used todetermine whether Syzygium caryophyllatum fruits andleaves are potential sources of dietary supplement inmanaging DM and its complications

2 Materials and Methods

21 Chemical and Reagents Folin-Ciocalteu reagent (FCReagent) gallic acid quercetin acarbose 6-hydroxy-2-5-7-8-tetramethylchroman-2carboxylic acid (Trolox) ethyl-enediaminetetraacetic acid (EDTA) 11-diphenyl-2-picryl-hydrazyl (DPPH) 2 2-azino-bis (3ethylbenzothiazoline-6-sul-fonic acid) diammonium salt (ABTS) potassium persulphate 22prime-azobis (2-amidinopropane) dihydrochloride (AAPH) so-dium fluorescein 246 tripyridyl-s-triazine (TPTZ) andα-amylase (Bacillus amyloliquefaciens) were purchased fromRoche Diagnostics (USA) and 35-dinitrosalycylic acid 4 4prime-disulfonic acid sodium salt (ferrozine) soluble starch bovineserum albumin (BSA) D-glucose and trichloro-acetic acid(TCA) were purchased from Sigma-Aldrich (USA) All otherchemicals used for the preparation of buffers and solvents werein the analytical grade Spectrophotometric assays were con-ducted using 96-well microplate readers (Spectra Max Plus384Molecular Devices USA and SPECTRAmax-Gemini EMMolecular Devices Inc USA) (e mineral analysis was doneusing microwave digester CEM Mar 5 USA using the EasyPrep digestion program and ICP-MS (Agilent 7900 USA)

22 PlantMaterials Fresh leaves and ripened fruits (Figure 1)were collected into sterile polypropylene bags from the areas ofHomagama and Malabe (Colombo district Sri Lanka) duringthe period from April to May 2016 (e plant materials werebotanically identified by the botanist Dr Chandima Wije-siriwardena Principle Research Scientist Industrial Tech-nology Institute Sri Lanka and voucher specimens (leavesWF10-1 and fruits-WF10-2) were deposited at the HerbalTechnology Section (HTS) Industrial Technology Institute(ITI) Sri Lanka (e collected materials were washed withrunning water followed by distilled water cleaned air driedand ground to obtain a fine powder(e powderwas packed inpolypropylene bags and stored at minus20degC for further analyses

23 Preparation of Extracts

231 Preparation of Leaf Crude Extract of Syzygiumcaryophyllatum (e moisture content of the powderedleaves was recorded bymoisture analyzer (moisture 1353)(e powdered sample (225 g dry weight of sample) wasextracted by the reflux apparatus for 3 hours and filteredusing a muslin cloth and later withWhatman number 1 filterpaper(e filtrate was treated with 70 ethanol (1 3 extractethanol) and centrifuged to remove the precipitate (eextract was concentrated in a vacuum below 50degC andneutralized to pH 7 with 001M HCl

2 Evidence-Based Complementary and Alternative Medicine

232 Preparation of Fruit Crude Extract of Syzygiumcaryophyllatum (e ripen fruits (30 g dry weight of sample)without seed were extracted with ethanol by overnight coldextraction with stirring (e ethanol extract was concentratedin a vacuum at 40degC

(e crude extracts of leaves and fruits were fractionatedinto hexane and ethyl acetate by using separation funnels andall concentrated samples were stored at minus20degC prior to theanalyses (e extracts were evaporated to dryness by rotaryevaporator and a known amount of sample was dissolved inDMSO for further analyses

24 Compound Isolation from Hexane Fraction of Syzygiumcaryophyllatum Fruits (e fraction was separated usingcolumn chromatography with different organic solvents as themobile phase A column of 75 cm height and 25 cm diameterwas packed with silica gel 100ndash200 mesh with hexane as asolvent in the wet packing method Compound isolation wascarried out in two different methods by using different solventson the isolation procedure

241 Hexane and Ethyl Acetate Solvents on Isolation Pro-cedure (Hex and EA) (e 50mg of Hex fraction of fruitswas separated by column by increasing polarity of eluenthexane and ethyl acetate 100 0 and 0 100 respectively(e two separated isolates were tested for α-amylase in-hibition activity

242 Hexane Dichloromethane and Ethyl Acetate Solventson Isolation Procedure (Hex DCM and EA) Columnchromatography was performed using 200mg of Hexfraction of fruits to obtain fractions by increasing polarityusing a gradient elution technique as follows Fractions(FR) 1ndash10 (Hex) FR 11ndash12 (Hex DCM 3 1) FR 13(Hex DCM 2 1) FR 14ndash19 (DCM) and FR 20ndash22(DCM EA 95 5) to obtain 518mg (C1 combinedfractions 1ndash13) 957 mg (C2 combined fractions 14ndash19)and 68mg (C3 combined fractions 20ndash22) respectivelyAll combined fractions were tested for α-amylase inhi-bition activity

Flowchart of the overall methodology is shown inFigure 2

Leaves and fruits of Syzygium caryophyllatum plantwere extracted fractionated and subjected for phyto-chemical analysis antioxidant antiamylase antiglycationpotentials analysis and compounds isolation Chemicalcompositions of Syzygium caryophyllatum leaf oil wereanalyzed by GC-MS and mineral composition of leavesand fruits were analyzed by ICP-MS

25 Estimation of Phytochemical Compounds

251 Determination of Total Polyphenol Content (TPC)(e total polyphenol content (TPC) was determined using theFolin-Ciocalteu (FC) reagent method [17] with minor modi-fication using 96-well microplates Freshly prepared 110μL of

(a)

(b) (c)

Figure 1 Different parts of the Syzygium caryophyllatum plant (a) leaf (b) flowers and (c) fruits

Evidence-Based Complementary and Alternative Medicine 3

FC Reagent was added to 20μL of the sample (e mixture wastreated with 10 sodium carbonate and incubated at roomtemperature for 30min and absorbance was measured at765nm Gallic acid was used as a reference standard and theresults were expressed as milligram gallic acid equivalent pergram of sample (mg GAEg of sample)

252 Determination of Total Flavonoid Content (TFC)(e total flavonoid content (TFC) was determined using analuminum chloride colorimetric method using 96-wellmicroplates [18] Extracts were dissolved in methanol and100 μL of the sample wasmixed with 100 μL of 2 aluminum

chloride (e mixture was incubated at room temperaturefor 10min and absorbance was measured at 415nm Quercetinwas used as a reference standard and the results wereexpressed as milligram quercetin equivalent per gram ofsample (mg QEg of sample)

26 Evaluation of Antioxidant Activity

261 Ferric Reducing Antioxidant Power (FRAP) Assay(e assay method was based on the reduction of Fe3+ to Fe2+by the electron donation ability of antioxidants resulting inan intense blue-colored complex (Fe2+-tripyridyltriazine)

Syzygium caryophyllatum

Leaves Fruits

Distillation Reflux Cold extraction

Crude extract

Fractionation

Hexane

Aqueous fractionEthyl acetate

Crude extract Aqueous fraction Ethyl acetate fraction Hexane fraction

AntioxidantAntiamylase

Antiglycation

Hexane(100)

EA(100)

Hexane (100) Hex DCM (3 1) Hex DCM (2 1) DCM (100) DCM EA (95 5)

Fr 1-10 Fr 11-12 Fr 13 Fr 14-19 Fr 20-22

C1 C3C2

Anti-amylase

Chemical composition analysis-GC-MS

Mineral composition analysisndashICP-MS

Solvent system IIHex amp EA

Silica gel column chromatography

Oil

Hexane fraction offruit

Solvent system IHex DCM amp EA

Figure 2 Flow chart depicting the methodology of the study

4 Evidence-Based Complementary and Alternative Medicine

[18] (e FRAP reagent was prepared freshly by mixingacetate buffer (300mM pH 36) solution of TPTZ in HCland FeCl3 (20mM) in 10 1 1 ratio and incubated at 37degCTwo hundred microliters of the reaction mixture (FRAPreagent and sample) was incubated at room temperatureand absorbance was measured at 600 nm Vitamin E ana-logue Trolox was used as the reference standard where theresults were expressed as mg of Trolox equivalent per gramof extract (mg TEg of extract)

262 DPPH Radical Scavenging Assay (e radical scav-enging activity was determined by using 1 1-diphenyl-2-picryl-hydrazyl (DPPH) stable radical method [18] (e method isbased on the reduction of purple-colored 1 1-diphenyl-2-picrylhydrazyl to a yellow-colored diphenylpicrylhydrazine(ereaction volume of 200μL containing sample (50μL)methanolandDPPHwere incubated at room temperature for 10min andabsorbance was measured at 517nm A percentage inhibitionversus concentration was plotted and the concentration of thesample required for 50 inhibition was determined and rep-resented as EC50 value (e following formula was used tocalculate the percent inhibition

Radical scavenging activity CAB minus SAB( 1113857

CABtimes 100 (1)

where CAB is the absorbance of the control and SAB is theabsorbance of the sample

263 ABTS Radical Scavenging Activity (e ABTS+ radicalcation (ABTS+) scavenging activity assay was performed [19]with some modifications in 96-well microplates Plant ex-tracts were prepared in phosphate buffer solutions and wereallowed to react with ABTS+ radicals in a microwell plate for10min Absorbance wasmeasured in 734 nm and Trolox wasused as the standard (e following formula was used tocalculate the percent inhibition

Radical scavenging activity CAB minus SAB( 1113857

CABtimes 100 (2)

where CAB is the absorbance of the control and SAB is theabsorbance of the sample

(e antiradical activity was expressed as EC50 theconcentration required to cause 50 inhibition of ABTS+radicals

264 Oxygen Radical Absorbance Capacity (ORAC) Assay(eORAC assay is a hydrogen atom transfer- (HAT-) basedassay which measures the depletion of fluorescence intensityof a fluorescent probe upon the generation of peroxy radicalsdue to the oxidation of azo compounds In the presence of anantioxidant compound the decaying of fluorescent intensityis retarded (e overall decay is calculated by the differencebetween the area under the fluorescence decay curve (AUC)of the sample of interest and AUC of blank [20](e reactionvolume of 200 μL (pH 74) containing fluorescein (100 μL)and sample (50 μL) was preincubated at 37degC for 5min (e22prime-azobis (2-amidinopropane) dihydrochloride (AAPH)

solution was added (50 μL) to the mixture and the plate(black 96 well plates) was immediately placed in a fluo-rescent microplate reader (SPECTRAmax-Gemini EMMolecular Devices Inc USA) (e decay of fluorescence wasrecorded every minute for 35min and the excitation andemission wavelengths were obtained at 494 nm and 535 nmrespectively Phosphate buffer (75mM pH 74) replacing thesample was used as the blank and Trolox was used as thestandard antioxidant Results were expressed as mg of Troloxequivalent per gram of extract

265 Ferrous Ion Chelating Activity (e ferrous ion che-lating (FIC) activity was measured by the decrease in theabsorbance at 562 nm of the iron (II)-ferrozine complex [21]EDTA was used as the reference standard and all the samplesand standards were prepared in distilled water (e samplewas mixed with 1mM ferrous sulfate and distilled waterfollowed by 1mM ferrozine solution(e ability of the sampleto chelate ferrous ions was calculated relative to the control(distilled water instead of the sample) by the following for-mula (e results were expressed as mg EDTA equivalent pergram of extract

Chelating activity CAB minus SAB( 1113857

CABtimes 100 (3)

where SAB is the absorbance of the sample and CAB is theabsorbance of the control

27 Determination of α-Amylase Inhibition Activity (eα-amylase inhibition activity was carried out using thedinitrosalicylic (DNS) method [22] where the inhibitionactivity was measured by quantifying the maltose liberatedunder the assay conditions (e enzyme inhibitory activitywas expressed as a decrease in units of maltose liberated Amodified dinitrosalicylic acid (DNS) method was adopted toestimate the maltose equivalent (e reaction volume con-taining 40 μL of 1 (wv) starch solution and 775 μL of100mM sodium acetate buffer was preincubated with 135 μLof the sample at 40degC shaking water bath for 10min (emixture was further incubated with 50 μL of the α-amylaseenzyme (5 μgmL) at 40degC shaking water bath for 15min(e reaction was terminated by adding 500 μL DNS reagentand the mixture was placed in a boiling water bath for 5minfollowed by cooling in an ice bath Absorbance was mea-sured at 540 nm using a 96-well microplate reader Anti-amylase activity (inhibition ) was calculated using thefollowing equation

Inhibition() AC minus AS minus Ab( 11138571113858 1113859

ACtimes 100 (4)

where AC is the absorbance of the control AS is the ab-sorbance of the sample and Ab is the absorbance of thesample blank

28 BSA-Glucose Glycation Inhibitory Activity AssayAntiglycation activity assay was performed with somemodifications to the previous methodology [23] for CR

Evidence-Based Complementary and Alternative Medicine 5

extracts of leaves and fruits of Syzygium caryophyllatum Amixture of 800 μgmL BSA 400mM glucose and 80 μL ofthe sample in 50mM phosphate buffer (pH 74) containingsodium azide (002) was incubated at 60degC for 40 h (e600 μL of the reaction mixture was transferred to 15mLmicrocentrifuge tubes and 60 μL of 100 (wv) TCA wasadded and sample mixtures were centrifuged at 15000 rpmat 4degC for 4min (e resultant advanced glycation endproducts-BSA (AGEs-BSA) precipitate was then dissolved in1mL of phosphate buffer saline (pH 10) and the fluores-cence intensity was measured at an excitation and emissionwavelengths of 370 nm and 440 nm using a 96-well fluo-rescence microplate reader (SpectraMax Gemini EMMolecular Devices Inc USA) Rutin was used as thepositive control and antiglycation activity ( inhibition) wascalculated using the following equation followed by thecalculation of IC50 value

Inhibition() Fc ndash Fb1113858 1113859 ndash Fs ndash Fsb1113858 1113859

Fc ndash Fb1113858 1113859times 100 (5)

where Fc is the fluorescence of the control Fb is the fluo-rescence of BSA Fs is the fluorescence of the sample andFsb is the fluorescence of the sample blank

All the above in vitro bioassay procedures were validatedat Herbal Technology Section Industrial Technology In-stitute Sri Lanka (ISO 9005 certified Laboratory)

29 Gas Chromatography-Mass Spectrometry (GC-MS)Analysis (e extracted essential oil from the leaves wassubjected to GC-MS analysis to identify volatile compounds(e analysis was carried out by using TRACE 1300 coupledto ISQ QD mass spectrophotometer instrument ((ermoFisher Scientific Milan Italy) (e sample was injected into(ermo-scientific TG-WAX capillary column(30mtimes 025mmtimes 025 μm) fused with silica having poly-ethylene glycol as the stationary phase with helium as thecarrier gas (1mLmin) (e temperature of the injector wasmaintained at 250degC (e GC oven temperature was initiallyprogrammed at a temperature of 60degC and was ramped up to220degC at the rate of 5degCmin GC-MS interface temperaturewas maintained at 250degC (e mass detector was operated inEI mode in a scan mass range of mz 40ndash450 (e identi-fication of compounds was ascertained by comparing themass spectral values with the known compounds in NIST11USA mass spectral database

210 Preparation of Plant Extracts for Mineral ContentDetermination Approximately 05 g of homogenized sam-ple was weighed into easy prep high-pressure microwavevessel and 100mL of 65 nitric acid was added (e easyprep microwave digestion program was followed to digestthe samples by using a microwave digester (CEM MARS 5USA) (e digested sample was quantitatively transferredand filtered using No 542 Whatman filter paper followed bywashing with deionized water and volume up to 25mL (eprepared solution was used to analyze mineral content byICP-MS (Agilent 7900 USA)

211 Statistical Analysis Statistical analysis was performedusing Graph Pad Prism (version 7) All experimental datawas analyzed by one-way analysis of variance (ANOVA)ANOVArsquos Turkey multiple comparison test was used tocompare the mean values Pearsonlsquos correlation coefficientwas used for the correlation analysis and in all cases p

values less than 005 (plt 005) were considered statisticallysignificant (e quantitative results of phytochemicals an-tioxidant potentials and enzyme inhibition values wereexpressed as meanplusmn standard deviation (SD)

3 Results

S caryophyllatum fruits and leaves were extractedaccording to the method described above and fractionatedinto nonpolar and polar solvents with increasing polarityhexane and ethyl acetate respectively (is solventndashsolventpartitioning procedure was allowed to generate hexane(Hex) fraction ethyl acetate (EA) fraction and aqueous(AQ) fraction which was labelled and stored in minus20degC priorto analysis (e yield was estimated to the dry weight whichranged from 06mg to 312 g where the highest yield wasrecorded in crude (CR) extract of fruits (312 g) and thelowest was recorded in Hex fraction of leaves (06mg) (eweights of the CR extracts Hex EA and AQ fractions aregiven in Table 1 and the yield was calculated as a percentage

All the crude extracts and fractions were subjected tofurther analysis except the hexane fraction of leaves ofSyzygium caryophyllatum due to the unavailability of ade-quate sample yield

31 Total Polyphenol Content (TPC) and Total FlavonoidContent (TFC) of Leaves and Fruit Extracts of Syzygiumcaryophyllatum Plant Extracts Total polyphenol contentwas expressed as milligram gallic acid equivalent per gram ofsample (mg GAEg) and the total flavonoid content asmilligram quercetin equivalent per gram of sample (mg QEg)Total polyphenol values varied from 00923mg GAEg ofsample to 818mg GAEg of sample and from 172mgGAEg of sample to 892mg GAEg of sample for leaves andfruits respectively Polyphenol contents of samples werediffered significantly (plt 005) Table 2 presents the TPCand TFC values for crude and fractions of samples (ehighest TPC value was recorded in CR extract of fruitsfollowed by CR extract of leaves and AQ fraction of leavesFlavonoid content of the tested samples varied significantly(plt 005) among extracts TFC ranged from 0001mg QEgof sample to 203mg QEg of sample and the highest TFCvalue was reported in CR extract of fruits with value as202plusmn 019mg QEg of the sample followed by Hex fractionand EA fraction of fruits with values 080plusmn 005mg QEg ofsample and 037plusmn 001mg QEg of sample respectively

32 In Vitro Antioxidant Properties of Different Extracts ofSyzygium caryophyllatum (e antioxidant properties ofcrudes and fractions of leaves and fruits of Syzygium car-yophyllatum were determined using DPPH ABTS ORACFRAP and iron chelation assay methods (e results

6 Evidence-Based Complementary and Alternative Medicine

obtained are depicted in Table 3 where the significant dif-ferences (plt 005) were observed between CR extracts ofleaves and fruits and among different fractions of leavesand fruits

321 DPPH Radical Scavenging Activity Results revealedthat the crude extract and fractions of S caryophyllatumhave the ability to scavenge radicals and depicted a dose-response relationship DPPH radical scavenging activityof different fractions of S caryophyllatum differed sig-nificantly (plt 005) (e EA fraction of leaves exhibitedthe lowest EC50 value followed by Hex and EA fraction offruits with values 912 plusmn 428 μgmL 10097 plusmn 819 μgmLand 13553 plusmn 983 μgmL respectively (e results ob-tained from the fractions showed that EA fractions ofleaves and Hex fractions of fruits exhibited high antiox-idant activities compared to other fractions It shows thatthe accumulation of a variety of antioxidant compoundsdepends on the polarity of the solvent (erefore thecompounds responsible for the high radical scavengingactivity of S caryophyllatum leaves are mainly mediumpolarity compounds (e dose-response relationships ofS caryophyllatum leaves and fruits are given inFigures 3(a) and 3(b)

322 ABTS+ Radical Scavenging Activity (e highestradical scavenging activity was reported in Hex fraction offruits with EC50 value 1501plusmn 065 μgmL followed by AQfraction of S caryophyllatum leaves and AQ fraction of Scaryophyllatum fruits and the EC50 values were 2703plusmn 206and 3145plusmn 143 μgmL respectively It was found that fruitsand leaves showed a significant difference (plt 005) among

fractions (e CR extracts and fractions of leaves and fruitsshowed significant difference and dose-dependent inhibitionon ABTS radical scavenging activity (Figures 4(a) and 4(b))

323 Ferric Reducing Antioxidant Power (FRAP) (e CRextracts of leaves and fruits differed significantly (plt 005) intheir FRAP values CR extract of leaves 4827plusmn 811mgTEgof extract and CR extract of fruits 681plusmn 117mgTEg ofextract (e FRAP values of the fractions ranged from1481mgTEg of the extract to 27041mgTEg of extract EAfraction of leaves was reported as the highest among thetested fractions in leaves and fruits where the AQ fraction offruits exhibited the lowest values (ese results suggestedthat EA fraction of leaves and fruits exhibited the highestferric reducing property among tested fractions whichexplains the important behavior of the polar compounds inits reducing property

324 Oxygen Radical Absorbance Capacity (ORAC) (eORAC values of fractions ranged from 1723mgTEg of theextract to 34732mgTEg of extract (e highest activity wasrecorded in EA fraction of leaves following the AQ fractionof leaves and the lowest was recorded in AQ fraction infruits (e CR extract of leaves exhibited a significantlyhigher (plt 005) activity compared to the CR extract offruits with values 22100plusmn 3496 and 4598plusmn 258mgTEg ofextract respectively

325 Iron Chelating Activity (e highest iron chelatingability of CR extract was reported in leaves with values332plusmn 168mg EDTAg of extract and fruits were reported as1373plusmn 069mg EDTAg of extract (e Hex fraction of

Table 1 (e yield of Syzygium caryophyllatum plant extracts

Scientific name Part of plant Extract Weight of concentrated extract (g) Yield

Syzygium caryophyllatum

Leaves

CR 17500 70Hex lt0001 lt01EA 0114 01AQ 15984 71

Fruits

CR 31200 620Hex 1950 26EA 1531 20AQ 10165 136

CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction

Table 2 Total polyphenol content (TPC) and total flavonoid content (TFC) in different extracts of Syzygium caryophyllatum

Scientific name Part of plant Extract TPC (mg GAEg of sample) TFC (mg QEg of sample)

Syzygium caryophyllatum

LeavesCR 812plusmn 046a 016plusmn 001aEA 009plusmn 001b lt001AQ 712plusmn 016c 018plusmn 002ab

Fruits

CR 883plusmn 062ad 202plusmn 019cHex 342plusmn 029e 080plusmn 005dEA 173plusmn 076f 037plusmn 001eAQ 216plusmn 012g 010plusmn 001af

Results expressed as meanplusmn standard deviation CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction Mean valueswithin a column superscripted by different letters are significantly different at plt 005

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Antioxidant potentials of leaves and fruits of Syzygium caryophyllatum

Scientific name Part of plant Extract ORAC mg TEgof extract

FRAP mg TEgof extract

EC50 DPPHμgmL

EC50 ABTSμgmL

Iron chelation mgEDTAg of extract

Syzygiumcaryophyllatum

LeavesCR 22100plusmn 3496a 4827plusmn 811a 50020plusmn 4433a 10793plusmn 1507a 332plusmn 168aEA 34431plusmn 337b 26226plusmn 4393b 9120plusmn 428b 4061plusmn 276b 3601plusmn 698aAQ 32097plusmn 2062bc 7342plusmn 1258c 25105plusmn 1752c 2704plusmn 206c 5617plusmn 798b

Fruit

CR 4598plusmn 258d 681plusmn 117d 87372plusmn 1252d 12808plusmn 612d 1373plusmn 069cHex 3277plusmn 141e 5391plusmn 1230a 10097plusmn 819e 1501plusmn 065e 7482plusmn 266dEA 11865plusmn 760f 6307plusmn 1017e 13553plusmn 983f 8954plusmn 388f 828plusmn 040eAQ 2093plusmn 449g 1545plusmn 068f 94642plusmn 6504g 3145plusmn 145g mdash

Results are expressed as meanplusmn standard deviation n 3 Mean values within a column superscripted by different letters are significantly different at plt 005CR crude Hex hexane EA ethyl acetate AQ aqueous ORAC oxygen radical absorbance capacity FRAP ferric reducing antioxidant power ABTSABTS radical scavenging activity DPPH DPPH radical scavenging activity

0

20

40

60

80

100

0 200 400 600 800

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

00 05 10 15 20

Inhi

bitio

n

Concentration (mgmL)

CRHex

EAAQ

(b)

Figure 3 Dose-response curves for DPPH radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

120

0 50 100 150 200 250

Inhi

bitio

n

Concentration (microgmL)

CRHex

EAAQ

(b)

Figure 4 Dose-response curves for ABTS+ radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

8 Evidence-Based Complementary and Alternative Medicine

fruits exhibited the highest iron-chelating ability among thetested fractions with value 7482plusmn 266mg EDTAg of ex-tract and the lowest was reported in EA fraction of fruitswith value 828plusmn 040mg EDTAg of extract respectively(e AQ fraction of Syzygium caryophyllatum fruits did notshow a significant chelating activity where the highestconcentration (215mgmL) exhibited 1605 inhibition

33 Correlation Analysis between Polyphenol Content andDPPH Radical Scavenging Activity of Syzygium car-yophyllatum Fruits (e correlation coefficient betweenTPC content and DPPH of Syzygium caryophyllatum fruitsexhibited a significant (plt 005) positive correlation withPearson r value 09921 (Figure 5)

34 Ce α-Amylase Inhibitory Activity (e α-amylase inhi-bition potentials of CR extract Hex EA and AQ fraction ofleaves and fruits were analyzed Fruits exhibited significantinhibition of α-amylase activities where the leaves did notexhibit significant inhibitory activities (e Hex fraction ofS caryophyllatum fruits was reported to have the highestinhibitory activity with IC50 value 4720 plusmn 03 μgmL fol-lowed by CR extract EA fraction and AQ fraction withIC50 values 21826 plusmn 538 μgmL 61885plusmn 1855 μgmL and101455plusmn 8555 μgmL respectively Interestingly the Hexfraction of fruits demonstrated potent α-amylase inhibitionactivity (IC50 4720 plusmn 03 μgmL) compared to the standarddrug acarbose (IC50 8796 plusmn 143 μgmL)(e percentage ofα-amylase inhibitions of crudes and fractions was plottedagainst concentration (Figure 6) where it showed a dose-dependent increase in percentage activity of CR extract andthree types of fractions of fruits

341 Ce α-Amylase Inhibitory Activity of Isolated Fractionsof Hexane Fraction of Syzygium caryophyllatum Fruits

(1) Hexane and Ethyl Acetate Solvents on Isolation Procedure(e inhibition potential of compound mixtures of EA fractionwas evaluated for α-amylase inhibitory activity (e highesttested concentration was 200μgmL followed by 100μgmL50μgmL and 25μgmL(e similar inhibition potentials wereobserved for the first three concentrations and the inhibitionvalues were 5763plusmn018 5599plusmn160 and 5730plusmn018respectively where the inhibition potential of 25μgmL wasobserved as a rapid drop to the inhibition 1278plusmn045Further inhibition potentials were evaluated between 50μgmLand 25μgmL (Table 4)(e α-amylase inhibitory activity of thetested fraction showed a poor linear response (R2 06384)hence IC50 was not calculated

(2) Dichloromethane and Ethyl Acetate Solvents on Iso-lation Procedure (e α-amylase inhibitory activity ofdifferent combined fractions was analyzed (C1 C2 andC3)(e inhibitory activities of C1 and C2 fractions did notexhibit significant inhibitory activities where the fractionC3 showed significant (plt 005) inhibitory activity for the

screened concentrations (e fraction C3 was dose-de-pendent and IC50 value was 227 plusmn 181 μgmL

(e summary of α-amylase inhibitory activity of hexanefraction and its chromatographic fraction is given in Table 5

35 Antiglycation Activity (e antiglycation activity ofcrude extracts of leaves and fruits was screened for threeconcentrations 200 100 and 50 μgmL (Table 6) (e CRextract of leaves exhibited the highest inhibitory activity(9361 plusmn 853 at a concentration of 200 μgmL) followedby CR extract of S caryophyllatum fruits (4797 plusmn 263)(e standard drug rutin exhibited antiglycation activitywith IC50 value as 3423 plusmn 318 μgmL (e IC50 value ofCR extract of leaves was 5783 plusmn 791 μgmL Howeverfor fruits similar inhibitions were observed for differentdoses hence IC50 value could not be calculated

36 Chemical Compositions of Syzygium caryophyllatum LeafEssential Oil Syzygium caryophyllatum leaves yieldedessential oil upon hydrodistillation GC-MS analysisidentified 58 compounds from the essential oil and theretention time and area of the identified major peaks arelisted in Table 7 (e major compounds identified in theleaves were phytol with a 2466 peak area (retentiontime 3444) α-cadinol with a 387 peak area (retentiontime 2781) α-guaiene with a 384 peak area (retentiontime 2629) cyclosiolongifolene 910-dehydro with a382 peak area (retention time 2826) humulene with a336 peak area (retention time 1645) and car-yophyllene with a 321 peak area (retention time 1479)respectively (e GC-MS chromatogram of Syzygiumcaryophyllatum leaf oil is presented in Figure 7

37 Mineral Compositions Functionally important micro-minerals such as selenium (Se) iron (Fe) strontium (Sr)manganese (Mn) cobalt (Co) copper (Cu) and the toxicheavymetals such as arsenic (As) cadmium (Cd) chromium(Cr) and lead (Pb) were evaluated (Table 8) (e ICP-MS

0

20

40

60

80

100

0 50 100 150

DPP

H v

alue

(mg

TEg

of e

xtra

ct)

Total phenol content (mg GAEg of extract)

Figure 5 Correlation of Syzygium caryophyllatum fruits TPCversus DPPH values (plt 005)

Evidence-Based Complementary and Alternative Medicine 9

data ranged from 009 to 45370mgkg of sample Sodium(Na) was found to be the highest (leaves 435768mgkg ofthe sample) while Se was found to be the lowest (fruits009mgkg of the sample) (e leaves were found to have thehighest concentration of the tested minerals compared to thefruits (e second richest macroelement was found to be K(leaves 355925mgkg and fruits 294428mgkg) followedby Mg (leaves 258040mgkg) and Ca (leaves 184352mgkg)(e maximum amount of Al Mn Fe and Zn was found

to be in leaves with values 52445mgkg 29882mgkg22198mgkg and 2160mgkg respectively Among thetested microelements Sr was found to be the richest with avalue of 32889mgkg in leaves which is significantly(plt 005) different from fruits (270mgkg) (e Cu and Crwere found in similar small concentrations in leaves (Cu491mgkg and Cr 403mgkg) and fruits (Cu 523mgkgand Cr 465mgkg) (e maximum concentration of Sewas determined to be 085mgkg in leaves and the fruitsexhibited the lowest concentration with a value of 009mgkg(e investigation of the level of Co revealed that the maxi-mum concentration of the element exists as 0128mgkg inleaves while in the fruits it was not detected at the detectionlimit of 005mgkg

(e analysis of toxic heavy metals revealed the presenceof a maximum concentration of Pb in leaves (102mgkg)and the lowest in fruits (015mgkg) Besides the concen-trations of toxic heavy metals such as Cd and As were notdetected in leaves and fruits (limit of detection 005mgkg)

4 Discussion

Diabetes mellitus (DM) is a complex metabolic disordercharacterized by hyperglycemia which is mainly associatedwith malfunctions and abnormalities in carbohydrate pro-tein and fat metabolism Digestion and absorption of car-bohydrates to the blood stream also have a significantinfluence on DM Further the increased production of re-active oxygen species (ROS) under DM accelerates the proteinglycation process [24] (e production and accumulation ofglycated proteins have shown a strong association withcomplications of DM [25] (us the management of DM isdirected toward addressing multiple targets including re-duction of postprandial carbohydrate levels in the bloodimprovement in the homeostatic and metabolic process ofcarbohydrates and lipids an improvement on the efficiency ofantioxidant defense and control of protein glycation [26]

In this regard WHO recommends functional ingredi-ents and supplements with multiple functional propertiesincluding antidiabetic and the antiradical properties in thecontrol of the DM [27]

In this study functional properties including α-amylaseinhibition protein glycation inhibition and antioxidantactivities of S caryophyllatum fruit and leaf extracts havebeen investigated along with chemical constituents andmineral analyses (e results revealed the potential of Scaryophyllatum as a source of supplement for DM man-agement To the best of our knowledge the present study isthe first study reported in Sri Lanka on functional activitiesof S caryophyllatum fruit and leaf extracts Crude extracts ofboth fruits and leaves showed significant antioxidant andmoderate antiglycation activities However only fruit ex-tracts showed α-amylase inhibitory activity Even thoughboth fruits and leaves CR were tested against α-glucosidaseenzyme inhibitory activity none of them showed a signif-icant inhibitory activity at a maximum dose of 400 μgmL(data not shown) Solventndashsolvent partitioning of both CRwas used to separate and valuate the most potent fractionswith α-amylase inhibition activity

0

20

40

60

80

100

0 200 400 600 800 1000

Inhi

bitio

n

Concentration (μgmL)

CRHex

EAAQ

Figure 6 Dose-response relationship of crude and fractionsof Syzygium caryophyllatum fruits for alpha-amylase inhibi-tory activity (CR crude Hex hexane EA ethyl acetateAQ aqueous)

Table 4 (e α-amylase inhibitory activity of isolated compoundmixtures of Hex fraction of Syzygium caryophyllatum fruits (hexaneand ethyl acetate solvents on isolation procedure)

Assay concentration μgmL Inhibition 200 5763plusmn 018a100 5599plusmn 160a50 5730plusmn 018a45 2339plusmn 021b40 2245plusmn 079b35 2143plusmn 019b30 1433plusmn 083c25 1278plusmn 045c

Results are expressed as meanplusmn standard deviation n 3lowast Mean valueswithin a column superscripted by different letters are significantly differentat plt 005

Table 5 (e α-amylase inhibitory activity of hexane fraction andits chromatographic fraction of Syzygium caryophyllatum fruits

Sample IC50 (μgmL)Hex fraction 4720aplusmn 030Hex EA (0 100) NLRC3 DCM EA (95 5) 227bplusmn 181Standard drug acarbose 8796cplusmn 143Results are expressed as meanplusmn standard deviation n 3lowast NLRno linearresponse Mean values within a column superscripted by different lettersare significantly different at plt 005 Hex hexane EA ethyl acetateDCM dichloromethane C3 combined fraction 3

10 Evidence-Based Complementary and Alternative Medicine

Hexane fraction of fruit extract exhibited the highestinhibition activity with IC50 value 4720plusmn 037 μgmL and issignificantly higher compared to acarbose (IC508796plusmn 143 μgmL) (erefore it was further separated bycolumn chromatography (e fraction C3 which was iso-lated on elution with DCM EA 95 5 exhibited a significant(plt 005) α-amylase inhibitory activity with an IC50 value of227plusmn 181 μgmL (e compound or the mixture of

compounds responsible for the α-amylase inhibition activitymay be in the C3 compound mixture which is required to besubjected to further isolation and identification Inhibitionmay be due to the presence of potential alpha-amylase in-hibitors such as alkaloids tannin polyphenols and flavo-noids (ese inhibitors can also act as starch blockers as theyblock the hydrolysis of 14-glycosidic linkages of starch andoligosaccharides [28] Hex fraction of S caryophyllatum fruit

Table 6 Antiglycation activity of crude extracts of Syzygium caryophyllatum

Crude extract Inhibition

IC50 (μgmL)Concentration (μgmL)50 100 200

Leaves 5488plusmn 310a 7987plusmn 594a 9361plusmn 850a 5783plusmn 791Fruits 4131plusmn 789b 5666plusmn 836b 4797plusmn 263b mdashStandard rutin IC50 3423plusmn 318 μgmLResults are expressed as meanplusmn standard deviation n 3lowast Mean values within a column superscripted by different letters are significantly different atplt 005

Table 7 Major compounds detected in Syzygium caryophyllatum leaf essential oil

Peak Compound Formula and lowastmol wt RT Area SI RSI1 Caryophyllene C15H24 204 1479 321 954 9542 Humulene C15H24 204 1645 336 907 9073 α-Cadinol C15H26O 222 2781 387 923 9284 Cyclosiolongifolene 910-dehydro C17H26O2 202 2826 382 790 8005 Phytol C20H40O 296 3444 2466 927 927lowastMol wtmolecular weight

2781

RT 000ndash4200100

90

80

70

60

50

40

30

20

10

0 5 10 15 20 25 30 35 40Retention time (min)

3444

2826

Rela

tive a

bund

ance

1 24

3

5

1479 1645

Figure 7 GC-MS chromatogram of Syzygium caryophyllatum leaf essential oil

Table 8 Mineral compositions of leaves and fruits of Syzygium caryophyllatum

Mineral elements (mgkg per sample)K Fe Na Ca Mg Mn Cu Zn Cr Zn Sr Se Pb As Cd

Syzygiumcaryophyllatum

Leaves 355925 22198 435768 184352 258039 29882 491 2160 403 2159 3289 085 102 ND NDFruits 294428 4955 75621 14927 85555 1814 523 775 465 775 270 009 015 ND ND

RDA 35 g 9ndash15mg 15 g 1 g 280ndash350mg 50ndash60 μglowastND not detected limit of detection 005mgkg RDA recommended daily intake

Evidence-Based Complementary and Alternative Medicine 11

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

232 Preparation of Fruit Crude Extract of Syzygiumcaryophyllatum (e ripen fruits (30 g dry weight of sample)without seed were extracted with ethanol by overnight coldextraction with stirring (e ethanol extract was concentratedin a vacuum at 40degC

(e crude extracts of leaves and fruits were fractionatedinto hexane and ethyl acetate by using separation funnels andall concentrated samples were stored at minus20degC prior to theanalyses (e extracts were evaporated to dryness by rotaryevaporator and a known amount of sample was dissolved inDMSO for further analyses

24 Compound Isolation from Hexane Fraction of Syzygiumcaryophyllatum Fruits (e fraction was separated usingcolumn chromatography with different organic solvents as themobile phase A column of 75 cm height and 25 cm diameterwas packed with silica gel 100ndash200 mesh with hexane as asolvent in the wet packing method Compound isolation wascarried out in two different methods by using different solventson the isolation procedure

241 Hexane and Ethyl Acetate Solvents on Isolation Pro-cedure (Hex and EA) (e 50mg of Hex fraction of fruitswas separated by column by increasing polarity of eluenthexane and ethyl acetate 100 0 and 0 100 respectively(e two separated isolates were tested for α-amylase in-hibition activity

242 Hexane Dichloromethane and Ethyl Acetate Solventson Isolation Procedure (Hex DCM and EA) Columnchromatography was performed using 200mg of Hexfraction of fruits to obtain fractions by increasing polarityusing a gradient elution technique as follows Fractions(FR) 1ndash10 (Hex) FR 11ndash12 (Hex DCM 3 1) FR 13(Hex DCM 2 1) FR 14ndash19 (DCM) and FR 20ndash22(DCM EA 95 5) to obtain 518mg (C1 combinedfractions 1ndash13) 957 mg (C2 combined fractions 14ndash19)and 68mg (C3 combined fractions 20ndash22) respectivelyAll combined fractions were tested for α-amylase inhi-bition activity

Flowchart of the overall methodology is shown inFigure 2

Leaves and fruits of Syzygium caryophyllatum plantwere extracted fractionated and subjected for phyto-chemical analysis antioxidant antiamylase antiglycationpotentials analysis and compounds isolation Chemicalcompositions of Syzygium caryophyllatum leaf oil wereanalyzed by GC-MS and mineral composition of leavesand fruits were analyzed by ICP-MS

25 Estimation of Phytochemical Compounds

251 Determination of Total Polyphenol Content (TPC)(e total polyphenol content (TPC) was determined using theFolin-Ciocalteu (FC) reagent method [17] with minor modi-fication using 96-well microplates Freshly prepared 110μL of

(a)

(b) (c)

Figure 1 Different parts of the Syzygium caryophyllatum plant (a) leaf (b) flowers and (c) fruits

Evidence-Based Complementary and Alternative Medicine 3

FC Reagent was added to 20μL of the sample (e mixture wastreated with 10 sodium carbonate and incubated at roomtemperature for 30min and absorbance was measured at765nm Gallic acid was used as a reference standard and theresults were expressed as milligram gallic acid equivalent pergram of sample (mg GAEg of sample)

252 Determination of Total Flavonoid Content (TFC)(e total flavonoid content (TFC) was determined using analuminum chloride colorimetric method using 96-wellmicroplates [18] Extracts were dissolved in methanol and100 μL of the sample wasmixed with 100 μL of 2 aluminum

chloride (e mixture was incubated at room temperaturefor 10min and absorbance was measured at 415nm Quercetinwas used as a reference standard and the results wereexpressed as milligram quercetin equivalent per gram ofsample (mg QEg of sample)

26 Evaluation of Antioxidant Activity

261 Ferric Reducing Antioxidant Power (FRAP) Assay(e assay method was based on the reduction of Fe3+ to Fe2+by the electron donation ability of antioxidants resulting inan intense blue-colored complex (Fe2+-tripyridyltriazine)

Syzygium caryophyllatum

Leaves Fruits

Distillation Reflux Cold extraction

Crude extract

Fractionation

Hexane

Aqueous fractionEthyl acetate

Crude extract Aqueous fraction Ethyl acetate fraction Hexane fraction

AntioxidantAntiamylase

Antiglycation

Hexane(100)

EA(100)

Hexane (100) Hex DCM (3 1) Hex DCM (2 1) DCM (100) DCM EA (95 5)

Fr 1-10 Fr 11-12 Fr 13 Fr 14-19 Fr 20-22

C1 C3C2

Anti-amylase

Chemical composition analysis-GC-MS

Mineral composition analysisndashICP-MS

Solvent system IIHex amp EA

Silica gel column chromatography

Oil

Hexane fraction offruit

Solvent system IHex DCM amp EA

Figure 2 Flow chart depicting the methodology of the study

4 Evidence-Based Complementary and Alternative Medicine

[18] (e FRAP reagent was prepared freshly by mixingacetate buffer (300mM pH 36) solution of TPTZ in HCland FeCl3 (20mM) in 10 1 1 ratio and incubated at 37degCTwo hundred microliters of the reaction mixture (FRAPreagent and sample) was incubated at room temperatureand absorbance was measured at 600 nm Vitamin E ana-logue Trolox was used as the reference standard where theresults were expressed as mg of Trolox equivalent per gramof extract (mg TEg of extract)

262 DPPH Radical Scavenging Assay (e radical scav-enging activity was determined by using 1 1-diphenyl-2-picryl-hydrazyl (DPPH) stable radical method [18] (e method isbased on the reduction of purple-colored 1 1-diphenyl-2-picrylhydrazyl to a yellow-colored diphenylpicrylhydrazine(ereaction volume of 200μL containing sample (50μL)methanolandDPPHwere incubated at room temperature for 10min andabsorbance was measured at 517nm A percentage inhibitionversus concentration was plotted and the concentration of thesample required for 50 inhibition was determined and rep-resented as EC50 value (e following formula was used tocalculate the percent inhibition

Radical scavenging activity CAB minus SAB( 1113857

CABtimes 100 (1)

where CAB is the absorbance of the control and SAB is theabsorbance of the sample

263 ABTS Radical Scavenging Activity (e ABTS+ radicalcation (ABTS+) scavenging activity assay was performed [19]with some modifications in 96-well microplates Plant ex-tracts were prepared in phosphate buffer solutions and wereallowed to react with ABTS+ radicals in a microwell plate for10min Absorbance wasmeasured in 734 nm and Trolox wasused as the standard (e following formula was used tocalculate the percent inhibition

Radical scavenging activity CAB minus SAB( 1113857

CABtimes 100 (2)

where CAB is the absorbance of the control and SAB is theabsorbance of the sample

(e antiradical activity was expressed as EC50 theconcentration required to cause 50 inhibition of ABTS+radicals

264 Oxygen Radical Absorbance Capacity (ORAC) Assay(eORAC assay is a hydrogen atom transfer- (HAT-) basedassay which measures the depletion of fluorescence intensityof a fluorescent probe upon the generation of peroxy radicalsdue to the oxidation of azo compounds In the presence of anantioxidant compound the decaying of fluorescent intensityis retarded (e overall decay is calculated by the differencebetween the area under the fluorescence decay curve (AUC)of the sample of interest and AUC of blank [20](e reactionvolume of 200 μL (pH 74) containing fluorescein (100 μL)and sample (50 μL) was preincubated at 37degC for 5min (e22prime-azobis (2-amidinopropane) dihydrochloride (AAPH)

solution was added (50 μL) to the mixture and the plate(black 96 well plates) was immediately placed in a fluo-rescent microplate reader (SPECTRAmax-Gemini EMMolecular Devices Inc USA) (e decay of fluorescence wasrecorded every minute for 35min and the excitation andemission wavelengths were obtained at 494 nm and 535 nmrespectively Phosphate buffer (75mM pH 74) replacing thesample was used as the blank and Trolox was used as thestandard antioxidant Results were expressed as mg of Troloxequivalent per gram of extract

265 Ferrous Ion Chelating Activity (e ferrous ion che-lating (FIC) activity was measured by the decrease in theabsorbance at 562 nm of the iron (II)-ferrozine complex [21]EDTA was used as the reference standard and all the samplesand standards were prepared in distilled water (e samplewas mixed with 1mM ferrous sulfate and distilled waterfollowed by 1mM ferrozine solution(e ability of the sampleto chelate ferrous ions was calculated relative to the control(distilled water instead of the sample) by the following for-mula (e results were expressed as mg EDTA equivalent pergram of extract

Chelating activity CAB minus SAB( 1113857

CABtimes 100 (3)

where SAB is the absorbance of the sample and CAB is theabsorbance of the control

27 Determination of α-Amylase Inhibition Activity (eα-amylase inhibition activity was carried out using thedinitrosalicylic (DNS) method [22] where the inhibitionactivity was measured by quantifying the maltose liberatedunder the assay conditions (e enzyme inhibitory activitywas expressed as a decrease in units of maltose liberated Amodified dinitrosalicylic acid (DNS) method was adopted toestimate the maltose equivalent (e reaction volume con-taining 40 μL of 1 (wv) starch solution and 775 μL of100mM sodium acetate buffer was preincubated with 135 μLof the sample at 40degC shaking water bath for 10min (emixture was further incubated with 50 μL of the α-amylaseenzyme (5 μgmL) at 40degC shaking water bath for 15min(e reaction was terminated by adding 500 μL DNS reagentand the mixture was placed in a boiling water bath for 5minfollowed by cooling in an ice bath Absorbance was mea-sured at 540 nm using a 96-well microplate reader Anti-amylase activity (inhibition ) was calculated using thefollowing equation

Inhibition() AC minus AS minus Ab( 11138571113858 1113859

ACtimes 100 (4)

where AC is the absorbance of the control AS is the ab-sorbance of the sample and Ab is the absorbance of thesample blank

28 BSA-Glucose Glycation Inhibitory Activity AssayAntiglycation activity assay was performed with somemodifications to the previous methodology [23] for CR

Evidence-Based Complementary and Alternative Medicine 5

extracts of leaves and fruits of Syzygium caryophyllatum Amixture of 800 μgmL BSA 400mM glucose and 80 μL ofthe sample in 50mM phosphate buffer (pH 74) containingsodium azide (002) was incubated at 60degC for 40 h (e600 μL of the reaction mixture was transferred to 15mLmicrocentrifuge tubes and 60 μL of 100 (wv) TCA wasadded and sample mixtures were centrifuged at 15000 rpmat 4degC for 4min (e resultant advanced glycation endproducts-BSA (AGEs-BSA) precipitate was then dissolved in1mL of phosphate buffer saline (pH 10) and the fluores-cence intensity was measured at an excitation and emissionwavelengths of 370 nm and 440 nm using a 96-well fluo-rescence microplate reader (SpectraMax Gemini EMMolecular Devices Inc USA) Rutin was used as thepositive control and antiglycation activity ( inhibition) wascalculated using the following equation followed by thecalculation of IC50 value

Inhibition() Fc ndash Fb1113858 1113859 ndash Fs ndash Fsb1113858 1113859

Fc ndash Fb1113858 1113859times 100 (5)

where Fc is the fluorescence of the control Fb is the fluo-rescence of BSA Fs is the fluorescence of the sample andFsb is the fluorescence of the sample blank

All the above in vitro bioassay procedures were validatedat Herbal Technology Section Industrial Technology In-stitute Sri Lanka (ISO 9005 certified Laboratory)

29 Gas Chromatography-Mass Spectrometry (GC-MS)Analysis (e extracted essential oil from the leaves wassubjected to GC-MS analysis to identify volatile compounds(e analysis was carried out by using TRACE 1300 coupledto ISQ QD mass spectrophotometer instrument ((ermoFisher Scientific Milan Italy) (e sample was injected into(ermo-scientific TG-WAX capillary column(30mtimes 025mmtimes 025 μm) fused with silica having poly-ethylene glycol as the stationary phase with helium as thecarrier gas (1mLmin) (e temperature of the injector wasmaintained at 250degC (e GC oven temperature was initiallyprogrammed at a temperature of 60degC and was ramped up to220degC at the rate of 5degCmin GC-MS interface temperaturewas maintained at 250degC (e mass detector was operated inEI mode in a scan mass range of mz 40ndash450 (e identi-fication of compounds was ascertained by comparing themass spectral values with the known compounds in NIST11USA mass spectral database

210 Preparation of Plant Extracts for Mineral ContentDetermination Approximately 05 g of homogenized sam-ple was weighed into easy prep high-pressure microwavevessel and 100mL of 65 nitric acid was added (e easyprep microwave digestion program was followed to digestthe samples by using a microwave digester (CEM MARS 5USA) (e digested sample was quantitatively transferredand filtered using No 542 Whatman filter paper followed bywashing with deionized water and volume up to 25mL (eprepared solution was used to analyze mineral content byICP-MS (Agilent 7900 USA)

211 Statistical Analysis Statistical analysis was performedusing Graph Pad Prism (version 7) All experimental datawas analyzed by one-way analysis of variance (ANOVA)ANOVArsquos Turkey multiple comparison test was used tocompare the mean values Pearsonlsquos correlation coefficientwas used for the correlation analysis and in all cases p

values less than 005 (plt 005) were considered statisticallysignificant (e quantitative results of phytochemicals an-tioxidant potentials and enzyme inhibition values wereexpressed as meanplusmn standard deviation (SD)

3 Results

S caryophyllatum fruits and leaves were extractedaccording to the method described above and fractionatedinto nonpolar and polar solvents with increasing polarityhexane and ethyl acetate respectively (is solventndashsolventpartitioning procedure was allowed to generate hexane(Hex) fraction ethyl acetate (EA) fraction and aqueous(AQ) fraction which was labelled and stored in minus20degC priorto analysis (e yield was estimated to the dry weight whichranged from 06mg to 312 g where the highest yield wasrecorded in crude (CR) extract of fruits (312 g) and thelowest was recorded in Hex fraction of leaves (06mg) (eweights of the CR extracts Hex EA and AQ fractions aregiven in Table 1 and the yield was calculated as a percentage

All the crude extracts and fractions were subjected tofurther analysis except the hexane fraction of leaves ofSyzygium caryophyllatum due to the unavailability of ade-quate sample yield

31 Total Polyphenol Content (TPC) and Total FlavonoidContent (TFC) of Leaves and Fruit Extracts of Syzygiumcaryophyllatum Plant Extracts Total polyphenol contentwas expressed as milligram gallic acid equivalent per gram ofsample (mg GAEg) and the total flavonoid content asmilligram quercetin equivalent per gram of sample (mg QEg)Total polyphenol values varied from 00923mg GAEg ofsample to 818mg GAEg of sample and from 172mgGAEg of sample to 892mg GAEg of sample for leaves andfruits respectively Polyphenol contents of samples werediffered significantly (plt 005) Table 2 presents the TPCand TFC values for crude and fractions of samples (ehighest TPC value was recorded in CR extract of fruitsfollowed by CR extract of leaves and AQ fraction of leavesFlavonoid content of the tested samples varied significantly(plt 005) among extracts TFC ranged from 0001mg QEgof sample to 203mg QEg of sample and the highest TFCvalue was reported in CR extract of fruits with value as202plusmn 019mg QEg of the sample followed by Hex fractionand EA fraction of fruits with values 080plusmn 005mg QEg ofsample and 037plusmn 001mg QEg of sample respectively

32 In Vitro Antioxidant Properties of Different Extracts ofSyzygium caryophyllatum (e antioxidant properties ofcrudes and fractions of leaves and fruits of Syzygium car-yophyllatum were determined using DPPH ABTS ORACFRAP and iron chelation assay methods (e results

6 Evidence-Based Complementary and Alternative Medicine

obtained are depicted in Table 3 where the significant dif-ferences (plt 005) were observed between CR extracts ofleaves and fruits and among different fractions of leavesand fruits

321 DPPH Radical Scavenging Activity Results revealedthat the crude extract and fractions of S caryophyllatumhave the ability to scavenge radicals and depicted a dose-response relationship DPPH radical scavenging activityof different fractions of S caryophyllatum differed sig-nificantly (plt 005) (e EA fraction of leaves exhibitedthe lowest EC50 value followed by Hex and EA fraction offruits with values 912 plusmn 428 μgmL 10097 plusmn 819 μgmLand 13553 plusmn 983 μgmL respectively (e results ob-tained from the fractions showed that EA fractions ofleaves and Hex fractions of fruits exhibited high antiox-idant activities compared to other fractions It shows thatthe accumulation of a variety of antioxidant compoundsdepends on the polarity of the solvent (erefore thecompounds responsible for the high radical scavengingactivity of S caryophyllatum leaves are mainly mediumpolarity compounds (e dose-response relationships ofS caryophyllatum leaves and fruits are given inFigures 3(a) and 3(b)

322 ABTS+ Radical Scavenging Activity (e highestradical scavenging activity was reported in Hex fraction offruits with EC50 value 1501plusmn 065 μgmL followed by AQfraction of S caryophyllatum leaves and AQ fraction of Scaryophyllatum fruits and the EC50 values were 2703plusmn 206and 3145plusmn 143 μgmL respectively It was found that fruitsand leaves showed a significant difference (plt 005) among

fractions (e CR extracts and fractions of leaves and fruitsshowed significant difference and dose-dependent inhibitionon ABTS radical scavenging activity (Figures 4(a) and 4(b))

323 Ferric Reducing Antioxidant Power (FRAP) (e CRextracts of leaves and fruits differed significantly (plt 005) intheir FRAP values CR extract of leaves 4827plusmn 811mgTEgof extract and CR extract of fruits 681plusmn 117mgTEg ofextract (e FRAP values of the fractions ranged from1481mgTEg of the extract to 27041mgTEg of extract EAfraction of leaves was reported as the highest among thetested fractions in leaves and fruits where the AQ fraction offruits exhibited the lowest values (ese results suggestedthat EA fraction of leaves and fruits exhibited the highestferric reducing property among tested fractions whichexplains the important behavior of the polar compounds inits reducing property

324 Oxygen Radical Absorbance Capacity (ORAC) (eORAC values of fractions ranged from 1723mgTEg of theextract to 34732mgTEg of extract (e highest activity wasrecorded in EA fraction of leaves following the AQ fractionof leaves and the lowest was recorded in AQ fraction infruits (e CR extract of leaves exhibited a significantlyhigher (plt 005) activity compared to the CR extract offruits with values 22100plusmn 3496 and 4598plusmn 258mgTEg ofextract respectively

325 Iron Chelating Activity (e highest iron chelatingability of CR extract was reported in leaves with values332plusmn 168mg EDTAg of extract and fruits were reported as1373plusmn 069mg EDTAg of extract (e Hex fraction of

Table 1 (e yield of Syzygium caryophyllatum plant extracts

Scientific name Part of plant Extract Weight of concentrated extract (g) Yield

Syzygium caryophyllatum

Leaves

CR 17500 70Hex lt0001 lt01EA 0114 01AQ 15984 71

Fruits

CR 31200 620Hex 1950 26EA 1531 20AQ 10165 136

CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction

Table 2 Total polyphenol content (TPC) and total flavonoid content (TFC) in different extracts of Syzygium caryophyllatum

Scientific name Part of plant Extract TPC (mg GAEg of sample) TFC (mg QEg of sample)

Syzygium caryophyllatum

LeavesCR 812plusmn 046a 016plusmn 001aEA 009plusmn 001b lt001AQ 712plusmn 016c 018plusmn 002ab

Fruits

CR 883plusmn 062ad 202plusmn 019cHex 342plusmn 029e 080plusmn 005dEA 173plusmn 076f 037plusmn 001eAQ 216plusmn 012g 010plusmn 001af

Results expressed as meanplusmn standard deviation CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction Mean valueswithin a column superscripted by different letters are significantly different at plt 005

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Antioxidant potentials of leaves and fruits of Syzygium caryophyllatum

Scientific name Part of plant Extract ORAC mg TEgof extract

FRAP mg TEgof extract

EC50 DPPHμgmL

EC50 ABTSμgmL

Iron chelation mgEDTAg of extract

Syzygiumcaryophyllatum

LeavesCR 22100plusmn 3496a 4827plusmn 811a 50020plusmn 4433a 10793plusmn 1507a 332plusmn 168aEA 34431plusmn 337b 26226plusmn 4393b 9120plusmn 428b 4061plusmn 276b 3601plusmn 698aAQ 32097plusmn 2062bc 7342plusmn 1258c 25105plusmn 1752c 2704plusmn 206c 5617plusmn 798b

Fruit

CR 4598plusmn 258d 681plusmn 117d 87372plusmn 1252d 12808plusmn 612d 1373plusmn 069cHex 3277plusmn 141e 5391plusmn 1230a 10097plusmn 819e 1501plusmn 065e 7482plusmn 266dEA 11865plusmn 760f 6307plusmn 1017e 13553plusmn 983f 8954plusmn 388f 828plusmn 040eAQ 2093plusmn 449g 1545plusmn 068f 94642plusmn 6504g 3145plusmn 145g mdash

Results are expressed as meanplusmn standard deviation n 3 Mean values within a column superscripted by different letters are significantly different at plt 005CR crude Hex hexane EA ethyl acetate AQ aqueous ORAC oxygen radical absorbance capacity FRAP ferric reducing antioxidant power ABTSABTS radical scavenging activity DPPH DPPH radical scavenging activity

0

20

40

60

80

100

0 200 400 600 800

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

00 05 10 15 20

Inhi

bitio

n

Concentration (mgmL)

CRHex

EAAQ

(b)

Figure 3 Dose-response curves for DPPH radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

120

0 50 100 150 200 250

Inhi

bitio

n

Concentration (microgmL)

CRHex

EAAQ

(b)

Figure 4 Dose-response curves for ABTS+ radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

8 Evidence-Based Complementary and Alternative Medicine

fruits exhibited the highest iron-chelating ability among thetested fractions with value 7482plusmn 266mg EDTAg of ex-tract and the lowest was reported in EA fraction of fruitswith value 828plusmn 040mg EDTAg of extract respectively(e AQ fraction of Syzygium caryophyllatum fruits did notshow a significant chelating activity where the highestconcentration (215mgmL) exhibited 1605 inhibition

33 Correlation Analysis between Polyphenol Content andDPPH Radical Scavenging Activity of Syzygium car-yophyllatum Fruits (e correlation coefficient betweenTPC content and DPPH of Syzygium caryophyllatum fruitsexhibited a significant (plt 005) positive correlation withPearson r value 09921 (Figure 5)

34 Ce α-Amylase Inhibitory Activity (e α-amylase inhi-bition potentials of CR extract Hex EA and AQ fraction ofleaves and fruits were analyzed Fruits exhibited significantinhibition of α-amylase activities where the leaves did notexhibit significant inhibitory activities (e Hex fraction ofS caryophyllatum fruits was reported to have the highestinhibitory activity with IC50 value 4720 plusmn 03 μgmL fol-lowed by CR extract EA fraction and AQ fraction withIC50 values 21826 plusmn 538 μgmL 61885plusmn 1855 μgmL and101455plusmn 8555 μgmL respectively Interestingly the Hexfraction of fruits demonstrated potent α-amylase inhibitionactivity (IC50 4720 plusmn 03 μgmL) compared to the standarddrug acarbose (IC50 8796 plusmn 143 μgmL)(e percentage ofα-amylase inhibitions of crudes and fractions was plottedagainst concentration (Figure 6) where it showed a dose-dependent increase in percentage activity of CR extract andthree types of fractions of fruits

341 Ce α-Amylase Inhibitory Activity of Isolated Fractionsof Hexane Fraction of Syzygium caryophyllatum Fruits

(1) Hexane and Ethyl Acetate Solvents on Isolation Procedure(e inhibition potential of compound mixtures of EA fractionwas evaluated for α-amylase inhibitory activity (e highesttested concentration was 200μgmL followed by 100μgmL50μgmL and 25μgmL(e similar inhibition potentials wereobserved for the first three concentrations and the inhibitionvalues were 5763plusmn018 5599plusmn160 and 5730plusmn018respectively where the inhibition potential of 25μgmL wasobserved as a rapid drop to the inhibition 1278plusmn045Further inhibition potentials were evaluated between 50μgmLand 25μgmL (Table 4)(e α-amylase inhibitory activity of thetested fraction showed a poor linear response (R2 06384)hence IC50 was not calculated

(2) Dichloromethane and Ethyl Acetate Solvents on Iso-lation Procedure (e α-amylase inhibitory activity ofdifferent combined fractions was analyzed (C1 C2 andC3)(e inhibitory activities of C1 and C2 fractions did notexhibit significant inhibitory activities where the fractionC3 showed significant (plt 005) inhibitory activity for the

screened concentrations (e fraction C3 was dose-de-pendent and IC50 value was 227 plusmn 181 μgmL

(e summary of α-amylase inhibitory activity of hexanefraction and its chromatographic fraction is given in Table 5

35 Antiglycation Activity (e antiglycation activity ofcrude extracts of leaves and fruits was screened for threeconcentrations 200 100 and 50 μgmL (Table 6) (e CRextract of leaves exhibited the highest inhibitory activity(9361 plusmn 853 at a concentration of 200 μgmL) followedby CR extract of S caryophyllatum fruits (4797 plusmn 263)(e standard drug rutin exhibited antiglycation activitywith IC50 value as 3423 plusmn 318 μgmL (e IC50 value ofCR extract of leaves was 5783 plusmn 791 μgmL Howeverfor fruits similar inhibitions were observed for differentdoses hence IC50 value could not be calculated

36 Chemical Compositions of Syzygium caryophyllatum LeafEssential Oil Syzygium caryophyllatum leaves yieldedessential oil upon hydrodistillation GC-MS analysisidentified 58 compounds from the essential oil and theretention time and area of the identified major peaks arelisted in Table 7 (e major compounds identified in theleaves were phytol with a 2466 peak area (retentiontime 3444) α-cadinol with a 387 peak area (retentiontime 2781) α-guaiene with a 384 peak area (retentiontime 2629) cyclosiolongifolene 910-dehydro with a382 peak area (retention time 2826) humulene with a336 peak area (retention time 1645) and car-yophyllene with a 321 peak area (retention time 1479)respectively (e GC-MS chromatogram of Syzygiumcaryophyllatum leaf oil is presented in Figure 7

37 Mineral Compositions Functionally important micro-minerals such as selenium (Se) iron (Fe) strontium (Sr)manganese (Mn) cobalt (Co) copper (Cu) and the toxicheavymetals such as arsenic (As) cadmium (Cd) chromium(Cr) and lead (Pb) were evaluated (Table 8) (e ICP-MS

0

20

40

60

80

100

0 50 100 150

DPP

H v

alue

(mg

TEg

of e

xtra

ct)

Total phenol content (mg GAEg of extract)

Figure 5 Correlation of Syzygium caryophyllatum fruits TPCversus DPPH values (plt 005)

Evidence-Based Complementary and Alternative Medicine 9

data ranged from 009 to 45370mgkg of sample Sodium(Na) was found to be the highest (leaves 435768mgkg ofthe sample) while Se was found to be the lowest (fruits009mgkg of the sample) (e leaves were found to have thehighest concentration of the tested minerals compared to thefruits (e second richest macroelement was found to be K(leaves 355925mgkg and fruits 294428mgkg) followedby Mg (leaves 258040mgkg) and Ca (leaves 184352mgkg)(e maximum amount of Al Mn Fe and Zn was found

to be in leaves with values 52445mgkg 29882mgkg22198mgkg and 2160mgkg respectively Among thetested microelements Sr was found to be the richest with avalue of 32889mgkg in leaves which is significantly(plt 005) different from fruits (270mgkg) (e Cu and Crwere found in similar small concentrations in leaves (Cu491mgkg and Cr 403mgkg) and fruits (Cu 523mgkgand Cr 465mgkg) (e maximum concentration of Sewas determined to be 085mgkg in leaves and the fruitsexhibited the lowest concentration with a value of 009mgkg(e investigation of the level of Co revealed that the maxi-mum concentration of the element exists as 0128mgkg inleaves while in the fruits it was not detected at the detectionlimit of 005mgkg

(e analysis of toxic heavy metals revealed the presenceof a maximum concentration of Pb in leaves (102mgkg)and the lowest in fruits (015mgkg) Besides the concen-trations of toxic heavy metals such as Cd and As were notdetected in leaves and fruits (limit of detection 005mgkg)

4 Discussion

Diabetes mellitus (DM) is a complex metabolic disordercharacterized by hyperglycemia which is mainly associatedwith malfunctions and abnormalities in carbohydrate pro-tein and fat metabolism Digestion and absorption of car-bohydrates to the blood stream also have a significantinfluence on DM Further the increased production of re-active oxygen species (ROS) under DM accelerates the proteinglycation process [24] (e production and accumulation ofglycated proteins have shown a strong association withcomplications of DM [25] (us the management of DM isdirected toward addressing multiple targets including re-duction of postprandial carbohydrate levels in the bloodimprovement in the homeostatic and metabolic process ofcarbohydrates and lipids an improvement on the efficiency ofantioxidant defense and control of protein glycation [26]

In this regard WHO recommends functional ingredi-ents and supplements with multiple functional propertiesincluding antidiabetic and the antiradical properties in thecontrol of the DM [27]

In this study functional properties including α-amylaseinhibition protein glycation inhibition and antioxidantactivities of S caryophyllatum fruit and leaf extracts havebeen investigated along with chemical constituents andmineral analyses (e results revealed the potential of Scaryophyllatum as a source of supplement for DM man-agement To the best of our knowledge the present study isthe first study reported in Sri Lanka on functional activitiesof S caryophyllatum fruit and leaf extracts Crude extracts ofboth fruits and leaves showed significant antioxidant andmoderate antiglycation activities However only fruit ex-tracts showed α-amylase inhibitory activity Even thoughboth fruits and leaves CR were tested against α-glucosidaseenzyme inhibitory activity none of them showed a signif-icant inhibitory activity at a maximum dose of 400 μgmL(data not shown) Solventndashsolvent partitioning of both CRwas used to separate and valuate the most potent fractionswith α-amylase inhibition activity

0

20

40

60

80

100

0 200 400 600 800 1000

Inhi

bitio

n

Concentration (μgmL)

CRHex

EAAQ

Figure 6 Dose-response relationship of crude and fractionsof Syzygium caryophyllatum fruits for alpha-amylase inhibi-tory activity (CR crude Hex hexane EA ethyl acetateAQ aqueous)

Table 4 (e α-amylase inhibitory activity of isolated compoundmixtures of Hex fraction of Syzygium caryophyllatum fruits (hexaneand ethyl acetate solvents on isolation procedure)

Assay concentration μgmL Inhibition 200 5763plusmn 018a100 5599plusmn 160a50 5730plusmn 018a45 2339plusmn 021b40 2245plusmn 079b35 2143plusmn 019b30 1433plusmn 083c25 1278plusmn 045c

Results are expressed as meanplusmn standard deviation n 3lowast Mean valueswithin a column superscripted by different letters are significantly differentat plt 005

Table 5 (e α-amylase inhibitory activity of hexane fraction andits chromatographic fraction of Syzygium caryophyllatum fruits

Sample IC50 (μgmL)Hex fraction 4720aplusmn 030Hex EA (0 100) NLRC3 DCM EA (95 5) 227bplusmn 181Standard drug acarbose 8796cplusmn 143Results are expressed as meanplusmn standard deviation n 3lowast NLRno linearresponse Mean values within a column superscripted by different lettersare significantly different at plt 005 Hex hexane EA ethyl acetateDCM dichloromethane C3 combined fraction 3

10 Evidence-Based Complementary and Alternative Medicine

Hexane fraction of fruit extract exhibited the highestinhibition activity with IC50 value 4720plusmn 037 μgmL and issignificantly higher compared to acarbose (IC508796plusmn 143 μgmL) (erefore it was further separated bycolumn chromatography (e fraction C3 which was iso-lated on elution with DCM EA 95 5 exhibited a significant(plt 005) α-amylase inhibitory activity with an IC50 value of227plusmn 181 μgmL (e compound or the mixture of

compounds responsible for the α-amylase inhibition activitymay be in the C3 compound mixture which is required to besubjected to further isolation and identification Inhibitionmay be due to the presence of potential alpha-amylase in-hibitors such as alkaloids tannin polyphenols and flavo-noids (ese inhibitors can also act as starch blockers as theyblock the hydrolysis of 14-glycosidic linkages of starch andoligosaccharides [28] Hex fraction of S caryophyllatum fruit

Table 6 Antiglycation activity of crude extracts of Syzygium caryophyllatum

Crude extract Inhibition

IC50 (μgmL)Concentration (μgmL)50 100 200

Leaves 5488plusmn 310a 7987plusmn 594a 9361plusmn 850a 5783plusmn 791Fruits 4131plusmn 789b 5666plusmn 836b 4797plusmn 263b mdashStandard rutin IC50 3423plusmn 318 μgmLResults are expressed as meanplusmn standard deviation n 3lowast Mean values within a column superscripted by different letters are significantly different atplt 005

Table 7 Major compounds detected in Syzygium caryophyllatum leaf essential oil

Peak Compound Formula and lowastmol wt RT Area SI RSI1 Caryophyllene C15H24 204 1479 321 954 9542 Humulene C15H24 204 1645 336 907 9073 α-Cadinol C15H26O 222 2781 387 923 9284 Cyclosiolongifolene 910-dehydro C17H26O2 202 2826 382 790 8005 Phytol C20H40O 296 3444 2466 927 927lowastMol wtmolecular weight

2781

RT 000ndash4200100

90

80

70

60

50

40

30

20

10

0 5 10 15 20 25 30 35 40Retention time (min)

3444

2826

Rela

tive a

bund

ance

1 24

3

5

1479 1645

Figure 7 GC-MS chromatogram of Syzygium caryophyllatum leaf essential oil

Table 8 Mineral compositions of leaves and fruits of Syzygium caryophyllatum

Mineral elements (mgkg per sample)K Fe Na Ca Mg Mn Cu Zn Cr Zn Sr Se Pb As Cd

Syzygiumcaryophyllatum

Leaves 355925 22198 435768 184352 258039 29882 491 2160 403 2159 3289 085 102 ND NDFruits 294428 4955 75621 14927 85555 1814 523 775 465 775 270 009 015 ND ND

RDA 35 g 9ndash15mg 15 g 1 g 280ndash350mg 50ndash60 μglowastND not detected limit of detection 005mgkg RDA recommended daily intake

Evidence-Based Complementary and Alternative Medicine 11

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

FC Reagent was added to 20μL of the sample (e mixture wastreated with 10 sodium carbonate and incubated at roomtemperature for 30min and absorbance was measured at765nm Gallic acid was used as a reference standard and theresults were expressed as milligram gallic acid equivalent pergram of sample (mg GAEg of sample)

252 Determination of Total Flavonoid Content (TFC)(e total flavonoid content (TFC) was determined using analuminum chloride colorimetric method using 96-wellmicroplates [18] Extracts were dissolved in methanol and100 μL of the sample wasmixed with 100 μL of 2 aluminum

chloride (e mixture was incubated at room temperaturefor 10min and absorbance was measured at 415nm Quercetinwas used as a reference standard and the results wereexpressed as milligram quercetin equivalent per gram ofsample (mg QEg of sample)

26 Evaluation of Antioxidant Activity

261 Ferric Reducing Antioxidant Power (FRAP) Assay(e assay method was based on the reduction of Fe3+ to Fe2+by the electron donation ability of antioxidants resulting inan intense blue-colored complex (Fe2+-tripyridyltriazine)

Syzygium caryophyllatum

Leaves Fruits

Distillation Reflux Cold extraction

Crude extract

Fractionation

Hexane

Aqueous fractionEthyl acetate

Crude extract Aqueous fraction Ethyl acetate fraction Hexane fraction

AntioxidantAntiamylase

Antiglycation

Hexane(100)

EA(100)

Hexane (100) Hex DCM (3 1) Hex DCM (2 1) DCM (100) DCM EA (95 5)

Fr 1-10 Fr 11-12 Fr 13 Fr 14-19 Fr 20-22

C1 C3C2

Anti-amylase

Chemical composition analysis-GC-MS

Mineral composition analysisndashICP-MS

Solvent system IIHex amp EA

Silica gel column chromatography

Oil

Hexane fraction offruit

Solvent system IHex DCM amp EA

Figure 2 Flow chart depicting the methodology of the study

4 Evidence-Based Complementary and Alternative Medicine

[18] (e FRAP reagent was prepared freshly by mixingacetate buffer (300mM pH 36) solution of TPTZ in HCland FeCl3 (20mM) in 10 1 1 ratio and incubated at 37degCTwo hundred microliters of the reaction mixture (FRAPreagent and sample) was incubated at room temperatureand absorbance was measured at 600 nm Vitamin E ana-logue Trolox was used as the reference standard where theresults were expressed as mg of Trolox equivalent per gramof extract (mg TEg of extract)

262 DPPH Radical Scavenging Assay (e radical scav-enging activity was determined by using 1 1-diphenyl-2-picryl-hydrazyl (DPPH) stable radical method [18] (e method isbased on the reduction of purple-colored 1 1-diphenyl-2-picrylhydrazyl to a yellow-colored diphenylpicrylhydrazine(ereaction volume of 200μL containing sample (50μL)methanolandDPPHwere incubated at room temperature for 10min andabsorbance was measured at 517nm A percentage inhibitionversus concentration was plotted and the concentration of thesample required for 50 inhibition was determined and rep-resented as EC50 value (e following formula was used tocalculate the percent inhibition

Radical scavenging activity CAB minus SAB( 1113857

CABtimes 100 (1)

where CAB is the absorbance of the control and SAB is theabsorbance of the sample

263 ABTS Radical Scavenging Activity (e ABTS+ radicalcation (ABTS+) scavenging activity assay was performed [19]with some modifications in 96-well microplates Plant ex-tracts were prepared in phosphate buffer solutions and wereallowed to react with ABTS+ radicals in a microwell plate for10min Absorbance wasmeasured in 734 nm and Trolox wasused as the standard (e following formula was used tocalculate the percent inhibition

Radical scavenging activity CAB minus SAB( 1113857

CABtimes 100 (2)

where CAB is the absorbance of the control and SAB is theabsorbance of the sample

(e antiradical activity was expressed as EC50 theconcentration required to cause 50 inhibition of ABTS+radicals

264 Oxygen Radical Absorbance Capacity (ORAC) Assay(eORAC assay is a hydrogen atom transfer- (HAT-) basedassay which measures the depletion of fluorescence intensityof a fluorescent probe upon the generation of peroxy radicalsdue to the oxidation of azo compounds In the presence of anantioxidant compound the decaying of fluorescent intensityis retarded (e overall decay is calculated by the differencebetween the area under the fluorescence decay curve (AUC)of the sample of interest and AUC of blank [20](e reactionvolume of 200 μL (pH 74) containing fluorescein (100 μL)and sample (50 μL) was preincubated at 37degC for 5min (e22prime-azobis (2-amidinopropane) dihydrochloride (AAPH)

solution was added (50 μL) to the mixture and the plate(black 96 well plates) was immediately placed in a fluo-rescent microplate reader (SPECTRAmax-Gemini EMMolecular Devices Inc USA) (e decay of fluorescence wasrecorded every minute for 35min and the excitation andemission wavelengths were obtained at 494 nm and 535 nmrespectively Phosphate buffer (75mM pH 74) replacing thesample was used as the blank and Trolox was used as thestandard antioxidant Results were expressed as mg of Troloxequivalent per gram of extract

265 Ferrous Ion Chelating Activity (e ferrous ion che-lating (FIC) activity was measured by the decrease in theabsorbance at 562 nm of the iron (II)-ferrozine complex [21]EDTA was used as the reference standard and all the samplesand standards were prepared in distilled water (e samplewas mixed with 1mM ferrous sulfate and distilled waterfollowed by 1mM ferrozine solution(e ability of the sampleto chelate ferrous ions was calculated relative to the control(distilled water instead of the sample) by the following for-mula (e results were expressed as mg EDTA equivalent pergram of extract

Chelating activity CAB minus SAB( 1113857

CABtimes 100 (3)

where SAB is the absorbance of the sample and CAB is theabsorbance of the control

27 Determination of α-Amylase Inhibition Activity (eα-amylase inhibition activity was carried out using thedinitrosalicylic (DNS) method [22] where the inhibitionactivity was measured by quantifying the maltose liberatedunder the assay conditions (e enzyme inhibitory activitywas expressed as a decrease in units of maltose liberated Amodified dinitrosalicylic acid (DNS) method was adopted toestimate the maltose equivalent (e reaction volume con-taining 40 μL of 1 (wv) starch solution and 775 μL of100mM sodium acetate buffer was preincubated with 135 μLof the sample at 40degC shaking water bath for 10min (emixture was further incubated with 50 μL of the α-amylaseenzyme (5 μgmL) at 40degC shaking water bath for 15min(e reaction was terminated by adding 500 μL DNS reagentand the mixture was placed in a boiling water bath for 5minfollowed by cooling in an ice bath Absorbance was mea-sured at 540 nm using a 96-well microplate reader Anti-amylase activity (inhibition ) was calculated using thefollowing equation

Inhibition() AC minus AS minus Ab( 11138571113858 1113859

ACtimes 100 (4)

where AC is the absorbance of the control AS is the ab-sorbance of the sample and Ab is the absorbance of thesample blank

28 BSA-Glucose Glycation Inhibitory Activity AssayAntiglycation activity assay was performed with somemodifications to the previous methodology [23] for CR

Evidence-Based Complementary and Alternative Medicine 5

extracts of leaves and fruits of Syzygium caryophyllatum Amixture of 800 μgmL BSA 400mM glucose and 80 μL ofthe sample in 50mM phosphate buffer (pH 74) containingsodium azide (002) was incubated at 60degC for 40 h (e600 μL of the reaction mixture was transferred to 15mLmicrocentrifuge tubes and 60 μL of 100 (wv) TCA wasadded and sample mixtures were centrifuged at 15000 rpmat 4degC for 4min (e resultant advanced glycation endproducts-BSA (AGEs-BSA) precipitate was then dissolved in1mL of phosphate buffer saline (pH 10) and the fluores-cence intensity was measured at an excitation and emissionwavelengths of 370 nm and 440 nm using a 96-well fluo-rescence microplate reader (SpectraMax Gemini EMMolecular Devices Inc USA) Rutin was used as thepositive control and antiglycation activity ( inhibition) wascalculated using the following equation followed by thecalculation of IC50 value

Inhibition() Fc ndash Fb1113858 1113859 ndash Fs ndash Fsb1113858 1113859

Fc ndash Fb1113858 1113859times 100 (5)

where Fc is the fluorescence of the control Fb is the fluo-rescence of BSA Fs is the fluorescence of the sample andFsb is the fluorescence of the sample blank

All the above in vitro bioassay procedures were validatedat Herbal Technology Section Industrial Technology In-stitute Sri Lanka (ISO 9005 certified Laboratory)

29 Gas Chromatography-Mass Spectrometry (GC-MS)Analysis (e extracted essential oil from the leaves wassubjected to GC-MS analysis to identify volatile compounds(e analysis was carried out by using TRACE 1300 coupledto ISQ QD mass spectrophotometer instrument ((ermoFisher Scientific Milan Italy) (e sample was injected into(ermo-scientific TG-WAX capillary column(30mtimes 025mmtimes 025 μm) fused with silica having poly-ethylene glycol as the stationary phase with helium as thecarrier gas (1mLmin) (e temperature of the injector wasmaintained at 250degC (e GC oven temperature was initiallyprogrammed at a temperature of 60degC and was ramped up to220degC at the rate of 5degCmin GC-MS interface temperaturewas maintained at 250degC (e mass detector was operated inEI mode in a scan mass range of mz 40ndash450 (e identi-fication of compounds was ascertained by comparing themass spectral values with the known compounds in NIST11USA mass spectral database

210 Preparation of Plant Extracts for Mineral ContentDetermination Approximately 05 g of homogenized sam-ple was weighed into easy prep high-pressure microwavevessel and 100mL of 65 nitric acid was added (e easyprep microwave digestion program was followed to digestthe samples by using a microwave digester (CEM MARS 5USA) (e digested sample was quantitatively transferredand filtered using No 542 Whatman filter paper followed bywashing with deionized water and volume up to 25mL (eprepared solution was used to analyze mineral content byICP-MS (Agilent 7900 USA)

211 Statistical Analysis Statistical analysis was performedusing Graph Pad Prism (version 7) All experimental datawas analyzed by one-way analysis of variance (ANOVA)ANOVArsquos Turkey multiple comparison test was used tocompare the mean values Pearsonlsquos correlation coefficientwas used for the correlation analysis and in all cases p

values less than 005 (plt 005) were considered statisticallysignificant (e quantitative results of phytochemicals an-tioxidant potentials and enzyme inhibition values wereexpressed as meanplusmn standard deviation (SD)

3 Results

S caryophyllatum fruits and leaves were extractedaccording to the method described above and fractionatedinto nonpolar and polar solvents with increasing polarityhexane and ethyl acetate respectively (is solventndashsolventpartitioning procedure was allowed to generate hexane(Hex) fraction ethyl acetate (EA) fraction and aqueous(AQ) fraction which was labelled and stored in minus20degC priorto analysis (e yield was estimated to the dry weight whichranged from 06mg to 312 g where the highest yield wasrecorded in crude (CR) extract of fruits (312 g) and thelowest was recorded in Hex fraction of leaves (06mg) (eweights of the CR extracts Hex EA and AQ fractions aregiven in Table 1 and the yield was calculated as a percentage

All the crude extracts and fractions were subjected tofurther analysis except the hexane fraction of leaves ofSyzygium caryophyllatum due to the unavailability of ade-quate sample yield

31 Total Polyphenol Content (TPC) and Total FlavonoidContent (TFC) of Leaves and Fruit Extracts of Syzygiumcaryophyllatum Plant Extracts Total polyphenol contentwas expressed as milligram gallic acid equivalent per gram ofsample (mg GAEg) and the total flavonoid content asmilligram quercetin equivalent per gram of sample (mg QEg)Total polyphenol values varied from 00923mg GAEg ofsample to 818mg GAEg of sample and from 172mgGAEg of sample to 892mg GAEg of sample for leaves andfruits respectively Polyphenol contents of samples werediffered significantly (plt 005) Table 2 presents the TPCand TFC values for crude and fractions of samples (ehighest TPC value was recorded in CR extract of fruitsfollowed by CR extract of leaves and AQ fraction of leavesFlavonoid content of the tested samples varied significantly(plt 005) among extracts TFC ranged from 0001mg QEgof sample to 203mg QEg of sample and the highest TFCvalue was reported in CR extract of fruits with value as202plusmn 019mg QEg of the sample followed by Hex fractionand EA fraction of fruits with values 080plusmn 005mg QEg ofsample and 037plusmn 001mg QEg of sample respectively

32 In Vitro Antioxidant Properties of Different Extracts ofSyzygium caryophyllatum (e antioxidant properties ofcrudes and fractions of leaves and fruits of Syzygium car-yophyllatum were determined using DPPH ABTS ORACFRAP and iron chelation assay methods (e results

6 Evidence-Based Complementary and Alternative Medicine

obtained are depicted in Table 3 where the significant dif-ferences (plt 005) were observed between CR extracts ofleaves and fruits and among different fractions of leavesand fruits

321 DPPH Radical Scavenging Activity Results revealedthat the crude extract and fractions of S caryophyllatumhave the ability to scavenge radicals and depicted a dose-response relationship DPPH radical scavenging activityof different fractions of S caryophyllatum differed sig-nificantly (plt 005) (e EA fraction of leaves exhibitedthe lowest EC50 value followed by Hex and EA fraction offruits with values 912 plusmn 428 μgmL 10097 plusmn 819 μgmLand 13553 plusmn 983 μgmL respectively (e results ob-tained from the fractions showed that EA fractions ofleaves and Hex fractions of fruits exhibited high antiox-idant activities compared to other fractions It shows thatthe accumulation of a variety of antioxidant compoundsdepends on the polarity of the solvent (erefore thecompounds responsible for the high radical scavengingactivity of S caryophyllatum leaves are mainly mediumpolarity compounds (e dose-response relationships ofS caryophyllatum leaves and fruits are given inFigures 3(a) and 3(b)

322 ABTS+ Radical Scavenging Activity (e highestradical scavenging activity was reported in Hex fraction offruits with EC50 value 1501plusmn 065 μgmL followed by AQfraction of S caryophyllatum leaves and AQ fraction of Scaryophyllatum fruits and the EC50 values were 2703plusmn 206and 3145plusmn 143 μgmL respectively It was found that fruitsand leaves showed a significant difference (plt 005) among

fractions (e CR extracts and fractions of leaves and fruitsshowed significant difference and dose-dependent inhibitionon ABTS radical scavenging activity (Figures 4(a) and 4(b))

323 Ferric Reducing Antioxidant Power (FRAP) (e CRextracts of leaves and fruits differed significantly (plt 005) intheir FRAP values CR extract of leaves 4827plusmn 811mgTEgof extract and CR extract of fruits 681plusmn 117mgTEg ofextract (e FRAP values of the fractions ranged from1481mgTEg of the extract to 27041mgTEg of extract EAfraction of leaves was reported as the highest among thetested fractions in leaves and fruits where the AQ fraction offruits exhibited the lowest values (ese results suggestedthat EA fraction of leaves and fruits exhibited the highestferric reducing property among tested fractions whichexplains the important behavior of the polar compounds inits reducing property

324 Oxygen Radical Absorbance Capacity (ORAC) (eORAC values of fractions ranged from 1723mgTEg of theextract to 34732mgTEg of extract (e highest activity wasrecorded in EA fraction of leaves following the AQ fractionof leaves and the lowest was recorded in AQ fraction infruits (e CR extract of leaves exhibited a significantlyhigher (plt 005) activity compared to the CR extract offruits with values 22100plusmn 3496 and 4598plusmn 258mgTEg ofextract respectively

325 Iron Chelating Activity (e highest iron chelatingability of CR extract was reported in leaves with values332plusmn 168mg EDTAg of extract and fruits were reported as1373plusmn 069mg EDTAg of extract (e Hex fraction of

Table 1 (e yield of Syzygium caryophyllatum plant extracts

Scientific name Part of plant Extract Weight of concentrated extract (g) Yield

Syzygium caryophyllatum

Leaves

CR 17500 70Hex lt0001 lt01EA 0114 01AQ 15984 71

Fruits

CR 31200 620Hex 1950 26EA 1531 20AQ 10165 136

CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction

Table 2 Total polyphenol content (TPC) and total flavonoid content (TFC) in different extracts of Syzygium caryophyllatum

Scientific name Part of plant Extract TPC (mg GAEg of sample) TFC (mg QEg of sample)

Syzygium caryophyllatum

LeavesCR 812plusmn 046a 016plusmn 001aEA 009plusmn 001b lt001AQ 712plusmn 016c 018plusmn 002ab

Fruits

CR 883plusmn 062ad 202plusmn 019cHex 342plusmn 029e 080plusmn 005dEA 173plusmn 076f 037plusmn 001eAQ 216plusmn 012g 010plusmn 001af

Results expressed as meanplusmn standard deviation CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction Mean valueswithin a column superscripted by different letters are significantly different at plt 005

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Antioxidant potentials of leaves and fruits of Syzygium caryophyllatum

Scientific name Part of plant Extract ORAC mg TEgof extract

FRAP mg TEgof extract

EC50 DPPHμgmL

EC50 ABTSμgmL

Iron chelation mgEDTAg of extract

Syzygiumcaryophyllatum

LeavesCR 22100plusmn 3496a 4827plusmn 811a 50020plusmn 4433a 10793plusmn 1507a 332plusmn 168aEA 34431plusmn 337b 26226plusmn 4393b 9120plusmn 428b 4061plusmn 276b 3601plusmn 698aAQ 32097plusmn 2062bc 7342plusmn 1258c 25105plusmn 1752c 2704plusmn 206c 5617plusmn 798b

Fruit

CR 4598plusmn 258d 681plusmn 117d 87372plusmn 1252d 12808plusmn 612d 1373plusmn 069cHex 3277plusmn 141e 5391plusmn 1230a 10097plusmn 819e 1501plusmn 065e 7482plusmn 266dEA 11865plusmn 760f 6307plusmn 1017e 13553plusmn 983f 8954plusmn 388f 828plusmn 040eAQ 2093plusmn 449g 1545plusmn 068f 94642plusmn 6504g 3145plusmn 145g mdash

Results are expressed as meanplusmn standard deviation n 3 Mean values within a column superscripted by different letters are significantly different at plt 005CR crude Hex hexane EA ethyl acetate AQ aqueous ORAC oxygen radical absorbance capacity FRAP ferric reducing antioxidant power ABTSABTS radical scavenging activity DPPH DPPH radical scavenging activity

0

20

40

60

80

100

0 200 400 600 800

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

00 05 10 15 20

Inhi

bitio

n

Concentration (mgmL)

CRHex

EAAQ

(b)

Figure 3 Dose-response curves for DPPH radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

120

0 50 100 150 200 250

Inhi

bitio

n

Concentration (microgmL)

CRHex

EAAQ

(b)

Figure 4 Dose-response curves for ABTS+ radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

8 Evidence-Based Complementary and Alternative Medicine

fruits exhibited the highest iron-chelating ability among thetested fractions with value 7482plusmn 266mg EDTAg of ex-tract and the lowest was reported in EA fraction of fruitswith value 828plusmn 040mg EDTAg of extract respectively(e AQ fraction of Syzygium caryophyllatum fruits did notshow a significant chelating activity where the highestconcentration (215mgmL) exhibited 1605 inhibition

33 Correlation Analysis between Polyphenol Content andDPPH Radical Scavenging Activity of Syzygium car-yophyllatum Fruits (e correlation coefficient betweenTPC content and DPPH of Syzygium caryophyllatum fruitsexhibited a significant (plt 005) positive correlation withPearson r value 09921 (Figure 5)

34 Ce α-Amylase Inhibitory Activity (e α-amylase inhi-bition potentials of CR extract Hex EA and AQ fraction ofleaves and fruits were analyzed Fruits exhibited significantinhibition of α-amylase activities where the leaves did notexhibit significant inhibitory activities (e Hex fraction ofS caryophyllatum fruits was reported to have the highestinhibitory activity with IC50 value 4720 plusmn 03 μgmL fol-lowed by CR extract EA fraction and AQ fraction withIC50 values 21826 plusmn 538 μgmL 61885plusmn 1855 μgmL and101455plusmn 8555 μgmL respectively Interestingly the Hexfraction of fruits demonstrated potent α-amylase inhibitionactivity (IC50 4720 plusmn 03 μgmL) compared to the standarddrug acarbose (IC50 8796 plusmn 143 μgmL)(e percentage ofα-amylase inhibitions of crudes and fractions was plottedagainst concentration (Figure 6) where it showed a dose-dependent increase in percentage activity of CR extract andthree types of fractions of fruits

341 Ce α-Amylase Inhibitory Activity of Isolated Fractionsof Hexane Fraction of Syzygium caryophyllatum Fruits

(1) Hexane and Ethyl Acetate Solvents on Isolation Procedure(e inhibition potential of compound mixtures of EA fractionwas evaluated for α-amylase inhibitory activity (e highesttested concentration was 200μgmL followed by 100μgmL50μgmL and 25μgmL(e similar inhibition potentials wereobserved for the first three concentrations and the inhibitionvalues were 5763plusmn018 5599plusmn160 and 5730plusmn018respectively where the inhibition potential of 25μgmL wasobserved as a rapid drop to the inhibition 1278plusmn045Further inhibition potentials were evaluated between 50μgmLand 25μgmL (Table 4)(e α-amylase inhibitory activity of thetested fraction showed a poor linear response (R2 06384)hence IC50 was not calculated

(2) Dichloromethane and Ethyl Acetate Solvents on Iso-lation Procedure (e α-amylase inhibitory activity ofdifferent combined fractions was analyzed (C1 C2 andC3)(e inhibitory activities of C1 and C2 fractions did notexhibit significant inhibitory activities where the fractionC3 showed significant (plt 005) inhibitory activity for the

screened concentrations (e fraction C3 was dose-de-pendent and IC50 value was 227 plusmn 181 μgmL

(e summary of α-amylase inhibitory activity of hexanefraction and its chromatographic fraction is given in Table 5

35 Antiglycation Activity (e antiglycation activity ofcrude extracts of leaves and fruits was screened for threeconcentrations 200 100 and 50 μgmL (Table 6) (e CRextract of leaves exhibited the highest inhibitory activity(9361 plusmn 853 at a concentration of 200 μgmL) followedby CR extract of S caryophyllatum fruits (4797 plusmn 263)(e standard drug rutin exhibited antiglycation activitywith IC50 value as 3423 plusmn 318 μgmL (e IC50 value ofCR extract of leaves was 5783 plusmn 791 μgmL Howeverfor fruits similar inhibitions were observed for differentdoses hence IC50 value could not be calculated

36 Chemical Compositions of Syzygium caryophyllatum LeafEssential Oil Syzygium caryophyllatum leaves yieldedessential oil upon hydrodistillation GC-MS analysisidentified 58 compounds from the essential oil and theretention time and area of the identified major peaks arelisted in Table 7 (e major compounds identified in theleaves were phytol with a 2466 peak area (retentiontime 3444) α-cadinol with a 387 peak area (retentiontime 2781) α-guaiene with a 384 peak area (retentiontime 2629) cyclosiolongifolene 910-dehydro with a382 peak area (retention time 2826) humulene with a336 peak area (retention time 1645) and car-yophyllene with a 321 peak area (retention time 1479)respectively (e GC-MS chromatogram of Syzygiumcaryophyllatum leaf oil is presented in Figure 7

37 Mineral Compositions Functionally important micro-minerals such as selenium (Se) iron (Fe) strontium (Sr)manganese (Mn) cobalt (Co) copper (Cu) and the toxicheavymetals such as arsenic (As) cadmium (Cd) chromium(Cr) and lead (Pb) were evaluated (Table 8) (e ICP-MS

0

20

40

60

80

100

0 50 100 150

DPP

H v

alue

(mg

TEg

of e

xtra

ct)

Total phenol content (mg GAEg of extract)

Figure 5 Correlation of Syzygium caryophyllatum fruits TPCversus DPPH values (plt 005)

Evidence-Based Complementary and Alternative Medicine 9

data ranged from 009 to 45370mgkg of sample Sodium(Na) was found to be the highest (leaves 435768mgkg ofthe sample) while Se was found to be the lowest (fruits009mgkg of the sample) (e leaves were found to have thehighest concentration of the tested minerals compared to thefruits (e second richest macroelement was found to be K(leaves 355925mgkg and fruits 294428mgkg) followedby Mg (leaves 258040mgkg) and Ca (leaves 184352mgkg)(e maximum amount of Al Mn Fe and Zn was found

to be in leaves with values 52445mgkg 29882mgkg22198mgkg and 2160mgkg respectively Among thetested microelements Sr was found to be the richest with avalue of 32889mgkg in leaves which is significantly(plt 005) different from fruits (270mgkg) (e Cu and Crwere found in similar small concentrations in leaves (Cu491mgkg and Cr 403mgkg) and fruits (Cu 523mgkgand Cr 465mgkg) (e maximum concentration of Sewas determined to be 085mgkg in leaves and the fruitsexhibited the lowest concentration with a value of 009mgkg(e investigation of the level of Co revealed that the maxi-mum concentration of the element exists as 0128mgkg inleaves while in the fruits it was not detected at the detectionlimit of 005mgkg

(e analysis of toxic heavy metals revealed the presenceof a maximum concentration of Pb in leaves (102mgkg)and the lowest in fruits (015mgkg) Besides the concen-trations of toxic heavy metals such as Cd and As were notdetected in leaves and fruits (limit of detection 005mgkg)

4 Discussion

Diabetes mellitus (DM) is a complex metabolic disordercharacterized by hyperglycemia which is mainly associatedwith malfunctions and abnormalities in carbohydrate pro-tein and fat metabolism Digestion and absorption of car-bohydrates to the blood stream also have a significantinfluence on DM Further the increased production of re-active oxygen species (ROS) under DM accelerates the proteinglycation process [24] (e production and accumulation ofglycated proteins have shown a strong association withcomplications of DM [25] (us the management of DM isdirected toward addressing multiple targets including re-duction of postprandial carbohydrate levels in the bloodimprovement in the homeostatic and metabolic process ofcarbohydrates and lipids an improvement on the efficiency ofantioxidant defense and control of protein glycation [26]

In this regard WHO recommends functional ingredi-ents and supplements with multiple functional propertiesincluding antidiabetic and the antiradical properties in thecontrol of the DM [27]

In this study functional properties including α-amylaseinhibition protein glycation inhibition and antioxidantactivities of S caryophyllatum fruit and leaf extracts havebeen investigated along with chemical constituents andmineral analyses (e results revealed the potential of Scaryophyllatum as a source of supplement for DM man-agement To the best of our knowledge the present study isthe first study reported in Sri Lanka on functional activitiesof S caryophyllatum fruit and leaf extracts Crude extracts ofboth fruits and leaves showed significant antioxidant andmoderate antiglycation activities However only fruit ex-tracts showed α-amylase inhibitory activity Even thoughboth fruits and leaves CR were tested against α-glucosidaseenzyme inhibitory activity none of them showed a signif-icant inhibitory activity at a maximum dose of 400 μgmL(data not shown) Solventndashsolvent partitioning of both CRwas used to separate and valuate the most potent fractionswith α-amylase inhibition activity

0

20

40

60

80

100

0 200 400 600 800 1000

Inhi

bitio

n

Concentration (μgmL)

CRHex

EAAQ

Figure 6 Dose-response relationship of crude and fractionsof Syzygium caryophyllatum fruits for alpha-amylase inhibi-tory activity (CR crude Hex hexane EA ethyl acetateAQ aqueous)

Table 4 (e α-amylase inhibitory activity of isolated compoundmixtures of Hex fraction of Syzygium caryophyllatum fruits (hexaneand ethyl acetate solvents on isolation procedure)

Assay concentration μgmL Inhibition 200 5763plusmn 018a100 5599plusmn 160a50 5730plusmn 018a45 2339plusmn 021b40 2245plusmn 079b35 2143plusmn 019b30 1433plusmn 083c25 1278plusmn 045c

Results are expressed as meanplusmn standard deviation n 3lowast Mean valueswithin a column superscripted by different letters are significantly differentat plt 005

Table 5 (e α-amylase inhibitory activity of hexane fraction andits chromatographic fraction of Syzygium caryophyllatum fruits

Sample IC50 (μgmL)Hex fraction 4720aplusmn 030Hex EA (0 100) NLRC3 DCM EA (95 5) 227bplusmn 181Standard drug acarbose 8796cplusmn 143Results are expressed as meanplusmn standard deviation n 3lowast NLRno linearresponse Mean values within a column superscripted by different lettersare significantly different at plt 005 Hex hexane EA ethyl acetateDCM dichloromethane C3 combined fraction 3

10 Evidence-Based Complementary and Alternative Medicine

Hexane fraction of fruit extract exhibited the highestinhibition activity with IC50 value 4720plusmn 037 μgmL and issignificantly higher compared to acarbose (IC508796plusmn 143 μgmL) (erefore it was further separated bycolumn chromatography (e fraction C3 which was iso-lated on elution with DCM EA 95 5 exhibited a significant(plt 005) α-amylase inhibitory activity with an IC50 value of227plusmn 181 μgmL (e compound or the mixture of

compounds responsible for the α-amylase inhibition activitymay be in the C3 compound mixture which is required to besubjected to further isolation and identification Inhibitionmay be due to the presence of potential alpha-amylase in-hibitors such as alkaloids tannin polyphenols and flavo-noids (ese inhibitors can also act as starch blockers as theyblock the hydrolysis of 14-glycosidic linkages of starch andoligosaccharides [28] Hex fraction of S caryophyllatum fruit

Table 6 Antiglycation activity of crude extracts of Syzygium caryophyllatum

Crude extract Inhibition

IC50 (μgmL)Concentration (μgmL)50 100 200

Leaves 5488plusmn 310a 7987plusmn 594a 9361plusmn 850a 5783plusmn 791Fruits 4131plusmn 789b 5666plusmn 836b 4797plusmn 263b mdashStandard rutin IC50 3423plusmn 318 μgmLResults are expressed as meanplusmn standard deviation n 3lowast Mean values within a column superscripted by different letters are significantly different atplt 005

Table 7 Major compounds detected in Syzygium caryophyllatum leaf essential oil

Peak Compound Formula and lowastmol wt RT Area SI RSI1 Caryophyllene C15H24 204 1479 321 954 9542 Humulene C15H24 204 1645 336 907 9073 α-Cadinol C15H26O 222 2781 387 923 9284 Cyclosiolongifolene 910-dehydro C17H26O2 202 2826 382 790 8005 Phytol C20H40O 296 3444 2466 927 927lowastMol wtmolecular weight

2781

RT 000ndash4200100

90

80

70

60

50

40

30

20

10

0 5 10 15 20 25 30 35 40Retention time (min)

3444

2826

Rela

tive a

bund

ance

1 24

3

5

1479 1645

Figure 7 GC-MS chromatogram of Syzygium caryophyllatum leaf essential oil

Table 8 Mineral compositions of leaves and fruits of Syzygium caryophyllatum

Mineral elements (mgkg per sample)K Fe Na Ca Mg Mn Cu Zn Cr Zn Sr Se Pb As Cd

Syzygiumcaryophyllatum

Leaves 355925 22198 435768 184352 258039 29882 491 2160 403 2159 3289 085 102 ND NDFruits 294428 4955 75621 14927 85555 1814 523 775 465 775 270 009 015 ND ND

RDA 35 g 9ndash15mg 15 g 1 g 280ndash350mg 50ndash60 μglowastND not detected limit of detection 005mgkg RDA recommended daily intake

Evidence-Based Complementary and Alternative Medicine 11

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

[18] (e FRAP reagent was prepared freshly by mixingacetate buffer (300mM pH 36) solution of TPTZ in HCland FeCl3 (20mM) in 10 1 1 ratio and incubated at 37degCTwo hundred microliters of the reaction mixture (FRAPreagent and sample) was incubated at room temperatureand absorbance was measured at 600 nm Vitamin E ana-logue Trolox was used as the reference standard where theresults were expressed as mg of Trolox equivalent per gramof extract (mg TEg of extract)

262 DPPH Radical Scavenging Assay (e radical scav-enging activity was determined by using 1 1-diphenyl-2-picryl-hydrazyl (DPPH) stable radical method [18] (e method isbased on the reduction of purple-colored 1 1-diphenyl-2-picrylhydrazyl to a yellow-colored diphenylpicrylhydrazine(ereaction volume of 200μL containing sample (50μL)methanolandDPPHwere incubated at room temperature for 10min andabsorbance was measured at 517nm A percentage inhibitionversus concentration was plotted and the concentration of thesample required for 50 inhibition was determined and rep-resented as EC50 value (e following formula was used tocalculate the percent inhibition

Radical scavenging activity CAB minus SAB( 1113857

CABtimes 100 (1)

where CAB is the absorbance of the control and SAB is theabsorbance of the sample

263 ABTS Radical Scavenging Activity (e ABTS+ radicalcation (ABTS+) scavenging activity assay was performed [19]with some modifications in 96-well microplates Plant ex-tracts were prepared in phosphate buffer solutions and wereallowed to react with ABTS+ radicals in a microwell plate for10min Absorbance wasmeasured in 734 nm and Trolox wasused as the standard (e following formula was used tocalculate the percent inhibition

Radical scavenging activity CAB minus SAB( 1113857

CABtimes 100 (2)

where CAB is the absorbance of the control and SAB is theabsorbance of the sample

(e antiradical activity was expressed as EC50 theconcentration required to cause 50 inhibition of ABTS+radicals

264 Oxygen Radical Absorbance Capacity (ORAC) Assay(eORAC assay is a hydrogen atom transfer- (HAT-) basedassay which measures the depletion of fluorescence intensityof a fluorescent probe upon the generation of peroxy radicalsdue to the oxidation of azo compounds In the presence of anantioxidant compound the decaying of fluorescent intensityis retarded (e overall decay is calculated by the differencebetween the area under the fluorescence decay curve (AUC)of the sample of interest and AUC of blank [20](e reactionvolume of 200 μL (pH 74) containing fluorescein (100 μL)and sample (50 μL) was preincubated at 37degC for 5min (e22prime-azobis (2-amidinopropane) dihydrochloride (AAPH)

solution was added (50 μL) to the mixture and the plate(black 96 well plates) was immediately placed in a fluo-rescent microplate reader (SPECTRAmax-Gemini EMMolecular Devices Inc USA) (e decay of fluorescence wasrecorded every minute for 35min and the excitation andemission wavelengths were obtained at 494 nm and 535 nmrespectively Phosphate buffer (75mM pH 74) replacing thesample was used as the blank and Trolox was used as thestandard antioxidant Results were expressed as mg of Troloxequivalent per gram of extract

265 Ferrous Ion Chelating Activity (e ferrous ion che-lating (FIC) activity was measured by the decrease in theabsorbance at 562 nm of the iron (II)-ferrozine complex [21]EDTA was used as the reference standard and all the samplesand standards were prepared in distilled water (e samplewas mixed with 1mM ferrous sulfate and distilled waterfollowed by 1mM ferrozine solution(e ability of the sampleto chelate ferrous ions was calculated relative to the control(distilled water instead of the sample) by the following for-mula (e results were expressed as mg EDTA equivalent pergram of extract

Chelating activity CAB minus SAB( 1113857

CABtimes 100 (3)

where SAB is the absorbance of the sample and CAB is theabsorbance of the control

27 Determination of α-Amylase Inhibition Activity (eα-amylase inhibition activity was carried out using thedinitrosalicylic (DNS) method [22] where the inhibitionactivity was measured by quantifying the maltose liberatedunder the assay conditions (e enzyme inhibitory activitywas expressed as a decrease in units of maltose liberated Amodified dinitrosalicylic acid (DNS) method was adopted toestimate the maltose equivalent (e reaction volume con-taining 40 μL of 1 (wv) starch solution and 775 μL of100mM sodium acetate buffer was preincubated with 135 μLof the sample at 40degC shaking water bath for 10min (emixture was further incubated with 50 μL of the α-amylaseenzyme (5 μgmL) at 40degC shaking water bath for 15min(e reaction was terminated by adding 500 μL DNS reagentand the mixture was placed in a boiling water bath for 5minfollowed by cooling in an ice bath Absorbance was mea-sured at 540 nm using a 96-well microplate reader Anti-amylase activity (inhibition ) was calculated using thefollowing equation

Inhibition() AC minus AS minus Ab( 11138571113858 1113859

ACtimes 100 (4)

where AC is the absorbance of the control AS is the ab-sorbance of the sample and Ab is the absorbance of thesample blank

28 BSA-Glucose Glycation Inhibitory Activity AssayAntiglycation activity assay was performed with somemodifications to the previous methodology [23] for CR

Evidence-Based Complementary and Alternative Medicine 5

extracts of leaves and fruits of Syzygium caryophyllatum Amixture of 800 μgmL BSA 400mM glucose and 80 μL ofthe sample in 50mM phosphate buffer (pH 74) containingsodium azide (002) was incubated at 60degC for 40 h (e600 μL of the reaction mixture was transferred to 15mLmicrocentrifuge tubes and 60 μL of 100 (wv) TCA wasadded and sample mixtures were centrifuged at 15000 rpmat 4degC for 4min (e resultant advanced glycation endproducts-BSA (AGEs-BSA) precipitate was then dissolved in1mL of phosphate buffer saline (pH 10) and the fluores-cence intensity was measured at an excitation and emissionwavelengths of 370 nm and 440 nm using a 96-well fluo-rescence microplate reader (SpectraMax Gemini EMMolecular Devices Inc USA) Rutin was used as thepositive control and antiglycation activity ( inhibition) wascalculated using the following equation followed by thecalculation of IC50 value

Inhibition() Fc ndash Fb1113858 1113859 ndash Fs ndash Fsb1113858 1113859

Fc ndash Fb1113858 1113859times 100 (5)

where Fc is the fluorescence of the control Fb is the fluo-rescence of BSA Fs is the fluorescence of the sample andFsb is the fluorescence of the sample blank

All the above in vitro bioassay procedures were validatedat Herbal Technology Section Industrial Technology In-stitute Sri Lanka (ISO 9005 certified Laboratory)

29 Gas Chromatography-Mass Spectrometry (GC-MS)Analysis (e extracted essential oil from the leaves wassubjected to GC-MS analysis to identify volatile compounds(e analysis was carried out by using TRACE 1300 coupledto ISQ QD mass spectrophotometer instrument ((ermoFisher Scientific Milan Italy) (e sample was injected into(ermo-scientific TG-WAX capillary column(30mtimes 025mmtimes 025 μm) fused with silica having poly-ethylene glycol as the stationary phase with helium as thecarrier gas (1mLmin) (e temperature of the injector wasmaintained at 250degC (e GC oven temperature was initiallyprogrammed at a temperature of 60degC and was ramped up to220degC at the rate of 5degCmin GC-MS interface temperaturewas maintained at 250degC (e mass detector was operated inEI mode in a scan mass range of mz 40ndash450 (e identi-fication of compounds was ascertained by comparing themass spectral values with the known compounds in NIST11USA mass spectral database

210 Preparation of Plant Extracts for Mineral ContentDetermination Approximately 05 g of homogenized sam-ple was weighed into easy prep high-pressure microwavevessel and 100mL of 65 nitric acid was added (e easyprep microwave digestion program was followed to digestthe samples by using a microwave digester (CEM MARS 5USA) (e digested sample was quantitatively transferredand filtered using No 542 Whatman filter paper followed bywashing with deionized water and volume up to 25mL (eprepared solution was used to analyze mineral content byICP-MS (Agilent 7900 USA)

211 Statistical Analysis Statistical analysis was performedusing Graph Pad Prism (version 7) All experimental datawas analyzed by one-way analysis of variance (ANOVA)ANOVArsquos Turkey multiple comparison test was used tocompare the mean values Pearsonlsquos correlation coefficientwas used for the correlation analysis and in all cases p

values less than 005 (plt 005) were considered statisticallysignificant (e quantitative results of phytochemicals an-tioxidant potentials and enzyme inhibition values wereexpressed as meanplusmn standard deviation (SD)

3 Results

S caryophyllatum fruits and leaves were extractedaccording to the method described above and fractionatedinto nonpolar and polar solvents with increasing polarityhexane and ethyl acetate respectively (is solventndashsolventpartitioning procedure was allowed to generate hexane(Hex) fraction ethyl acetate (EA) fraction and aqueous(AQ) fraction which was labelled and stored in minus20degC priorto analysis (e yield was estimated to the dry weight whichranged from 06mg to 312 g where the highest yield wasrecorded in crude (CR) extract of fruits (312 g) and thelowest was recorded in Hex fraction of leaves (06mg) (eweights of the CR extracts Hex EA and AQ fractions aregiven in Table 1 and the yield was calculated as a percentage

All the crude extracts and fractions were subjected tofurther analysis except the hexane fraction of leaves ofSyzygium caryophyllatum due to the unavailability of ade-quate sample yield

31 Total Polyphenol Content (TPC) and Total FlavonoidContent (TFC) of Leaves and Fruit Extracts of Syzygiumcaryophyllatum Plant Extracts Total polyphenol contentwas expressed as milligram gallic acid equivalent per gram ofsample (mg GAEg) and the total flavonoid content asmilligram quercetin equivalent per gram of sample (mg QEg)Total polyphenol values varied from 00923mg GAEg ofsample to 818mg GAEg of sample and from 172mgGAEg of sample to 892mg GAEg of sample for leaves andfruits respectively Polyphenol contents of samples werediffered significantly (plt 005) Table 2 presents the TPCand TFC values for crude and fractions of samples (ehighest TPC value was recorded in CR extract of fruitsfollowed by CR extract of leaves and AQ fraction of leavesFlavonoid content of the tested samples varied significantly(plt 005) among extracts TFC ranged from 0001mg QEgof sample to 203mg QEg of sample and the highest TFCvalue was reported in CR extract of fruits with value as202plusmn 019mg QEg of the sample followed by Hex fractionand EA fraction of fruits with values 080plusmn 005mg QEg ofsample and 037plusmn 001mg QEg of sample respectively

32 In Vitro Antioxidant Properties of Different Extracts ofSyzygium caryophyllatum (e antioxidant properties ofcrudes and fractions of leaves and fruits of Syzygium car-yophyllatum were determined using DPPH ABTS ORACFRAP and iron chelation assay methods (e results

6 Evidence-Based Complementary and Alternative Medicine

obtained are depicted in Table 3 where the significant dif-ferences (plt 005) were observed between CR extracts ofleaves and fruits and among different fractions of leavesand fruits

321 DPPH Radical Scavenging Activity Results revealedthat the crude extract and fractions of S caryophyllatumhave the ability to scavenge radicals and depicted a dose-response relationship DPPH radical scavenging activityof different fractions of S caryophyllatum differed sig-nificantly (plt 005) (e EA fraction of leaves exhibitedthe lowest EC50 value followed by Hex and EA fraction offruits with values 912 plusmn 428 μgmL 10097 plusmn 819 μgmLand 13553 plusmn 983 μgmL respectively (e results ob-tained from the fractions showed that EA fractions ofleaves and Hex fractions of fruits exhibited high antiox-idant activities compared to other fractions It shows thatthe accumulation of a variety of antioxidant compoundsdepends on the polarity of the solvent (erefore thecompounds responsible for the high radical scavengingactivity of S caryophyllatum leaves are mainly mediumpolarity compounds (e dose-response relationships ofS caryophyllatum leaves and fruits are given inFigures 3(a) and 3(b)

322 ABTS+ Radical Scavenging Activity (e highestradical scavenging activity was reported in Hex fraction offruits with EC50 value 1501plusmn 065 μgmL followed by AQfraction of S caryophyllatum leaves and AQ fraction of Scaryophyllatum fruits and the EC50 values were 2703plusmn 206and 3145plusmn 143 μgmL respectively It was found that fruitsand leaves showed a significant difference (plt 005) among

fractions (e CR extracts and fractions of leaves and fruitsshowed significant difference and dose-dependent inhibitionon ABTS radical scavenging activity (Figures 4(a) and 4(b))

323 Ferric Reducing Antioxidant Power (FRAP) (e CRextracts of leaves and fruits differed significantly (plt 005) intheir FRAP values CR extract of leaves 4827plusmn 811mgTEgof extract and CR extract of fruits 681plusmn 117mgTEg ofextract (e FRAP values of the fractions ranged from1481mgTEg of the extract to 27041mgTEg of extract EAfraction of leaves was reported as the highest among thetested fractions in leaves and fruits where the AQ fraction offruits exhibited the lowest values (ese results suggestedthat EA fraction of leaves and fruits exhibited the highestferric reducing property among tested fractions whichexplains the important behavior of the polar compounds inits reducing property

324 Oxygen Radical Absorbance Capacity (ORAC) (eORAC values of fractions ranged from 1723mgTEg of theextract to 34732mgTEg of extract (e highest activity wasrecorded in EA fraction of leaves following the AQ fractionof leaves and the lowest was recorded in AQ fraction infruits (e CR extract of leaves exhibited a significantlyhigher (plt 005) activity compared to the CR extract offruits with values 22100plusmn 3496 and 4598plusmn 258mgTEg ofextract respectively

325 Iron Chelating Activity (e highest iron chelatingability of CR extract was reported in leaves with values332plusmn 168mg EDTAg of extract and fruits were reported as1373plusmn 069mg EDTAg of extract (e Hex fraction of

Table 1 (e yield of Syzygium caryophyllatum plant extracts

Scientific name Part of plant Extract Weight of concentrated extract (g) Yield

Syzygium caryophyllatum

Leaves

CR 17500 70Hex lt0001 lt01EA 0114 01AQ 15984 71

Fruits

CR 31200 620Hex 1950 26EA 1531 20AQ 10165 136

CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction

Table 2 Total polyphenol content (TPC) and total flavonoid content (TFC) in different extracts of Syzygium caryophyllatum

Scientific name Part of plant Extract TPC (mg GAEg of sample) TFC (mg QEg of sample)

Syzygium caryophyllatum

LeavesCR 812plusmn 046a 016plusmn 001aEA 009plusmn 001b lt001AQ 712plusmn 016c 018plusmn 002ab

Fruits

CR 883plusmn 062ad 202plusmn 019cHex 342plusmn 029e 080plusmn 005dEA 173plusmn 076f 037plusmn 001eAQ 216plusmn 012g 010plusmn 001af

Results expressed as meanplusmn standard deviation CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction Mean valueswithin a column superscripted by different letters are significantly different at plt 005

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Antioxidant potentials of leaves and fruits of Syzygium caryophyllatum

Scientific name Part of plant Extract ORAC mg TEgof extract

FRAP mg TEgof extract

EC50 DPPHμgmL

EC50 ABTSμgmL

Iron chelation mgEDTAg of extract

Syzygiumcaryophyllatum

LeavesCR 22100plusmn 3496a 4827plusmn 811a 50020plusmn 4433a 10793plusmn 1507a 332plusmn 168aEA 34431plusmn 337b 26226plusmn 4393b 9120plusmn 428b 4061plusmn 276b 3601plusmn 698aAQ 32097plusmn 2062bc 7342plusmn 1258c 25105plusmn 1752c 2704plusmn 206c 5617plusmn 798b

Fruit

CR 4598plusmn 258d 681plusmn 117d 87372plusmn 1252d 12808plusmn 612d 1373plusmn 069cHex 3277plusmn 141e 5391plusmn 1230a 10097plusmn 819e 1501plusmn 065e 7482plusmn 266dEA 11865plusmn 760f 6307plusmn 1017e 13553plusmn 983f 8954plusmn 388f 828plusmn 040eAQ 2093plusmn 449g 1545plusmn 068f 94642plusmn 6504g 3145plusmn 145g mdash

Results are expressed as meanplusmn standard deviation n 3 Mean values within a column superscripted by different letters are significantly different at plt 005CR crude Hex hexane EA ethyl acetate AQ aqueous ORAC oxygen radical absorbance capacity FRAP ferric reducing antioxidant power ABTSABTS radical scavenging activity DPPH DPPH radical scavenging activity

0

20

40

60

80

100

0 200 400 600 800

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

00 05 10 15 20

Inhi

bitio

n

Concentration (mgmL)

CRHex

EAAQ

(b)

Figure 3 Dose-response curves for DPPH radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

120

0 50 100 150 200 250

Inhi

bitio

n

Concentration (microgmL)

CRHex

EAAQ

(b)

Figure 4 Dose-response curves for ABTS+ radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

8 Evidence-Based Complementary and Alternative Medicine

fruits exhibited the highest iron-chelating ability among thetested fractions with value 7482plusmn 266mg EDTAg of ex-tract and the lowest was reported in EA fraction of fruitswith value 828plusmn 040mg EDTAg of extract respectively(e AQ fraction of Syzygium caryophyllatum fruits did notshow a significant chelating activity where the highestconcentration (215mgmL) exhibited 1605 inhibition

33 Correlation Analysis between Polyphenol Content andDPPH Radical Scavenging Activity of Syzygium car-yophyllatum Fruits (e correlation coefficient betweenTPC content and DPPH of Syzygium caryophyllatum fruitsexhibited a significant (plt 005) positive correlation withPearson r value 09921 (Figure 5)

34 Ce α-Amylase Inhibitory Activity (e α-amylase inhi-bition potentials of CR extract Hex EA and AQ fraction ofleaves and fruits were analyzed Fruits exhibited significantinhibition of α-amylase activities where the leaves did notexhibit significant inhibitory activities (e Hex fraction ofS caryophyllatum fruits was reported to have the highestinhibitory activity with IC50 value 4720 plusmn 03 μgmL fol-lowed by CR extract EA fraction and AQ fraction withIC50 values 21826 plusmn 538 μgmL 61885plusmn 1855 μgmL and101455plusmn 8555 μgmL respectively Interestingly the Hexfraction of fruits demonstrated potent α-amylase inhibitionactivity (IC50 4720 plusmn 03 μgmL) compared to the standarddrug acarbose (IC50 8796 plusmn 143 μgmL)(e percentage ofα-amylase inhibitions of crudes and fractions was plottedagainst concentration (Figure 6) where it showed a dose-dependent increase in percentage activity of CR extract andthree types of fractions of fruits

341 Ce α-Amylase Inhibitory Activity of Isolated Fractionsof Hexane Fraction of Syzygium caryophyllatum Fruits

(1) Hexane and Ethyl Acetate Solvents on Isolation Procedure(e inhibition potential of compound mixtures of EA fractionwas evaluated for α-amylase inhibitory activity (e highesttested concentration was 200μgmL followed by 100μgmL50μgmL and 25μgmL(e similar inhibition potentials wereobserved for the first three concentrations and the inhibitionvalues were 5763plusmn018 5599plusmn160 and 5730plusmn018respectively where the inhibition potential of 25μgmL wasobserved as a rapid drop to the inhibition 1278plusmn045Further inhibition potentials were evaluated between 50μgmLand 25μgmL (Table 4)(e α-amylase inhibitory activity of thetested fraction showed a poor linear response (R2 06384)hence IC50 was not calculated

(2) Dichloromethane and Ethyl Acetate Solvents on Iso-lation Procedure (e α-amylase inhibitory activity ofdifferent combined fractions was analyzed (C1 C2 andC3)(e inhibitory activities of C1 and C2 fractions did notexhibit significant inhibitory activities where the fractionC3 showed significant (plt 005) inhibitory activity for the

screened concentrations (e fraction C3 was dose-de-pendent and IC50 value was 227 plusmn 181 μgmL

(e summary of α-amylase inhibitory activity of hexanefraction and its chromatographic fraction is given in Table 5

35 Antiglycation Activity (e antiglycation activity ofcrude extracts of leaves and fruits was screened for threeconcentrations 200 100 and 50 μgmL (Table 6) (e CRextract of leaves exhibited the highest inhibitory activity(9361 plusmn 853 at a concentration of 200 μgmL) followedby CR extract of S caryophyllatum fruits (4797 plusmn 263)(e standard drug rutin exhibited antiglycation activitywith IC50 value as 3423 plusmn 318 μgmL (e IC50 value ofCR extract of leaves was 5783 plusmn 791 μgmL Howeverfor fruits similar inhibitions were observed for differentdoses hence IC50 value could not be calculated

36 Chemical Compositions of Syzygium caryophyllatum LeafEssential Oil Syzygium caryophyllatum leaves yieldedessential oil upon hydrodistillation GC-MS analysisidentified 58 compounds from the essential oil and theretention time and area of the identified major peaks arelisted in Table 7 (e major compounds identified in theleaves were phytol with a 2466 peak area (retentiontime 3444) α-cadinol with a 387 peak area (retentiontime 2781) α-guaiene with a 384 peak area (retentiontime 2629) cyclosiolongifolene 910-dehydro with a382 peak area (retention time 2826) humulene with a336 peak area (retention time 1645) and car-yophyllene with a 321 peak area (retention time 1479)respectively (e GC-MS chromatogram of Syzygiumcaryophyllatum leaf oil is presented in Figure 7

37 Mineral Compositions Functionally important micro-minerals such as selenium (Se) iron (Fe) strontium (Sr)manganese (Mn) cobalt (Co) copper (Cu) and the toxicheavymetals such as arsenic (As) cadmium (Cd) chromium(Cr) and lead (Pb) were evaluated (Table 8) (e ICP-MS

0

20

40

60

80

100

0 50 100 150

DPP

H v

alue

(mg

TEg

of e

xtra

ct)

Total phenol content (mg GAEg of extract)

Figure 5 Correlation of Syzygium caryophyllatum fruits TPCversus DPPH values (plt 005)

Evidence-Based Complementary and Alternative Medicine 9

data ranged from 009 to 45370mgkg of sample Sodium(Na) was found to be the highest (leaves 435768mgkg ofthe sample) while Se was found to be the lowest (fruits009mgkg of the sample) (e leaves were found to have thehighest concentration of the tested minerals compared to thefruits (e second richest macroelement was found to be K(leaves 355925mgkg and fruits 294428mgkg) followedby Mg (leaves 258040mgkg) and Ca (leaves 184352mgkg)(e maximum amount of Al Mn Fe and Zn was found

to be in leaves with values 52445mgkg 29882mgkg22198mgkg and 2160mgkg respectively Among thetested microelements Sr was found to be the richest with avalue of 32889mgkg in leaves which is significantly(plt 005) different from fruits (270mgkg) (e Cu and Crwere found in similar small concentrations in leaves (Cu491mgkg and Cr 403mgkg) and fruits (Cu 523mgkgand Cr 465mgkg) (e maximum concentration of Sewas determined to be 085mgkg in leaves and the fruitsexhibited the lowest concentration with a value of 009mgkg(e investigation of the level of Co revealed that the maxi-mum concentration of the element exists as 0128mgkg inleaves while in the fruits it was not detected at the detectionlimit of 005mgkg

(e analysis of toxic heavy metals revealed the presenceof a maximum concentration of Pb in leaves (102mgkg)and the lowest in fruits (015mgkg) Besides the concen-trations of toxic heavy metals such as Cd and As were notdetected in leaves and fruits (limit of detection 005mgkg)

4 Discussion

Diabetes mellitus (DM) is a complex metabolic disordercharacterized by hyperglycemia which is mainly associatedwith malfunctions and abnormalities in carbohydrate pro-tein and fat metabolism Digestion and absorption of car-bohydrates to the blood stream also have a significantinfluence on DM Further the increased production of re-active oxygen species (ROS) under DM accelerates the proteinglycation process [24] (e production and accumulation ofglycated proteins have shown a strong association withcomplications of DM [25] (us the management of DM isdirected toward addressing multiple targets including re-duction of postprandial carbohydrate levels in the bloodimprovement in the homeostatic and metabolic process ofcarbohydrates and lipids an improvement on the efficiency ofantioxidant defense and control of protein glycation [26]

In this regard WHO recommends functional ingredi-ents and supplements with multiple functional propertiesincluding antidiabetic and the antiradical properties in thecontrol of the DM [27]

In this study functional properties including α-amylaseinhibition protein glycation inhibition and antioxidantactivities of S caryophyllatum fruit and leaf extracts havebeen investigated along with chemical constituents andmineral analyses (e results revealed the potential of Scaryophyllatum as a source of supplement for DM man-agement To the best of our knowledge the present study isthe first study reported in Sri Lanka on functional activitiesof S caryophyllatum fruit and leaf extracts Crude extracts ofboth fruits and leaves showed significant antioxidant andmoderate antiglycation activities However only fruit ex-tracts showed α-amylase inhibitory activity Even thoughboth fruits and leaves CR were tested against α-glucosidaseenzyme inhibitory activity none of them showed a signif-icant inhibitory activity at a maximum dose of 400 μgmL(data not shown) Solventndashsolvent partitioning of both CRwas used to separate and valuate the most potent fractionswith α-amylase inhibition activity

0

20

40

60

80

100

0 200 400 600 800 1000

Inhi

bitio

n

Concentration (μgmL)

CRHex

EAAQ

Figure 6 Dose-response relationship of crude and fractionsof Syzygium caryophyllatum fruits for alpha-amylase inhibi-tory activity (CR crude Hex hexane EA ethyl acetateAQ aqueous)

Table 4 (e α-amylase inhibitory activity of isolated compoundmixtures of Hex fraction of Syzygium caryophyllatum fruits (hexaneand ethyl acetate solvents on isolation procedure)

Assay concentration μgmL Inhibition 200 5763plusmn 018a100 5599plusmn 160a50 5730plusmn 018a45 2339plusmn 021b40 2245plusmn 079b35 2143plusmn 019b30 1433plusmn 083c25 1278plusmn 045c

Results are expressed as meanplusmn standard deviation n 3lowast Mean valueswithin a column superscripted by different letters are significantly differentat plt 005

Table 5 (e α-amylase inhibitory activity of hexane fraction andits chromatographic fraction of Syzygium caryophyllatum fruits

Sample IC50 (μgmL)Hex fraction 4720aplusmn 030Hex EA (0 100) NLRC3 DCM EA (95 5) 227bplusmn 181Standard drug acarbose 8796cplusmn 143Results are expressed as meanplusmn standard deviation n 3lowast NLRno linearresponse Mean values within a column superscripted by different lettersare significantly different at plt 005 Hex hexane EA ethyl acetateDCM dichloromethane C3 combined fraction 3

10 Evidence-Based Complementary and Alternative Medicine

Hexane fraction of fruit extract exhibited the highestinhibition activity with IC50 value 4720plusmn 037 μgmL and issignificantly higher compared to acarbose (IC508796plusmn 143 μgmL) (erefore it was further separated bycolumn chromatography (e fraction C3 which was iso-lated on elution with DCM EA 95 5 exhibited a significant(plt 005) α-amylase inhibitory activity with an IC50 value of227plusmn 181 μgmL (e compound or the mixture of

compounds responsible for the α-amylase inhibition activitymay be in the C3 compound mixture which is required to besubjected to further isolation and identification Inhibitionmay be due to the presence of potential alpha-amylase in-hibitors such as alkaloids tannin polyphenols and flavo-noids (ese inhibitors can also act as starch blockers as theyblock the hydrolysis of 14-glycosidic linkages of starch andoligosaccharides [28] Hex fraction of S caryophyllatum fruit

Table 6 Antiglycation activity of crude extracts of Syzygium caryophyllatum

Crude extract Inhibition

IC50 (μgmL)Concentration (μgmL)50 100 200

Leaves 5488plusmn 310a 7987plusmn 594a 9361plusmn 850a 5783plusmn 791Fruits 4131plusmn 789b 5666plusmn 836b 4797plusmn 263b mdashStandard rutin IC50 3423plusmn 318 μgmLResults are expressed as meanplusmn standard deviation n 3lowast Mean values within a column superscripted by different letters are significantly different atplt 005

Table 7 Major compounds detected in Syzygium caryophyllatum leaf essential oil

Peak Compound Formula and lowastmol wt RT Area SI RSI1 Caryophyllene C15H24 204 1479 321 954 9542 Humulene C15H24 204 1645 336 907 9073 α-Cadinol C15H26O 222 2781 387 923 9284 Cyclosiolongifolene 910-dehydro C17H26O2 202 2826 382 790 8005 Phytol C20H40O 296 3444 2466 927 927lowastMol wtmolecular weight

2781

RT 000ndash4200100

90

80

70

60

50

40

30

20

10

0 5 10 15 20 25 30 35 40Retention time (min)

3444

2826

Rela

tive a

bund

ance

1 24

3

5

1479 1645

Figure 7 GC-MS chromatogram of Syzygium caryophyllatum leaf essential oil

Table 8 Mineral compositions of leaves and fruits of Syzygium caryophyllatum

Mineral elements (mgkg per sample)K Fe Na Ca Mg Mn Cu Zn Cr Zn Sr Se Pb As Cd

Syzygiumcaryophyllatum

Leaves 355925 22198 435768 184352 258039 29882 491 2160 403 2159 3289 085 102 ND NDFruits 294428 4955 75621 14927 85555 1814 523 775 465 775 270 009 015 ND ND

RDA 35 g 9ndash15mg 15 g 1 g 280ndash350mg 50ndash60 μglowastND not detected limit of detection 005mgkg RDA recommended daily intake

Evidence-Based Complementary and Alternative Medicine 11

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

extracts of leaves and fruits of Syzygium caryophyllatum Amixture of 800 μgmL BSA 400mM glucose and 80 μL ofthe sample in 50mM phosphate buffer (pH 74) containingsodium azide (002) was incubated at 60degC for 40 h (e600 μL of the reaction mixture was transferred to 15mLmicrocentrifuge tubes and 60 μL of 100 (wv) TCA wasadded and sample mixtures were centrifuged at 15000 rpmat 4degC for 4min (e resultant advanced glycation endproducts-BSA (AGEs-BSA) precipitate was then dissolved in1mL of phosphate buffer saline (pH 10) and the fluores-cence intensity was measured at an excitation and emissionwavelengths of 370 nm and 440 nm using a 96-well fluo-rescence microplate reader (SpectraMax Gemini EMMolecular Devices Inc USA) Rutin was used as thepositive control and antiglycation activity ( inhibition) wascalculated using the following equation followed by thecalculation of IC50 value

Inhibition() Fc ndash Fb1113858 1113859 ndash Fs ndash Fsb1113858 1113859

Fc ndash Fb1113858 1113859times 100 (5)

where Fc is the fluorescence of the control Fb is the fluo-rescence of BSA Fs is the fluorescence of the sample andFsb is the fluorescence of the sample blank

All the above in vitro bioassay procedures were validatedat Herbal Technology Section Industrial Technology In-stitute Sri Lanka (ISO 9005 certified Laboratory)

29 Gas Chromatography-Mass Spectrometry (GC-MS)Analysis (e extracted essential oil from the leaves wassubjected to GC-MS analysis to identify volatile compounds(e analysis was carried out by using TRACE 1300 coupledto ISQ QD mass spectrophotometer instrument ((ermoFisher Scientific Milan Italy) (e sample was injected into(ermo-scientific TG-WAX capillary column(30mtimes 025mmtimes 025 μm) fused with silica having poly-ethylene glycol as the stationary phase with helium as thecarrier gas (1mLmin) (e temperature of the injector wasmaintained at 250degC (e GC oven temperature was initiallyprogrammed at a temperature of 60degC and was ramped up to220degC at the rate of 5degCmin GC-MS interface temperaturewas maintained at 250degC (e mass detector was operated inEI mode in a scan mass range of mz 40ndash450 (e identi-fication of compounds was ascertained by comparing themass spectral values with the known compounds in NIST11USA mass spectral database

210 Preparation of Plant Extracts for Mineral ContentDetermination Approximately 05 g of homogenized sam-ple was weighed into easy prep high-pressure microwavevessel and 100mL of 65 nitric acid was added (e easyprep microwave digestion program was followed to digestthe samples by using a microwave digester (CEM MARS 5USA) (e digested sample was quantitatively transferredand filtered using No 542 Whatman filter paper followed bywashing with deionized water and volume up to 25mL (eprepared solution was used to analyze mineral content byICP-MS (Agilent 7900 USA)

211 Statistical Analysis Statistical analysis was performedusing Graph Pad Prism (version 7) All experimental datawas analyzed by one-way analysis of variance (ANOVA)ANOVArsquos Turkey multiple comparison test was used tocompare the mean values Pearsonlsquos correlation coefficientwas used for the correlation analysis and in all cases p

values less than 005 (plt 005) were considered statisticallysignificant (e quantitative results of phytochemicals an-tioxidant potentials and enzyme inhibition values wereexpressed as meanplusmn standard deviation (SD)

3 Results

S caryophyllatum fruits and leaves were extractedaccording to the method described above and fractionatedinto nonpolar and polar solvents with increasing polarityhexane and ethyl acetate respectively (is solventndashsolventpartitioning procedure was allowed to generate hexane(Hex) fraction ethyl acetate (EA) fraction and aqueous(AQ) fraction which was labelled and stored in minus20degC priorto analysis (e yield was estimated to the dry weight whichranged from 06mg to 312 g where the highest yield wasrecorded in crude (CR) extract of fruits (312 g) and thelowest was recorded in Hex fraction of leaves (06mg) (eweights of the CR extracts Hex EA and AQ fractions aregiven in Table 1 and the yield was calculated as a percentage

All the crude extracts and fractions were subjected tofurther analysis except the hexane fraction of leaves ofSyzygium caryophyllatum due to the unavailability of ade-quate sample yield

31 Total Polyphenol Content (TPC) and Total FlavonoidContent (TFC) of Leaves and Fruit Extracts of Syzygiumcaryophyllatum Plant Extracts Total polyphenol contentwas expressed as milligram gallic acid equivalent per gram ofsample (mg GAEg) and the total flavonoid content asmilligram quercetin equivalent per gram of sample (mg QEg)Total polyphenol values varied from 00923mg GAEg ofsample to 818mg GAEg of sample and from 172mgGAEg of sample to 892mg GAEg of sample for leaves andfruits respectively Polyphenol contents of samples werediffered significantly (plt 005) Table 2 presents the TPCand TFC values for crude and fractions of samples (ehighest TPC value was recorded in CR extract of fruitsfollowed by CR extract of leaves and AQ fraction of leavesFlavonoid content of the tested samples varied significantly(plt 005) among extracts TFC ranged from 0001mg QEgof sample to 203mg QEg of sample and the highest TFCvalue was reported in CR extract of fruits with value as202plusmn 019mg QEg of the sample followed by Hex fractionand EA fraction of fruits with values 080plusmn 005mg QEg ofsample and 037plusmn 001mg QEg of sample respectively

32 In Vitro Antioxidant Properties of Different Extracts ofSyzygium caryophyllatum (e antioxidant properties ofcrudes and fractions of leaves and fruits of Syzygium car-yophyllatum were determined using DPPH ABTS ORACFRAP and iron chelation assay methods (e results

6 Evidence-Based Complementary and Alternative Medicine

obtained are depicted in Table 3 where the significant dif-ferences (plt 005) were observed between CR extracts ofleaves and fruits and among different fractions of leavesand fruits

321 DPPH Radical Scavenging Activity Results revealedthat the crude extract and fractions of S caryophyllatumhave the ability to scavenge radicals and depicted a dose-response relationship DPPH radical scavenging activityof different fractions of S caryophyllatum differed sig-nificantly (plt 005) (e EA fraction of leaves exhibitedthe lowest EC50 value followed by Hex and EA fraction offruits with values 912 plusmn 428 μgmL 10097 plusmn 819 μgmLand 13553 plusmn 983 μgmL respectively (e results ob-tained from the fractions showed that EA fractions ofleaves and Hex fractions of fruits exhibited high antiox-idant activities compared to other fractions It shows thatthe accumulation of a variety of antioxidant compoundsdepends on the polarity of the solvent (erefore thecompounds responsible for the high radical scavengingactivity of S caryophyllatum leaves are mainly mediumpolarity compounds (e dose-response relationships ofS caryophyllatum leaves and fruits are given inFigures 3(a) and 3(b)

322 ABTS+ Radical Scavenging Activity (e highestradical scavenging activity was reported in Hex fraction offruits with EC50 value 1501plusmn 065 μgmL followed by AQfraction of S caryophyllatum leaves and AQ fraction of Scaryophyllatum fruits and the EC50 values were 2703plusmn 206and 3145plusmn 143 μgmL respectively It was found that fruitsand leaves showed a significant difference (plt 005) among

fractions (e CR extracts and fractions of leaves and fruitsshowed significant difference and dose-dependent inhibitionon ABTS radical scavenging activity (Figures 4(a) and 4(b))

323 Ferric Reducing Antioxidant Power (FRAP) (e CRextracts of leaves and fruits differed significantly (plt 005) intheir FRAP values CR extract of leaves 4827plusmn 811mgTEgof extract and CR extract of fruits 681plusmn 117mgTEg ofextract (e FRAP values of the fractions ranged from1481mgTEg of the extract to 27041mgTEg of extract EAfraction of leaves was reported as the highest among thetested fractions in leaves and fruits where the AQ fraction offruits exhibited the lowest values (ese results suggestedthat EA fraction of leaves and fruits exhibited the highestferric reducing property among tested fractions whichexplains the important behavior of the polar compounds inits reducing property

324 Oxygen Radical Absorbance Capacity (ORAC) (eORAC values of fractions ranged from 1723mgTEg of theextract to 34732mgTEg of extract (e highest activity wasrecorded in EA fraction of leaves following the AQ fractionof leaves and the lowest was recorded in AQ fraction infruits (e CR extract of leaves exhibited a significantlyhigher (plt 005) activity compared to the CR extract offruits with values 22100plusmn 3496 and 4598plusmn 258mgTEg ofextract respectively

325 Iron Chelating Activity (e highest iron chelatingability of CR extract was reported in leaves with values332plusmn 168mg EDTAg of extract and fruits were reported as1373plusmn 069mg EDTAg of extract (e Hex fraction of

Table 1 (e yield of Syzygium caryophyllatum plant extracts

Scientific name Part of plant Extract Weight of concentrated extract (g) Yield

Syzygium caryophyllatum

Leaves

CR 17500 70Hex lt0001 lt01EA 0114 01AQ 15984 71

Fruits

CR 31200 620Hex 1950 26EA 1531 20AQ 10165 136

CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction

Table 2 Total polyphenol content (TPC) and total flavonoid content (TFC) in different extracts of Syzygium caryophyllatum

Scientific name Part of plant Extract TPC (mg GAEg of sample) TFC (mg QEg of sample)

Syzygium caryophyllatum

LeavesCR 812plusmn 046a 016plusmn 001aEA 009plusmn 001b lt001AQ 712plusmn 016c 018plusmn 002ab

Fruits

CR 883plusmn 062ad 202plusmn 019cHex 342plusmn 029e 080plusmn 005dEA 173plusmn 076f 037plusmn 001eAQ 216plusmn 012g 010plusmn 001af

Results expressed as meanplusmn standard deviation CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction Mean valueswithin a column superscripted by different letters are significantly different at plt 005

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Antioxidant potentials of leaves and fruits of Syzygium caryophyllatum

Scientific name Part of plant Extract ORAC mg TEgof extract

FRAP mg TEgof extract

EC50 DPPHμgmL

EC50 ABTSμgmL

Iron chelation mgEDTAg of extract

Syzygiumcaryophyllatum

LeavesCR 22100plusmn 3496a 4827plusmn 811a 50020plusmn 4433a 10793plusmn 1507a 332plusmn 168aEA 34431plusmn 337b 26226plusmn 4393b 9120plusmn 428b 4061plusmn 276b 3601plusmn 698aAQ 32097plusmn 2062bc 7342plusmn 1258c 25105plusmn 1752c 2704plusmn 206c 5617plusmn 798b

Fruit

CR 4598plusmn 258d 681plusmn 117d 87372plusmn 1252d 12808plusmn 612d 1373plusmn 069cHex 3277plusmn 141e 5391plusmn 1230a 10097plusmn 819e 1501plusmn 065e 7482plusmn 266dEA 11865plusmn 760f 6307plusmn 1017e 13553plusmn 983f 8954plusmn 388f 828plusmn 040eAQ 2093plusmn 449g 1545plusmn 068f 94642plusmn 6504g 3145plusmn 145g mdash

Results are expressed as meanplusmn standard deviation n 3 Mean values within a column superscripted by different letters are significantly different at plt 005CR crude Hex hexane EA ethyl acetate AQ aqueous ORAC oxygen radical absorbance capacity FRAP ferric reducing antioxidant power ABTSABTS radical scavenging activity DPPH DPPH radical scavenging activity

0

20

40

60

80

100

0 200 400 600 800

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

00 05 10 15 20

Inhi

bitio

n

Concentration (mgmL)

CRHex

EAAQ

(b)

Figure 3 Dose-response curves for DPPH radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

120

0 50 100 150 200 250

Inhi

bitio

n

Concentration (microgmL)

CRHex

EAAQ

(b)

Figure 4 Dose-response curves for ABTS+ radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

8 Evidence-Based Complementary and Alternative Medicine

fruits exhibited the highest iron-chelating ability among thetested fractions with value 7482plusmn 266mg EDTAg of ex-tract and the lowest was reported in EA fraction of fruitswith value 828plusmn 040mg EDTAg of extract respectively(e AQ fraction of Syzygium caryophyllatum fruits did notshow a significant chelating activity where the highestconcentration (215mgmL) exhibited 1605 inhibition

33 Correlation Analysis between Polyphenol Content andDPPH Radical Scavenging Activity of Syzygium car-yophyllatum Fruits (e correlation coefficient betweenTPC content and DPPH of Syzygium caryophyllatum fruitsexhibited a significant (plt 005) positive correlation withPearson r value 09921 (Figure 5)

34 Ce α-Amylase Inhibitory Activity (e α-amylase inhi-bition potentials of CR extract Hex EA and AQ fraction ofleaves and fruits were analyzed Fruits exhibited significantinhibition of α-amylase activities where the leaves did notexhibit significant inhibitory activities (e Hex fraction ofS caryophyllatum fruits was reported to have the highestinhibitory activity with IC50 value 4720 plusmn 03 μgmL fol-lowed by CR extract EA fraction and AQ fraction withIC50 values 21826 plusmn 538 μgmL 61885plusmn 1855 μgmL and101455plusmn 8555 μgmL respectively Interestingly the Hexfraction of fruits demonstrated potent α-amylase inhibitionactivity (IC50 4720 plusmn 03 μgmL) compared to the standarddrug acarbose (IC50 8796 plusmn 143 μgmL)(e percentage ofα-amylase inhibitions of crudes and fractions was plottedagainst concentration (Figure 6) where it showed a dose-dependent increase in percentage activity of CR extract andthree types of fractions of fruits

341 Ce α-Amylase Inhibitory Activity of Isolated Fractionsof Hexane Fraction of Syzygium caryophyllatum Fruits

(1) Hexane and Ethyl Acetate Solvents on Isolation Procedure(e inhibition potential of compound mixtures of EA fractionwas evaluated for α-amylase inhibitory activity (e highesttested concentration was 200μgmL followed by 100μgmL50μgmL and 25μgmL(e similar inhibition potentials wereobserved for the first three concentrations and the inhibitionvalues were 5763plusmn018 5599plusmn160 and 5730plusmn018respectively where the inhibition potential of 25μgmL wasobserved as a rapid drop to the inhibition 1278plusmn045Further inhibition potentials were evaluated between 50μgmLand 25μgmL (Table 4)(e α-amylase inhibitory activity of thetested fraction showed a poor linear response (R2 06384)hence IC50 was not calculated

(2) Dichloromethane and Ethyl Acetate Solvents on Iso-lation Procedure (e α-amylase inhibitory activity ofdifferent combined fractions was analyzed (C1 C2 andC3)(e inhibitory activities of C1 and C2 fractions did notexhibit significant inhibitory activities where the fractionC3 showed significant (plt 005) inhibitory activity for the

screened concentrations (e fraction C3 was dose-de-pendent and IC50 value was 227 plusmn 181 μgmL

(e summary of α-amylase inhibitory activity of hexanefraction and its chromatographic fraction is given in Table 5

35 Antiglycation Activity (e antiglycation activity ofcrude extracts of leaves and fruits was screened for threeconcentrations 200 100 and 50 μgmL (Table 6) (e CRextract of leaves exhibited the highest inhibitory activity(9361 plusmn 853 at a concentration of 200 μgmL) followedby CR extract of S caryophyllatum fruits (4797 plusmn 263)(e standard drug rutin exhibited antiglycation activitywith IC50 value as 3423 plusmn 318 μgmL (e IC50 value ofCR extract of leaves was 5783 plusmn 791 μgmL Howeverfor fruits similar inhibitions were observed for differentdoses hence IC50 value could not be calculated

36 Chemical Compositions of Syzygium caryophyllatum LeafEssential Oil Syzygium caryophyllatum leaves yieldedessential oil upon hydrodistillation GC-MS analysisidentified 58 compounds from the essential oil and theretention time and area of the identified major peaks arelisted in Table 7 (e major compounds identified in theleaves were phytol with a 2466 peak area (retentiontime 3444) α-cadinol with a 387 peak area (retentiontime 2781) α-guaiene with a 384 peak area (retentiontime 2629) cyclosiolongifolene 910-dehydro with a382 peak area (retention time 2826) humulene with a336 peak area (retention time 1645) and car-yophyllene with a 321 peak area (retention time 1479)respectively (e GC-MS chromatogram of Syzygiumcaryophyllatum leaf oil is presented in Figure 7

37 Mineral Compositions Functionally important micro-minerals such as selenium (Se) iron (Fe) strontium (Sr)manganese (Mn) cobalt (Co) copper (Cu) and the toxicheavymetals such as arsenic (As) cadmium (Cd) chromium(Cr) and lead (Pb) were evaluated (Table 8) (e ICP-MS

0

20

40

60

80

100

0 50 100 150

DPP

H v

alue

(mg

TEg

of e

xtra

ct)

Total phenol content (mg GAEg of extract)

Figure 5 Correlation of Syzygium caryophyllatum fruits TPCversus DPPH values (plt 005)

Evidence-Based Complementary and Alternative Medicine 9

data ranged from 009 to 45370mgkg of sample Sodium(Na) was found to be the highest (leaves 435768mgkg ofthe sample) while Se was found to be the lowest (fruits009mgkg of the sample) (e leaves were found to have thehighest concentration of the tested minerals compared to thefruits (e second richest macroelement was found to be K(leaves 355925mgkg and fruits 294428mgkg) followedby Mg (leaves 258040mgkg) and Ca (leaves 184352mgkg)(e maximum amount of Al Mn Fe and Zn was found

to be in leaves with values 52445mgkg 29882mgkg22198mgkg and 2160mgkg respectively Among thetested microelements Sr was found to be the richest with avalue of 32889mgkg in leaves which is significantly(plt 005) different from fruits (270mgkg) (e Cu and Crwere found in similar small concentrations in leaves (Cu491mgkg and Cr 403mgkg) and fruits (Cu 523mgkgand Cr 465mgkg) (e maximum concentration of Sewas determined to be 085mgkg in leaves and the fruitsexhibited the lowest concentration with a value of 009mgkg(e investigation of the level of Co revealed that the maxi-mum concentration of the element exists as 0128mgkg inleaves while in the fruits it was not detected at the detectionlimit of 005mgkg

(e analysis of toxic heavy metals revealed the presenceof a maximum concentration of Pb in leaves (102mgkg)and the lowest in fruits (015mgkg) Besides the concen-trations of toxic heavy metals such as Cd and As were notdetected in leaves and fruits (limit of detection 005mgkg)

4 Discussion

Diabetes mellitus (DM) is a complex metabolic disordercharacterized by hyperglycemia which is mainly associatedwith malfunctions and abnormalities in carbohydrate pro-tein and fat metabolism Digestion and absorption of car-bohydrates to the blood stream also have a significantinfluence on DM Further the increased production of re-active oxygen species (ROS) under DM accelerates the proteinglycation process [24] (e production and accumulation ofglycated proteins have shown a strong association withcomplications of DM [25] (us the management of DM isdirected toward addressing multiple targets including re-duction of postprandial carbohydrate levels in the bloodimprovement in the homeostatic and metabolic process ofcarbohydrates and lipids an improvement on the efficiency ofantioxidant defense and control of protein glycation [26]

In this regard WHO recommends functional ingredi-ents and supplements with multiple functional propertiesincluding antidiabetic and the antiradical properties in thecontrol of the DM [27]

In this study functional properties including α-amylaseinhibition protein glycation inhibition and antioxidantactivities of S caryophyllatum fruit and leaf extracts havebeen investigated along with chemical constituents andmineral analyses (e results revealed the potential of Scaryophyllatum as a source of supplement for DM man-agement To the best of our knowledge the present study isthe first study reported in Sri Lanka on functional activitiesof S caryophyllatum fruit and leaf extracts Crude extracts ofboth fruits and leaves showed significant antioxidant andmoderate antiglycation activities However only fruit ex-tracts showed α-amylase inhibitory activity Even thoughboth fruits and leaves CR were tested against α-glucosidaseenzyme inhibitory activity none of them showed a signif-icant inhibitory activity at a maximum dose of 400 μgmL(data not shown) Solventndashsolvent partitioning of both CRwas used to separate and valuate the most potent fractionswith α-amylase inhibition activity

0

20

40

60

80

100

0 200 400 600 800 1000

Inhi

bitio

n

Concentration (μgmL)

CRHex

EAAQ

Figure 6 Dose-response relationship of crude and fractionsof Syzygium caryophyllatum fruits for alpha-amylase inhibi-tory activity (CR crude Hex hexane EA ethyl acetateAQ aqueous)

Table 4 (e α-amylase inhibitory activity of isolated compoundmixtures of Hex fraction of Syzygium caryophyllatum fruits (hexaneand ethyl acetate solvents on isolation procedure)

Assay concentration μgmL Inhibition 200 5763plusmn 018a100 5599plusmn 160a50 5730plusmn 018a45 2339plusmn 021b40 2245plusmn 079b35 2143plusmn 019b30 1433plusmn 083c25 1278plusmn 045c

Results are expressed as meanplusmn standard deviation n 3lowast Mean valueswithin a column superscripted by different letters are significantly differentat plt 005

Table 5 (e α-amylase inhibitory activity of hexane fraction andits chromatographic fraction of Syzygium caryophyllatum fruits

Sample IC50 (μgmL)Hex fraction 4720aplusmn 030Hex EA (0 100) NLRC3 DCM EA (95 5) 227bplusmn 181Standard drug acarbose 8796cplusmn 143Results are expressed as meanplusmn standard deviation n 3lowast NLRno linearresponse Mean values within a column superscripted by different lettersare significantly different at plt 005 Hex hexane EA ethyl acetateDCM dichloromethane C3 combined fraction 3

10 Evidence-Based Complementary and Alternative Medicine

Hexane fraction of fruit extract exhibited the highestinhibition activity with IC50 value 4720plusmn 037 μgmL and issignificantly higher compared to acarbose (IC508796plusmn 143 μgmL) (erefore it was further separated bycolumn chromatography (e fraction C3 which was iso-lated on elution with DCM EA 95 5 exhibited a significant(plt 005) α-amylase inhibitory activity with an IC50 value of227plusmn 181 μgmL (e compound or the mixture of

compounds responsible for the α-amylase inhibition activitymay be in the C3 compound mixture which is required to besubjected to further isolation and identification Inhibitionmay be due to the presence of potential alpha-amylase in-hibitors such as alkaloids tannin polyphenols and flavo-noids (ese inhibitors can also act as starch blockers as theyblock the hydrolysis of 14-glycosidic linkages of starch andoligosaccharides [28] Hex fraction of S caryophyllatum fruit

Table 6 Antiglycation activity of crude extracts of Syzygium caryophyllatum

Crude extract Inhibition

IC50 (μgmL)Concentration (μgmL)50 100 200

Leaves 5488plusmn 310a 7987plusmn 594a 9361plusmn 850a 5783plusmn 791Fruits 4131plusmn 789b 5666plusmn 836b 4797plusmn 263b mdashStandard rutin IC50 3423plusmn 318 μgmLResults are expressed as meanplusmn standard deviation n 3lowast Mean values within a column superscripted by different letters are significantly different atplt 005

Table 7 Major compounds detected in Syzygium caryophyllatum leaf essential oil

Peak Compound Formula and lowastmol wt RT Area SI RSI1 Caryophyllene C15H24 204 1479 321 954 9542 Humulene C15H24 204 1645 336 907 9073 α-Cadinol C15H26O 222 2781 387 923 9284 Cyclosiolongifolene 910-dehydro C17H26O2 202 2826 382 790 8005 Phytol C20H40O 296 3444 2466 927 927lowastMol wtmolecular weight

2781

RT 000ndash4200100

90

80

70

60

50

40

30

20

10

0 5 10 15 20 25 30 35 40Retention time (min)

3444

2826

Rela

tive a

bund

ance

1 24

3

5

1479 1645

Figure 7 GC-MS chromatogram of Syzygium caryophyllatum leaf essential oil

Table 8 Mineral compositions of leaves and fruits of Syzygium caryophyllatum

Mineral elements (mgkg per sample)K Fe Na Ca Mg Mn Cu Zn Cr Zn Sr Se Pb As Cd

Syzygiumcaryophyllatum

Leaves 355925 22198 435768 184352 258039 29882 491 2160 403 2159 3289 085 102 ND NDFruits 294428 4955 75621 14927 85555 1814 523 775 465 775 270 009 015 ND ND

RDA 35 g 9ndash15mg 15 g 1 g 280ndash350mg 50ndash60 μglowastND not detected limit of detection 005mgkg RDA recommended daily intake

Evidence-Based Complementary and Alternative Medicine 11

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

obtained are depicted in Table 3 where the significant dif-ferences (plt 005) were observed between CR extracts ofleaves and fruits and among different fractions of leavesand fruits

321 DPPH Radical Scavenging Activity Results revealedthat the crude extract and fractions of S caryophyllatumhave the ability to scavenge radicals and depicted a dose-response relationship DPPH radical scavenging activityof different fractions of S caryophyllatum differed sig-nificantly (plt 005) (e EA fraction of leaves exhibitedthe lowest EC50 value followed by Hex and EA fraction offruits with values 912 plusmn 428 μgmL 10097 plusmn 819 μgmLand 13553 plusmn 983 μgmL respectively (e results ob-tained from the fractions showed that EA fractions ofleaves and Hex fractions of fruits exhibited high antiox-idant activities compared to other fractions It shows thatthe accumulation of a variety of antioxidant compoundsdepends on the polarity of the solvent (erefore thecompounds responsible for the high radical scavengingactivity of S caryophyllatum leaves are mainly mediumpolarity compounds (e dose-response relationships ofS caryophyllatum leaves and fruits are given inFigures 3(a) and 3(b)

322 ABTS+ Radical Scavenging Activity (e highestradical scavenging activity was reported in Hex fraction offruits with EC50 value 1501plusmn 065 μgmL followed by AQfraction of S caryophyllatum leaves and AQ fraction of Scaryophyllatum fruits and the EC50 values were 2703plusmn 206and 3145plusmn 143 μgmL respectively It was found that fruitsand leaves showed a significant difference (plt 005) among

fractions (e CR extracts and fractions of leaves and fruitsshowed significant difference and dose-dependent inhibitionon ABTS radical scavenging activity (Figures 4(a) and 4(b))

323 Ferric Reducing Antioxidant Power (FRAP) (e CRextracts of leaves and fruits differed significantly (plt 005) intheir FRAP values CR extract of leaves 4827plusmn 811mgTEgof extract and CR extract of fruits 681plusmn 117mgTEg ofextract (e FRAP values of the fractions ranged from1481mgTEg of the extract to 27041mgTEg of extract EAfraction of leaves was reported as the highest among thetested fractions in leaves and fruits where the AQ fraction offruits exhibited the lowest values (ese results suggestedthat EA fraction of leaves and fruits exhibited the highestferric reducing property among tested fractions whichexplains the important behavior of the polar compounds inits reducing property

324 Oxygen Radical Absorbance Capacity (ORAC) (eORAC values of fractions ranged from 1723mgTEg of theextract to 34732mgTEg of extract (e highest activity wasrecorded in EA fraction of leaves following the AQ fractionof leaves and the lowest was recorded in AQ fraction infruits (e CR extract of leaves exhibited a significantlyhigher (plt 005) activity compared to the CR extract offruits with values 22100plusmn 3496 and 4598plusmn 258mgTEg ofextract respectively

325 Iron Chelating Activity (e highest iron chelatingability of CR extract was reported in leaves with values332plusmn 168mg EDTAg of extract and fruits were reported as1373plusmn 069mg EDTAg of extract (e Hex fraction of

Table 1 (e yield of Syzygium caryophyllatum plant extracts

Scientific name Part of plant Extract Weight of concentrated extract (g) Yield

Syzygium caryophyllatum

Leaves

CR 17500 70Hex lt0001 lt01EA 0114 01AQ 15984 71

Fruits

CR 31200 620Hex 1950 26EA 1531 20AQ 10165 136

CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction

Table 2 Total polyphenol content (TPC) and total flavonoid content (TFC) in different extracts of Syzygium caryophyllatum

Scientific name Part of plant Extract TPC (mg GAEg of sample) TFC (mg QEg of sample)

Syzygium caryophyllatum

LeavesCR 812plusmn 046a 016plusmn 001aEA 009plusmn 001b lt001AQ 712plusmn 016c 018plusmn 002ab

Fruits

CR 883plusmn 062ad 202plusmn 019cHex 342plusmn 029e 080plusmn 005dEA 173plusmn 076f 037plusmn 001eAQ 216plusmn 012g 010plusmn 001af

Results expressed as meanplusmn standard deviation CR crude extract Hex hexane fraction EA ethyl acetate fraction AQ aqueous fraction Mean valueswithin a column superscripted by different letters are significantly different at plt 005

Evidence-Based Complementary and Alternative Medicine 7

Table 3 Antioxidant potentials of leaves and fruits of Syzygium caryophyllatum

Scientific name Part of plant Extract ORAC mg TEgof extract

FRAP mg TEgof extract

EC50 DPPHμgmL

EC50 ABTSμgmL

Iron chelation mgEDTAg of extract

Syzygiumcaryophyllatum

LeavesCR 22100plusmn 3496a 4827plusmn 811a 50020plusmn 4433a 10793plusmn 1507a 332plusmn 168aEA 34431plusmn 337b 26226plusmn 4393b 9120plusmn 428b 4061plusmn 276b 3601plusmn 698aAQ 32097plusmn 2062bc 7342plusmn 1258c 25105plusmn 1752c 2704plusmn 206c 5617plusmn 798b

Fruit

CR 4598plusmn 258d 681plusmn 117d 87372plusmn 1252d 12808plusmn 612d 1373plusmn 069cHex 3277plusmn 141e 5391plusmn 1230a 10097plusmn 819e 1501plusmn 065e 7482plusmn 266dEA 11865plusmn 760f 6307plusmn 1017e 13553plusmn 983f 8954plusmn 388f 828plusmn 040eAQ 2093plusmn 449g 1545plusmn 068f 94642plusmn 6504g 3145plusmn 145g mdash

Results are expressed as meanplusmn standard deviation n 3 Mean values within a column superscripted by different letters are significantly different at plt 005CR crude Hex hexane EA ethyl acetate AQ aqueous ORAC oxygen radical absorbance capacity FRAP ferric reducing antioxidant power ABTSABTS radical scavenging activity DPPH DPPH radical scavenging activity

0

20

40

60

80

100

0 200 400 600 800

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

00 05 10 15 20

Inhi

bitio

n

Concentration (mgmL)

CRHex

EAAQ

(b)

Figure 3 Dose-response curves for DPPH radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

120

0 50 100 150 200 250

Inhi

bitio

n

Concentration (microgmL)

CRHex

EAAQ

(b)

Figure 4 Dose-response curves for ABTS+ radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

8 Evidence-Based Complementary and Alternative Medicine

fruits exhibited the highest iron-chelating ability among thetested fractions with value 7482plusmn 266mg EDTAg of ex-tract and the lowest was reported in EA fraction of fruitswith value 828plusmn 040mg EDTAg of extract respectively(e AQ fraction of Syzygium caryophyllatum fruits did notshow a significant chelating activity where the highestconcentration (215mgmL) exhibited 1605 inhibition

33 Correlation Analysis between Polyphenol Content andDPPH Radical Scavenging Activity of Syzygium car-yophyllatum Fruits (e correlation coefficient betweenTPC content and DPPH of Syzygium caryophyllatum fruitsexhibited a significant (plt 005) positive correlation withPearson r value 09921 (Figure 5)

34 Ce α-Amylase Inhibitory Activity (e α-amylase inhi-bition potentials of CR extract Hex EA and AQ fraction ofleaves and fruits were analyzed Fruits exhibited significantinhibition of α-amylase activities where the leaves did notexhibit significant inhibitory activities (e Hex fraction ofS caryophyllatum fruits was reported to have the highestinhibitory activity with IC50 value 4720 plusmn 03 μgmL fol-lowed by CR extract EA fraction and AQ fraction withIC50 values 21826 plusmn 538 μgmL 61885plusmn 1855 μgmL and101455plusmn 8555 μgmL respectively Interestingly the Hexfraction of fruits demonstrated potent α-amylase inhibitionactivity (IC50 4720 plusmn 03 μgmL) compared to the standarddrug acarbose (IC50 8796 plusmn 143 μgmL)(e percentage ofα-amylase inhibitions of crudes and fractions was plottedagainst concentration (Figure 6) where it showed a dose-dependent increase in percentage activity of CR extract andthree types of fractions of fruits

341 Ce α-Amylase Inhibitory Activity of Isolated Fractionsof Hexane Fraction of Syzygium caryophyllatum Fruits

(1) Hexane and Ethyl Acetate Solvents on Isolation Procedure(e inhibition potential of compound mixtures of EA fractionwas evaluated for α-amylase inhibitory activity (e highesttested concentration was 200μgmL followed by 100μgmL50μgmL and 25μgmL(e similar inhibition potentials wereobserved for the first three concentrations and the inhibitionvalues were 5763plusmn018 5599plusmn160 and 5730plusmn018respectively where the inhibition potential of 25μgmL wasobserved as a rapid drop to the inhibition 1278plusmn045Further inhibition potentials were evaluated between 50μgmLand 25μgmL (Table 4)(e α-amylase inhibitory activity of thetested fraction showed a poor linear response (R2 06384)hence IC50 was not calculated

(2) Dichloromethane and Ethyl Acetate Solvents on Iso-lation Procedure (e α-amylase inhibitory activity ofdifferent combined fractions was analyzed (C1 C2 andC3)(e inhibitory activities of C1 and C2 fractions did notexhibit significant inhibitory activities where the fractionC3 showed significant (plt 005) inhibitory activity for the

screened concentrations (e fraction C3 was dose-de-pendent and IC50 value was 227 plusmn 181 μgmL

(e summary of α-amylase inhibitory activity of hexanefraction and its chromatographic fraction is given in Table 5

35 Antiglycation Activity (e antiglycation activity ofcrude extracts of leaves and fruits was screened for threeconcentrations 200 100 and 50 μgmL (Table 6) (e CRextract of leaves exhibited the highest inhibitory activity(9361 plusmn 853 at a concentration of 200 μgmL) followedby CR extract of S caryophyllatum fruits (4797 plusmn 263)(e standard drug rutin exhibited antiglycation activitywith IC50 value as 3423 plusmn 318 μgmL (e IC50 value ofCR extract of leaves was 5783 plusmn 791 μgmL Howeverfor fruits similar inhibitions were observed for differentdoses hence IC50 value could not be calculated

36 Chemical Compositions of Syzygium caryophyllatum LeafEssential Oil Syzygium caryophyllatum leaves yieldedessential oil upon hydrodistillation GC-MS analysisidentified 58 compounds from the essential oil and theretention time and area of the identified major peaks arelisted in Table 7 (e major compounds identified in theleaves were phytol with a 2466 peak area (retentiontime 3444) α-cadinol with a 387 peak area (retentiontime 2781) α-guaiene with a 384 peak area (retentiontime 2629) cyclosiolongifolene 910-dehydro with a382 peak area (retention time 2826) humulene with a336 peak area (retention time 1645) and car-yophyllene with a 321 peak area (retention time 1479)respectively (e GC-MS chromatogram of Syzygiumcaryophyllatum leaf oil is presented in Figure 7

37 Mineral Compositions Functionally important micro-minerals such as selenium (Se) iron (Fe) strontium (Sr)manganese (Mn) cobalt (Co) copper (Cu) and the toxicheavymetals such as arsenic (As) cadmium (Cd) chromium(Cr) and lead (Pb) were evaluated (Table 8) (e ICP-MS

0

20

40

60

80

100

0 50 100 150

DPP

H v

alue

(mg

TEg

of e

xtra

ct)

Total phenol content (mg GAEg of extract)

Figure 5 Correlation of Syzygium caryophyllatum fruits TPCversus DPPH values (plt 005)

Evidence-Based Complementary and Alternative Medicine 9

data ranged from 009 to 45370mgkg of sample Sodium(Na) was found to be the highest (leaves 435768mgkg ofthe sample) while Se was found to be the lowest (fruits009mgkg of the sample) (e leaves were found to have thehighest concentration of the tested minerals compared to thefruits (e second richest macroelement was found to be K(leaves 355925mgkg and fruits 294428mgkg) followedby Mg (leaves 258040mgkg) and Ca (leaves 184352mgkg)(e maximum amount of Al Mn Fe and Zn was found

to be in leaves with values 52445mgkg 29882mgkg22198mgkg and 2160mgkg respectively Among thetested microelements Sr was found to be the richest with avalue of 32889mgkg in leaves which is significantly(plt 005) different from fruits (270mgkg) (e Cu and Crwere found in similar small concentrations in leaves (Cu491mgkg and Cr 403mgkg) and fruits (Cu 523mgkgand Cr 465mgkg) (e maximum concentration of Sewas determined to be 085mgkg in leaves and the fruitsexhibited the lowest concentration with a value of 009mgkg(e investigation of the level of Co revealed that the maxi-mum concentration of the element exists as 0128mgkg inleaves while in the fruits it was not detected at the detectionlimit of 005mgkg

(e analysis of toxic heavy metals revealed the presenceof a maximum concentration of Pb in leaves (102mgkg)and the lowest in fruits (015mgkg) Besides the concen-trations of toxic heavy metals such as Cd and As were notdetected in leaves and fruits (limit of detection 005mgkg)

4 Discussion

Diabetes mellitus (DM) is a complex metabolic disordercharacterized by hyperglycemia which is mainly associatedwith malfunctions and abnormalities in carbohydrate pro-tein and fat metabolism Digestion and absorption of car-bohydrates to the blood stream also have a significantinfluence on DM Further the increased production of re-active oxygen species (ROS) under DM accelerates the proteinglycation process [24] (e production and accumulation ofglycated proteins have shown a strong association withcomplications of DM [25] (us the management of DM isdirected toward addressing multiple targets including re-duction of postprandial carbohydrate levels in the bloodimprovement in the homeostatic and metabolic process ofcarbohydrates and lipids an improvement on the efficiency ofantioxidant defense and control of protein glycation [26]

In this regard WHO recommends functional ingredi-ents and supplements with multiple functional propertiesincluding antidiabetic and the antiradical properties in thecontrol of the DM [27]

In this study functional properties including α-amylaseinhibition protein glycation inhibition and antioxidantactivities of S caryophyllatum fruit and leaf extracts havebeen investigated along with chemical constituents andmineral analyses (e results revealed the potential of Scaryophyllatum as a source of supplement for DM man-agement To the best of our knowledge the present study isthe first study reported in Sri Lanka on functional activitiesof S caryophyllatum fruit and leaf extracts Crude extracts ofboth fruits and leaves showed significant antioxidant andmoderate antiglycation activities However only fruit ex-tracts showed α-amylase inhibitory activity Even thoughboth fruits and leaves CR were tested against α-glucosidaseenzyme inhibitory activity none of them showed a signif-icant inhibitory activity at a maximum dose of 400 μgmL(data not shown) Solventndashsolvent partitioning of both CRwas used to separate and valuate the most potent fractionswith α-amylase inhibition activity

0

20

40

60

80

100

0 200 400 600 800 1000

Inhi

bitio

n

Concentration (μgmL)

CRHex

EAAQ

Figure 6 Dose-response relationship of crude and fractionsof Syzygium caryophyllatum fruits for alpha-amylase inhibi-tory activity (CR crude Hex hexane EA ethyl acetateAQ aqueous)

Table 4 (e α-amylase inhibitory activity of isolated compoundmixtures of Hex fraction of Syzygium caryophyllatum fruits (hexaneand ethyl acetate solvents on isolation procedure)

Assay concentration μgmL Inhibition 200 5763plusmn 018a100 5599plusmn 160a50 5730plusmn 018a45 2339plusmn 021b40 2245plusmn 079b35 2143plusmn 019b30 1433plusmn 083c25 1278plusmn 045c

Results are expressed as meanplusmn standard deviation n 3lowast Mean valueswithin a column superscripted by different letters are significantly differentat plt 005

Table 5 (e α-amylase inhibitory activity of hexane fraction andits chromatographic fraction of Syzygium caryophyllatum fruits

Sample IC50 (μgmL)Hex fraction 4720aplusmn 030Hex EA (0 100) NLRC3 DCM EA (95 5) 227bplusmn 181Standard drug acarbose 8796cplusmn 143Results are expressed as meanplusmn standard deviation n 3lowast NLRno linearresponse Mean values within a column superscripted by different lettersare significantly different at plt 005 Hex hexane EA ethyl acetateDCM dichloromethane C3 combined fraction 3

10 Evidence-Based Complementary and Alternative Medicine

Hexane fraction of fruit extract exhibited the highestinhibition activity with IC50 value 4720plusmn 037 μgmL and issignificantly higher compared to acarbose (IC508796plusmn 143 μgmL) (erefore it was further separated bycolumn chromatography (e fraction C3 which was iso-lated on elution with DCM EA 95 5 exhibited a significant(plt 005) α-amylase inhibitory activity with an IC50 value of227plusmn 181 μgmL (e compound or the mixture of

compounds responsible for the α-amylase inhibition activitymay be in the C3 compound mixture which is required to besubjected to further isolation and identification Inhibitionmay be due to the presence of potential alpha-amylase in-hibitors such as alkaloids tannin polyphenols and flavo-noids (ese inhibitors can also act as starch blockers as theyblock the hydrolysis of 14-glycosidic linkages of starch andoligosaccharides [28] Hex fraction of S caryophyllatum fruit

Table 6 Antiglycation activity of crude extracts of Syzygium caryophyllatum

Crude extract Inhibition

IC50 (μgmL)Concentration (μgmL)50 100 200

Leaves 5488plusmn 310a 7987plusmn 594a 9361plusmn 850a 5783plusmn 791Fruits 4131plusmn 789b 5666plusmn 836b 4797plusmn 263b mdashStandard rutin IC50 3423plusmn 318 μgmLResults are expressed as meanplusmn standard deviation n 3lowast Mean values within a column superscripted by different letters are significantly different atplt 005

Table 7 Major compounds detected in Syzygium caryophyllatum leaf essential oil

Peak Compound Formula and lowastmol wt RT Area SI RSI1 Caryophyllene C15H24 204 1479 321 954 9542 Humulene C15H24 204 1645 336 907 9073 α-Cadinol C15H26O 222 2781 387 923 9284 Cyclosiolongifolene 910-dehydro C17H26O2 202 2826 382 790 8005 Phytol C20H40O 296 3444 2466 927 927lowastMol wtmolecular weight

2781

RT 000ndash4200100

90

80

70

60

50

40

30

20

10

0 5 10 15 20 25 30 35 40Retention time (min)

3444

2826

Rela

tive a

bund

ance

1 24

3

5

1479 1645

Figure 7 GC-MS chromatogram of Syzygium caryophyllatum leaf essential oil

Table 8 Mineral compositions of leaves and fruits of Syzygium caryophyllatum

Mineral elements (mgkg per sample)K Fe Na Ca Mg Mn Cu Zn Cr Zn Sr Se Pb As Cd

Syzygiumcaryophyllatum

Leaves 355925 22198 435768 184352 258039 29882 491 2160 403 2159 3289 085 102 ND NDFruits 294428 4955 75621 14927 85555 1814 523 775 465 775 270 009 015 ND ND

RDA 35 g 9ndash15mg 15 g 1 g 280ndash350mg 50ndash60 μglowastND not detected limit of detection 005mgkg RDA recommended daily intake

Evidence-Based Complementary and Alternative Medicine 11

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

Table 3 Antioxidant potentials of leaves and fruits of Syzygium caryophyllatum

Scientific name Part of plant Extract ORAC mg TEgof extract

FRAP mg TEgof extract

EC50 DPPHμgmL

EC50 ABTSμgmL

Iron chelation mgEDTAg of extract

Syzygiumcaryophyllatum

LeavesCR 22100plusmn 3496a 4827plusmn 811a 50020plusmn 4433a 10793plusmn 1507a 332plusmn 168aEA 34431plusmn 337b 26226plusmn 4393b 9120plusmn 428b 4061plusmn 276b 3601plusmn 698aAQ 32097plusmn 2062bc 7342plusmn 1258c 25105plusmn 1752c 2704plusmn 206c 5617plusmn 798b

Fruit

CR 4598plusmn 258d 681plusmn 117d 87372plusmn 1252d 12808plusmn 612d 1373plusmn 069cHex 3277plusmn 141e 5391plusmn 1230a 10097plusmn 819e 1501plusmn 065e 7482plusmn 266dEA 11865plusmn 760f 6307plusmn 1017e 13553plusmn 983f 8954plusmn 388f 828plusmn 040eAQ 2093plusmn 449g 1545plusmn 068f 94642plusmn 6504g 3145plusmn 145g mdash

Results are expressed as meanplusmn standard deviation n 3 Mean values within a column superscripted by different letters are significantly different at plt 005CR crude Hex hexane EA ethyl acetate AQ aqueous ORAC oxygen radical absorbance capacity FRAP ferric reducing antioxidant power ABTSABTS radical scavenging activity DPPH DPPH radical scavenging activity

0

20

40

60

80

100

0 200 400 600 800

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

00 05 10 15 20

Inhi

bitio

n

Concentration (mgmL)

CRHex

EAAQ

(b)

Figure 3 Dose-response curves for DPPH radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

0

20

40

60

80

100

0 20 40 60 80 100

Inhi

bitio

n

Concentration (microgmL)

CREAAQ

(a)

0

20

40

60

80

100

120

0 50 100 150 200 250

Inhi

bitio

n

Concentration (microgmL)

CRHex

EAAQ

(b)

Figure 4 Dose-response curves for ABTS+ radical scavenging activity of Syzygium caryophyllatum (a) leaves and (b) fruits (CR crudeHex hexane EA ethyl acetate AQ aqueous)

8 Evidence-Based Complementary and Alternative Medicine

fruits exhibited the highest iron-chelating ability among thetested fractions with value 7482plusmn 266mg EDTAg of ex-tract and the lowest was reported in EA fraction of fruitswith value 828plusmn 040mg EDTAg of extract respectively(e AQ fraction of Syzygium caryophyllatum fruits did notshow a significant chelating activity where the highestconcentration (215mgmL) exhibited 1605 inhibition

33 Correlation Analysis between Polyphenol Content andDPPH Radical Scavenging Activity of Syzygium car-yophyllatum Fruits (e correlation coefficient betweenTPC content and DPPH of Syzygium caryophyllatum fruitsexhibited a significant (plt 005) positive correlation withPearson r value 09921 (Figure 5)

34 Ce α-Amylase Inhibitory Activity (e α-amylase inhi-bition potentials of CR extract Hex EA and AQ fraction ofleaves and fruits were analyzed Fruits exhibited significantinhibition of α-amylase activities where the leaves did notexhibit significant inhibitory activities (e Hex fraction ofS caryophyllatum fruits was reported to have the highestinhibitory activity with IC50 value 4720 plusmn 03 μgmL fol-lowed by CR extract EA fraction and AQ fraction withIC50 values 21826 plusmn 538 μgmL 61885plusmn 1855 μgmL and101455plusmn 8555 μgmL respectively Interestingly the Hexfraction of fruits demonstrated potent α-amylase inhibitionactivity (IC50 4720 plusmn 03 μgmL) compared to the standarddrug acarbose (IC50 8796 plusmn 143 μgmL)(e percentage ofα-amylase inhibitions of crudes and fractions was plottedagainst concentration (Figure 6) where it showed a dose-dependent increase in percentage activity of CR extract andthree types of fractions of fruits

341 Ce α-Amylase Inhibitory Activity of Isolated Fractionsof Hexane Fraction of Syzygium caryophyllatum Fruits

(1) Hexane and Ethyl Acetate Solvents on Isolation Procedure(e inhibition potential of compound mixtures of EA fractionwas evaluated for α-amylase inhibitory activity (e highesttested concentration was 200μgmL followed by 100μgmL50μgmL and 25μgmL(e similar inhibition potentials wereobserved for the first three concentrations and the inhibitionvalues were 5763plusmn018 5599plusmn160 and 5730plusmn018respectively where the inhibition potential of 25μgmL wasobserved as a rapid drop to the inhibition 1278plusmn045Further inhibition potentials were evaluated between 50μgmLand 25μgmL (Table 4)(e α-amylase inhibitory activity of thetested fraction showed a poor linear response (R2 06384)hence IC50 was not calculated

(2) Dichloromethane and Ethyl Acetate Solvents on Iso-lation Procedure (e α-amylase inhibitory activity ofdifferent combined fractions was analyzed (C1 C2 andC3)(e inhibitory activities of C1 and C2 fractions did notexhibit significant inhibitory activities where the fractionC3 showed significant (plt 005) inhibitory activity for the

screened concentrations (e fraction C3 was dose-de-pendent and IC50 value was 227 plusmn 181 μgmL

(e summary of α-amylase inhibitory activity of hexanefraction and its chromatographic fraction is given in Table 5

35 Antiglycation Activity (e antiglycation activity ofcrude extracts of leaves and fruits was screened for threeconcentrations 200 100 and 50 μgmL (Table 6) (e CRextract of leaves exhibited the highest inhibitory activity(9361 plusmn 853 at a concentration of 200 μgmL) followedby CR extract of S caryophyllatum fruits (4797 plusmn 263)(e standard drug rutin exhibited antiglycation activitywith IC50 value as 3423 plusmn 318 μgmL (e IC50 value ofCR extract of leaves was 5783 plusmn 791 μgmL Howeverfor fruits similar inhibitions were observed for differentdoses hence IC50 value could not be calculated

36 Chemical Compositions of Syzygium caryophyllatum LeafEssential Oil Syzygium caryophyllatum leaves yieldedessential oil upon hydrodistillation GC-MS analysisidentified 58 compounds from the essential oil and theretention time and area of the identified major peaks arelisted in Table 7 (e major compounds identified in theleaves were phytol with a 2466 peak area (retentiontime 3444) α-cadinol with a 387 peak area (retentiontime 2781) α-guaiene with a 384 peak area (retentiontime 2629) cyclosiolongifolene 910-dehydro with a382 peak area (retention time 2826) humulene with a336 peak area (retention time 1645) and car-yophyllene with a 321 peak area (retention time 1479)respectively (e GC-MS chromatogram of Syzygiumcaryophyllatum leaf oil is presented in Figure 7

37 Mineral Compositions Functionally important micro-minerals such as selenium (Se) iron (Fe) strontium (Sr)manganese (Mn) cobalt (Co) copper (Cu) and the toxicheavymetals such as arsenic (As) cadmium (Cd) chromium(Cr) and lead (Pb) were evaluated (Table 8) (e ICP-MS

0

20

40

60

80

100

0 50 100 150

DPP

H v

alue

(mg

TEg

of e

xtra

ct)

Total phenol content (mg GAEg of extract)

Figure 5 Correlation of Syzygium caryophyllatum fruits TPCversus DPPH values (plt 005)

Evidence-Based Complementary and Alternative Medicine 9

data ranged from 009 to 45370mgkg of sample Sodium(Na) was found to be the highest (leaves 435768mgkg ofthe sample) while Se was found to be the lowest (fruits009mgkg of the sample) (e leaves were found to have thehighest concentration of the tested minerals compared to thefruits (e second richest macroelement was found to be K(leaves 355925mgkg and fruits 294428mgkg) followedby Mg (leaves 258040mgkg) and Ca (leaves 184352mgkg)(e maximum amount of Al Mn Fe and Zn was found

to be in leaves with values 52445mgkg 29882mgkg22198mgkg and 2160mgkg respectively Among thetested microelements Sr was found to be the richest with avalue of 32889mgkg in leaves which is significantly(plt 005) different from fruits (270mgkg) (e Cu and Crwere found in similar small concentrations in leaves (Cu491mgkg and Cr 403mgkg) and fruits (Cu 523mgkgand Cr 465mgkg) (e maximum concentration of Sewas determined to be 085mgkg in leaves and the fruitsexhibited the lowest concentration with a value of 009mgkg(e investigation of the level of Co revealed that the maxi-mum concentration of the element exists as 0128mgkg inleaves while in the fruits it was not detected at the detectionlimit of 005mgkg

(e analysis of toxic heavy metals revealed the presenceof a maximum concentration of Pb in leaves (102mgkg)and the lowest in fruits (015mgkg) Besides the concen-trations of toxic heavy metals such as Cd and As were notdetected in leaves and fruits (limit of detection 005mgkg)

4 Discussion

Diabetes mellitus (DM) is a complex metabolic disordercharacterized by hyperglycemia which is mainly associatedwith malfunctions and abnormalities in carbohydrate pro-tein and fat metabolism Digestion and absorption of car-bohydrates to the blood stream also have a significantinfluence on DM Further the increased production of re-active oxygen species (ROS) under DM accelerates the proteinglycation process [24] (e production and accumulation ofglycated proteins have shown a strong association withcomplications of DM [25] (us the management of DM isdirected toward addressing multiple targets including re-duction of postprandial carbohydrate levels in the bloodimprovement in the homeostatic and metabolic process ofcarbohydrates and lipids an improvement on the efficiency ofantioxidant defense and control of protein glycation [26]

In this regard WHO recommends functional ingredi-ents and supplements with multiple functional propertiesincluding antidiabetic and the antiradical properties in thecontrol of the DM [27]

In this study functional properties including α-amylaseinhibition protein glycation inhibition and antioxidantactivities of S caryophyllatum fruit and leaf extracts havebeen investigated along with chemical constituents andmineral analyses (e results revealed the potential of Scaryophyllatum as a source of supplement for DM man-agement To the best of our knowledge the present study isthe first study reported in Sri Lanka on functional activitiesof S caryophyllatum fruit and leaf extracts Crude extracts ofboth fruits and leaves showed significant antioxidant andmoderate antiglycation activities However only fruit ex-tracts showed α-amylase inhibitory activity Even thoughboth fruits and leaves CR were tested against α-glucosidaseenzyme inhibitory activity none of them showed a signif-icant inhibitory activity at a maximum dose of 400 μgmL(data not shown) Solventndashsolvent partitioning of both CRwas used to separate and valuate the most potent fractionswith α-amylase inhibition activity

0

20

40

60

80

100

0 200 400 600 800 1000

Inhi

bitio

n

Concentration (μgmL)

CRHex

EAAQ

Figure 6 Dose-response relationship of crude and fractionsof Syzygium caryophyllatum fruits for alpha-amylase inhibi-tory activity (CR crude Hex hexane EA ethyl acetateAQ aqueous)

Table 4 (e α-amylase inhibitory activity of isolated compoundmixtures of Hex fraction of Syzygium caryophyllatum fruits (hexaneand ethyl acetate solvents on isolation procedure)

Assay concentration μgmL Inhibition 200 5763plusmn 018a100 5599plusmn 160a50 5730plusmn 018a45 2339plusmn 021b40 2245plusmn 079b35 2143plusmn 019b30 1433plusmn 083c25 1278plusmn 045c

Results are expressed as meanplusmn standard deviation n 3lowast Mean valueswithin a column superscripted by different letters are significantly differentat plt 005

Table 5 (e α-amylase inhibitory activity of hexane fraction andits chromatographic fraction of Syzygium caryophyllatum fruits

Sample IC50 (μgmL)Hex fraction 4720aplusmn 030Hex EA (0 100) NLRC3 DCM EA (95 5) 227bplusmn 181Standard drug acarbose 8796cplusmn 143Results are expressed as meanplusmn standard deviation n 3lowast NLRno linearresponse Mean values within a column superscripted by different lettersare significantly different at plt 005 Hex hexane EA ethyl acetateDCM dichloromethane C3 combined fraction 3

10 Evidence-Based Complementary and Alternative Medicine

Hexane fraction of fruit extract exhibited the highestinhibition activity with IC50 value 4720plusmn 037 μgmL and issignificantly higher compared to acarbose (IC508796plusmn 143 μgmL) (erefore it was further separated bycolumn chromatography (e fraction C3 which was iso-lated on elution with DCM EA 95 5 exhibited a significant(plt 005) α-amylase inhibitory activity with an IC50 value of227plusmn 181 μgmL (e compound or the mixture of

compounds responsible for the α-amylase inhibition activitymay be in the C3 compound mixture which is required to besubjected to further isolation and identification Inhibitionmay be due to the presence of potential alpha-amylase in-hibitors such as alkaloids tannin polyphenols and flavo-noids (ese inhibitors can also act as starch blockers as theyblock the hydrolysis of 14-glycosidic linkages of starch andoligosaccharides [28] Hex fraction of S caryophyllatum fruit

Table 6 Antiglycation activity of crude extracts of Syzygium caryophyllatum

Crude extract Inhibition

IC50 (μgmL)Concentration (μgmL)50 100 200

Leaves 5488plusmn 310a 7987plusmn 594a 9361plusmn 850a 5783plusmn 791Fruits 4131plusmn 789b 5666plusmn 836b 4797plusmn 263b mdashStandard rutin IC50 3423plusmn 318 μgmLResults are expressed as meanplusmn standard deviation n 3lowast Mean values within a column superscripted by different letters are significantly different atplt 005

Table 7 Major compounds detected in Syzygium caryophyllatum leaf essential oil

Peak Compound Formula and lowastmol wt RT Area SI RSI1 Caryophyllene C15H24 204 1479 321 954 9542 Humulene C15H24 204 1645 336 907 9073 α-Cadinol C15H26O 222 2781 387 923 9284 Cyclosiolongifolene 910-dehydro C17H26O2 202 2826 382 790 8005 Phytol C20H40O 296 3444 2466 927 927lowastMol wtmolecular weight

2781

RT 000ndash4200100

90

80

70

60

50

40

30

20

10

0 5 10 15 20 25 30 35 40Retention time (min)

3444

2826

Rela

tive a

bund

ance

1 24

3

5

1479 1645

Figure 7 GC-MS chromatogram of Syzygium caryophyllatum leaf essential oil

Table 8 Mineral compositions of leaves and fruits of Syzygium caryophyllatum

Mineral elements (mgkg per sample)K Fe Na Ca Mg Mn Cu Zn Cr Zn Sr Se Pb As Cd

Syzygiumcaryophyllatum

Leaves 355925 22198 435768 184352 258039 29882 491 2160 403 2159 3289 085 102 ND NDFruits 294428 4955 75621 14927 85555 1814 523 775 465 775 270 009 015 ND ND

RDA 35 g 9ndash15mg 15 g 1 g 280ndash350mg 50ndash60 μglowastND not detected limit of detection 005mgkg RDA recommended daily intake

Evidence-Based Complementary and Alternative Medicine 11

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

fruits exhibited the highest iron-chelating ability among thetested fractions with value 7482plusmn 266mg EDTAg of ex-tract and the lowest was reported in EA fraction of fruitswith value 828plusmn 040mg EDTAg of extract respectively(e AQ fraction of Syzygium caryophyllatum fruits did notshow a significant chelating activity where the highestconcentration (215mgmL) exhibited 1605 inhibition

33 Correlation Analysis between Polyphenol Content andDPPH Radical Scavenging Activity of Syzygium car-yophyllatum Fruits (e correlation coefficient betweenTPC content and DPPH of Syzygium caryophyllatum fruitsexhibited a significant (plt 005) positive correlation withPearson r value 09921 (Figure 5)

34 Ce α-Amylase Inhibitory Activity (e α-amylase inhi-bition potentials of CR extract Hex EA and AQ fraction ofleaves and fruits were analyzed Fruits exhibited significantinhibition of α-amylase activities where the leaves did notexhibit significant inhibitory activities (e Hex fraction ofS caryophyllatum fruits was reported to have the highestinhibitory activity with IC50 value 4720 plusmn 03 μgmL fol-lowed by CR extract EA fraction and AQ fraction withIC50 values 21826 plusmn 538 μgmL 61885plusmn 1855 μgmL and101455plusmn 8555 μgmL respectively Interestingly the Hexfraction of fruits demonstrated potent α-amylase inhibitionactivity (IC50 4720 plusmn 03 μgmL) compared to the standarddrug acarbose (IC50 8796 plusmn 143 μgmL)(e percentage ofα-amylase inhibitions of crudes and fractions was plottedagainst concentration (Figure 6) where it showed a dose-dependent increase in percentage activity of CR extract andthree types of fractions of fruits

341 Ce α-Amylase Inhibitory Activity of Isolated Fractionsof Hexane Fraction of Syzygium caryophyllatum Fruits

(1) Hexane and Ethyl Acetate Solvents on Isolation Procedure(e inhibition potential of compound mixtures of EA fractionwas evaluated for α-amylase inhibitory activity (e highesttested concentration was 200μgmL followed by 100μgmL50μgmL and 25μgmL(e similar inhibition potentials wereobserved for the first three concentrations and the inhibitionvalues were 5763plusmn018 5599plusmn160 and 5730plusmn018respectively where the inhibition potential of 25μgmL wasobserved as a rapid drop to the inhibition 1278plusmn045Further inhibition potentials were evaluated between 50μgmLand 25μgmL (Table 4)(e α-amylase inhibitory activity of thetested fraction showed a poor linear response (R2 06384)hence IC50 was not calculated

(2) Dichloromethane and Ethyl Acetate Solvents on Iso-lation Procedure (e α-amylase inhibitory activity ofdifferent combined fractions was analyzed (C1 C2 andC3)(e inhibitory activities of C1 and C2 fractions did notexhibit significant inhibitory activities where the fractionC3 showed significant (plt 005) inhibitory activity for the

screened concentrations (e fraction C3 was dose-de-pendent and IC50 value was 227 plusmn 181 μgmL

(e summary of α-amylase inhibitory activity of hexanefraction and its chromatographic fraction is given in Table 5

35 Antiglycation Activity (e antiglycation activity ofcrude extracts of leaves and fruits was screened for threeconcentrations 200 100 and 50 μgmL (Table 6) (e CRextract of leaves exhibited the highest inhibitory activity(9361 plusmn 853 at a concentration of 200 μgmL) followedby CR extract of S caryophyllatum fruits (4797 plusmn 263)(e standard drug rutin exhibited antiglycation activitywith IC50 value as 3423 plusmn 318 μgmL (e IC50 value ofCR extract of leaves was 5783 plusmn 791 μgmL Howeverfor fruits similar inhibitions were observed for differentdoses hence IC50 value could not be calculated

36 Chemical Compositions of Syzygium caryophyllatum LeafEssential Oil Syzygium caryophyllatum leaves yieldedessential oil upon hydrodistillation GC-MS analysisidentified 58 compounds from the essential oil and theretention time and area of the identified major peaks arelisted in Table 7 (e major compounds identified in theleaves were phytol with a 2466 peak area (retentiontime 3444) α-cadinol with a 387 peak area (retentiontime 2781) α-guaiene with a 384 peak area (retentiontime 2629) cyclosiolongifolene 910-dehydro with a382 peak area (retention time 2826) humulene with a336 peak area (retention time 1645) and car-yophyllene with a 321 peak area (retention time 1479)respectively (e GC-MS chromatogram of Syzygiumcaryophyllatum leaf oil is presented in Figure 7

37 Mineral Compositions Functionally important micro-minerals such as selenium (Se) iron (Fe) strontium (Sr)manganese (Mn) cobalt (Co) copper (Cu) and the toxicheavymetals such as arsenic (As) cadmium (Cd) chromium(Cr) and lead (Pb) were evaluated (Table 8) (e ICP-MS

0

20

40

60

80

100

0 50 100 150

DPP

H v

alue

(mg

TEg

of e

xtra

ct)

Total phenol content (mg GAEg of extract)

Figure 5 Correlation of Syzygium caryophyllatum fruits TPCversus DPPH values (plt 005)

Evidence-Based Complementary and Alternative Medicine 9

data ranged from 009 to 45370mgkg of sample Sodium(Na) was found to be the highest (leaves 435768mgkg ofthe sample) while Se was found to be the lowest (fruits009mgkg of the sample) (e leaves were found to have thehighest concentration of the tested minerals compared to thefruits (e second richest macroelement was found to be K(leaves 355925mgkg and fruits 294428mgkg) followedby Mg (leaves 258040mgkg) and Ca (leaves 184352mgkg)(e maximum amount of Al Mn Fe and Zn was found

to be in leaves with values 52445mgkg 29882mgkg22198mgkg and 2160mgkg respectively Among thetested microelements Sr was found to be the richest with avalue of 32889mgkg in leaves which is significantly(plt 005) different from fruits (270mgkg) (e Cu and Crwere found in similar small concentrations in leaves (Cu491mgkg and Cr 403mgkg) and fruits (Cu 523mgkgand Cr 465mgkg) (e maximum concentration of Sewas determined to be 085mgkg in leaves and the fruitsexhibited the lowest concentration with a value of 009mgkg(e investigation of the level of Co revealed that the maxi-mum concentration of the element exists as 0128mgkg inleaves while in the fruits it was not detected at the detectionlimit of 005mgkg

(e analysis of toxic heavy metals revealed the presenceof a maximum concentration of Pb in leaves (102mgkg)and the lowest in fruits (015mgkg) Besides the concen-trations of toxic heavy metals such as Cd and As were notdetected in leaves and fruits (limit of detection 005mgkg)

4 Discussion

Diabetes mellitus (DM) is a complex metabolic disordercharacterized by hyperglycemia which is mainly associatedwith malfunctions and abnormalities in carbohydrate pro-tein and fat metabolism Digestion and absorption of car-bohydrates to the blood stream also have a significantinfluence on DM Further the increased production of re-active oxygen species (ROS) under DM accelerates the proteinglycation process [24] (e production and accumulation ofglycated proteins have shown a strong association withcomplications of DM [25] (us the management of DM isdirected toward addressing multiple targets including re-duction of postprandial carbohydrate levels in the bloodimprovement in the homeostatic and metabolic process ofcarbohydrates and lipids an improvement on the efficiency ofantioxidant defense and control of protein glycation [26]

In this regard WHO recommends functional ingredi-ents and supplements with multiple functional propertiesincluding antidiabetic and the antiradical properties in thecontrol of the DM [27]

In this study functional properties including α-amylaseinhibition protein glycation inhibition and antioxidantactivities of S caryophyllatum fruit and leaf extracts havebeen investigated along with chemical constituents andmineral analyses (e results revealed the potential of Scaryophyllatum as a source of supplement for DM man-agement To the best of our knowledge the present study isthe first study reported in Sri Lanka on functional activitiesof S caryophyllatum fruit and leaf extracts Crude extracts ofboth fruits and leaves showed significant antioxidant andmoderate antiglycation activities However only fruit ex-tracts showed α-amylase inhibitory activity Even thoughboth fruits and leaves CR were tested against α-glucosidaseenzyme inhibitory activity none of them showed a signif-icant inhibitory activity at a maximum dose of 400 μgmL(data not shown) Solventndashsolvent partitioning of both CRwas used to separate and valuate the most potent fractionswith α-amylase inhibition activity

0

20

40

60

80

100

0 200 400 600 800 1000

Inhi

bitio

n

Concentration (μgmL)

CRHex

EAAQ

Figure 6 Dose-response relationship of crude and fractionsof Syzygium caryophyllatum fruits for alpha-amylase inhibi-tory activity (CR crude Hex hexane EA ethyl acetateAQ aqueous)

Table 4 (e α-amylase inhibitory activity of isolated compoundmixtures of Hex fraction of Syzygium caryophyllatum fruits (hexaneand ethyl acetate solvents on isolation procedure)

Assay concentration μgmL Inhibition 200 5763plusmn 018a100 5599plusmn 160a50 5730plusmn 018a45 2339plusmn 021b40 2245plusmn 079b35 2143plusmn 019b30 1433plusmn 083c25 1278plusmn 045c

Results are expressed as meanplusmn standard deviation n 3lowast Mean valueswithin a column superscripted by different letters are significantly differentat plt 005

Table 5 (e α-amylase inhibitory activity of hexane fraction andits chromatographic fraction of Syzygium caryophyllatum fruits

Sample IC50 (μgmL)Hex fraction 4720aplusmn 030Hex EA (0 100) NLRC3 DCM EA (95 5) 227bplusmn 181Standard drug acarbose 8796cplusmn 143Results are expressed as meanplusmn standard deviation n 3lowast NLRno linearresponse Mean values within a column superscripted by different lettersare significantly different at plt 005 Hex hexane EA ethyl acetateDCM dichloromethane C3 combined fraction 3

10 Evidence-Based Complementary and Alternative Medicine

Hexane fraction of fruit extract exhibited the highestinhibition activity with IC50 value 4720plusmn 037 μgmL and issignificantly higher compared to acarbose (IC508796plusmn 143 μgmL) (erefore it was further separated bycolumn chromatography (e fraction C3 which was iso-lated on elution with DCM EA 95 5 exhibited a significant(plt 005) α-amylase inhibitory activity with an IC50 value of227plusmn 181 μgmL (e compound or the mixture of

compounds responsible for the α-amylase inhibition activitymay be in the C3 compound mixture which is required to besubjected to further isolation and identification Inhibitionmay be due to the presence of potential alpha-amylase in-hibitors such as alkaloids tannin polyphenols and flavo-noids (ese inhibitors can also act as starch blockers as theyblock the hydrolysis of 14-glycosidic linkages of starch andoligosaccharides [28] Hex fraction of S caryophyllatum fruit

Table 6 Antiglycation activity of crude extracts of Syzygium caryophyllatum

Crude extract Inhibition

IC50 (μgmL)Concentration (μgmL)50 100 200

Leaves 5488plusmn 310a 7987plusmn 594a 9361plusmn 850a 5783plusmn 791Fruits 4131plusmn 789b 5666plusmn 836b 4797plusmn 263b mdashStandard rutin IC50 3423plusmn 318 μgmLResults are expressed as meanplusmn standard deviation n 3lowast Mean values within a column superscripted by different letters are significantly different atplt 005

Table 7 Major compounds detected in Syzygium caryophyllatum leaf essential oil

Peak Compound Formula and lowastmol wt RT Area SI RSI1 Caryophyllene C15H24 204 1479 321 954 9542 Humulene C15H24 204 1645 336 907 9073 α-Cadinol C15H26O 222 2781 387 923 9284 Cyclosiolongifolene 910-dehydro C17H26O2 202 2826 382 790 8005 Phytol C20H40O 296 3444 2466 927 927lowastMol wtmolecular weight

2781

RT 000ndash4200100

90

80

70

60

50

40

30

20

10

0 5 10 15 20 25 30 35 40Retention time (min)

3444

2826

Rela

tive a

bund

ance

1 24

3

5

1479 1645

Figure 7 GC-MS chromatogram of Syzygium caryophyllatum leaf essential oil

Table 8 Mineral compositions of leaves and fruits of Syzygium caryophyllatum

Mineral elements (mgkg per sample)K Fe Na Ca Mg Mn Cu Zn Cr Zn Sr Se Pb As Cd

Syzygiumcaryophyllatum

Leaves 355925 22198 435768 184352 258039 29882 491 2160 403 2159 3289 085 102 ND NDFruits 294428 4955 75621 14927 85555 1814 523 775 465 775 270 009 015 ND ND

RDA 35 g 9ndash15mg 15 g 1 g 280ndash350mg 50ndash60 μglowastND not detected limit of detection 005mgkg RDA recommended daily intake

Evidence-Based Complementary and Alternative Medicine 11

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

data ranged from 009 to 45370mgkg of sample Sodium(Na) was found to be the highest (leaves 435768mgkg ofthe sample) while Se was found to be the lowest (fruits009mgkg of the sample) (e leaves were found to have thehighest concentration of the tested minerals compared to thefruits (e second richest macroelement was found to be K(leaves 355925mgkg and fruits 294428mgkg) followedby Mg (leaves 258040mgkg) and Ca (leaves 184352mgkg)(e maximum amount of Al Mn Fe and Zn was found

to be in leaves with values 52445mgkg 29882mgkg22198mgkg and 2160mgkg respectively Among thetested microelements Sr was found to be the richest with avalue of 32889mgkg in leaves which is significantly(plt 005) different from fruits (270mgkg) (e Cu and Crwere found in similar small concentrations in leaves (Cu491mgkg and Cr 403mgkg) and fruits (Cu 523mgkgand Cr 465mgkg) (e maximum concentration of Sewas determined to be 085mgkg in leaves and the fruitsexhibited the lowest concentration with a value of 009mgkg(e investigation of the level of Co revealed that the maxi-mum concentration of the element exists as 0128mgkg inleaves while in the fruits it was not detected at the detectionlimit of 005mgkg

(e analysis of toxic heavy metals revealed the presenceof a maximum concentration of Pb in leaves (102mgkg)and the lowest in fruits (015mgkg) Besides the concen-trations of toxic heavy metals such as Cd and As were notdetected in leaves and fruits (limit of detection 005mgkg)

4 Discussion

Diabetes mellitus (DM) is a complex metabolic disordercharacterized by hyperglycemia which is mainly associatedwith malfunctions and abnormalities in carbohydrate pro-tein and fat metabolism Digestion and absorption of car-bohydrates to the blood stream also have a significantinfluence on DM Further the increased production of re-active oxygen species (ROS) under DM accelerates the proteinglycation process [24] (e production and accumulation ofglycated proteins have shown a strong association withcomplications of DM [25] (us the management of DM isdirected toward addressing multiple targets including re-duction of postprandial carbohydrate levels in the bloodimprovement in the homeostatic and metabolic process ofcarbohydrates and lipids an improvement on the efficiency ofantioxidant defense and control of protein glycation [26]

In this regard WHO recommends functional ingredi-ents and supplements with multiple functional propertiesincluding antidiabetic and the antiradical properties in thecontrol of the DM [27]

In this study functional properties including α-amylaseinhibition protein glycation inhibition and antioxidantactivities of S caryophyllatum fruit and leaf extracts havebeen investigated along with chemical constituents andmineral analyses (e results revealed the potential of Scaryophyllatum as a source of supplement for DM man-agement To the best of our knowledge the present study isthe first study reported in Sri Lanka on functional activitiesof S caryophyllatum fruit and leaf extracts Crude extracts ofboth fruits and leaves showed significant antioxidant andmoderate antiglycation activities However only fruit ex-tracts showed α-amylase inhibitory activity Even thoughboth fruits and leaves CR were tested against α-glucosidaseenzyme inhibitory activity none of them showed a signif-icant inhibitory activity at a maximum dose of 400 μgmL(data not shown) Solventndashsolvent partitioning of both CRwas used to separate and valuate the most potent fractionswith α-amylase inhibition activity

0

20

40

60

80

100

0 200 400 600 800 1000

Inhi

bitio

n

Concentration (μgmL)

CRHex

EAAQ

Figure 6 Dose-response relationship of crude and fractionsof Syzygium caryophyllatum fruits for alpha-amylase inhibi-tory activity (CR crude Hex hexane EA ethyl acetateAQ aqueous)

Table 4 (e α-amylase inhibitory activity of isolated compoundmixtures of Hex fraction of Syzygium caryophyllatum fruits (hexaneand ethyl acetate solvents on isolation procedure)

Assay concentration μgmL Inhibition 200 5763plusmn 018a100 5599plusmn 160a50 5730plusmn 018a45 2339plusmn 021b40 2245plusmn 079b35 2143plusmn 019b30 1433plusmn 083c25 1278plusmn 045c

Results are expressed as meanplusmn standard deviation n 3lowast Mean valueswithin a column superscripted by different letters are significantly differentat plt 005

Table 5 (e α-amylase inhibitory activity of hexane fraction andits chromatographic fraction of Syzygium caryophyllatum fruits

Sample IC50 (μgmL)Hex fraction 4720aplusmn 030Hex EA (0 100) NLRC3 DCM EA (95 5) 227bplusmn 181Standard drug acarbose 8796cplusmn 143Results are expressed as meanplusmn standard deviation n 3lowast NLRno linearresponse Mean values within a column superscripted by different lettersare significantly different at plt 005 Hex hexane EA ethyl acetateDCM dichloromethane C3 combined fraction 3

10 Evidence-Based Complementary and Alternative Medicine

Hexane fraction of fruit extract exhibited the highestinhibition activity with IC50 value 4720plusmn 037 μgmL and issignificantly higher compared to acarbose (IC508796plusmn 143 μgmL) (erefore it was further separated bycolumn chromatography (e fraction C3 which was iso-lated on elution with DCM EA 95 5 exhibited a significant(plt 005) α-amylase inhibitory activity with an IC50 value of227plusmn 181 μgmL (e compound or the mixture of

compounds responsible for the α-amylase inhibition activitymay be in the C3 compound mixture which is required to besubjected to further isolation and identification Inhibitionmay be due to the presence of potential alpha-amylase in-hibitors such as alkaloids tannin polyphenols and flavo-noids (ese inhibitors can also act as starch blockers as theyblock the hydrolysis of 14-glycosidic linkages of starch andoligosaccharides [28] Hex fraction of S caryophyllatum fruit

Table 6 Antiglycation activity of crude extracts of Syzygium caryophyllatum

Crude extract Inhibition

IC50 (μgmL)Concentration (μgmL)50 100 200

Leaves 5488plusmn 310a 7987plusmn 594a 9361plusmn 850a 5783plusmn 791Fruits 4131plusmn 789b 5666plusmn 836b 4797plusmn 263b mdashStandard rutin IC50 3423plusmn 318 μgmLResults are expressed as meanplusmn standard deviation n 3lowast Mean values within a column superscripted by different letters are significantly different atplt 005

Table 7 Major compounds detected in Syzygium caryophyllatum leaf essential oil

Peak Compound Formula and lowastmol wt RT Area SI RSI1 Caryophyllene C15H24 204 1479 321 954 9542 Humulene C15H24 204 1645 336 907 9073 α-Cadinol C15H26O 222 2781 387 923 9284 Cyclosiolongifolene 910-dehydro C17H26O2 202 2826 382 790 8005 Phytol C20H40O 296 3444 2466 927 927lowastMol wtmolecular weight

2781

RT 000ndash4200100

90

80

70

60

50

40

30

20

10

0 5 10 15 20 25 30 35 40Retention time (min)

3444

2826

Rela

tive a

bund

ance

1 24

3

5

1479 1645

Figure 7 GC-MS chromatogram of Syzygium caryophyllatum leaf essential oil

Table 8 Mineral compositions of leaves and fruits of Syzygium caryophyllatum

Mineral elements (mgkg per sample)K Fe Na Ca Mg Mn Cu Zn Cr Zn Sr Se Pb As Cd

Syzygiumcaryophyllatum

Leaves 355925 22198 435768 184352 258039 29882 491 2160 403 2159 3289 085 102 ND NDFruits 294428 4955 75621 14927 85555 1814 523 775 465 775 270 009 015 ND ND

RDA 35 g 9ndash15mg 15 g 1 g 280ndash350mg 50ndash60 μglowastND not detected limit of detection 005mgkg RDA recommended daily intake

Evidence-Based Complementary and Alternative Medicine 11

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

Hexane fraction of fruit extract exhibited the highestinhibition activity with IC50 value 4720plusmn 037 μgmL and issignificantly higher compared to acarbose (IC508796plusmn 143 μgmL) (erefore it was further separated bycolumn chromatography (e fraction C3 which was iso-lated on elution with DCM EA 95 5 exhibited a significant(plt 005) α-amylase inhibitory activity with an IC50 value of227plusmn 181 μgmL (e compound or the mixture of

compounds responsible for the α-amylase inhibition activitymay be in the C3 compound mixture which is required to besubjected to further isolation and identification Inhibitionmay be due to the presence of potential alpha-amylase in-hibitors such as alkaloids tannin polyphenols and flavo-noids (ese inhibitors can also act as starch blockers as theyblock the hydrolysis of 14-glycosidic linkages of starch andoligosaccharides [28] Hex fraction of S caryophyllatum fruit

Table 6 Antiglycation activity of crude extracts of Syzygium caryophyllatum

Crude extract Inhibition

IC50 (μgmL)Concentration (μgmL)50 100 200

Leaves 5488plusmn 310a 7987plusmn 594a 9361plusmn 850a 5783plusmn 791Fruits 4131plusmn 789b 5666plusmn 836b 4797plusmn 263b mdashStandard rutin IC50 3423plusmn 318 μgmLResults are expressed as meanplusmn standard deviation n 3lowast Mean values within a column superscripted by different letters are significantly different atplt 005

Table 7 Major compounds detected in Syzygium caryophyllatum leaf essential oil

Peak Compound Formula and lowastmol wt RT Area SI RSI1 Caryophyllene C15H24 204 1479 321 954 9542 Humulene C15H24 204 1645 336 907 9073 α-Cadinol C15H26O 222 2781 387 923 9284 Cyclosiolongifolene 910-dehydro C17H26O2 202 2826 382 790 8005 Phytol C20H40O 296 3444 2466 927 927lowastMol wtmolecular weight

2781

RT 000ndash4200100

90

80

70

60

50

40

30

20

10

0 5 10 15 20 25 30 35 40Retention time (min)

3444

2826

Rela

tive a

bund

ance

1 24

3

5

1479 1645

Figure 7 GC-MS chromatogram of Syzygium caryophyllatum leaf essential oil

Table 8 Mineral compositions of leaves and fruits of Syzygium caryophyllatum

Mineral elements (mgkg per sample)K Fe Na Ca Mg Mn Cu Zn Cr Zn Sr Se Pb As Cd

Syzygiumcaryophyllatum

Leaves 355925 22198 435768 184352 258039 29882 491 2160 403 2159 3289 085 102 ND NDFruits 294428 4955 75621 14927 85555 1814 523 775 465 775 270 009 015 ND ND

RDA 35 g 9ndash15mg 15 g 1 g 280ndash350mg 50ndash60 μglowastND not detected limit of detection 005mgkg RDA recommended daily intake

Evidence-Based Complementary and Alternative Medicine 11

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

extract which can be easily prepared even at a commercialscale may be a very good source for the formulation ofsupplements for DM management

Hyperglycemic conditions in DM promote the forma-tion of AGEs that leads to the complications of DM In vivostudies have revealed the involvement of potent glycationinhibitors in the prevention of diabetic complications [29]In this regard studies show that pyridoxamine slows downnephropathy in DM and aminoguanidine reduces the de-velopment of albuminuria in diabetic rats [30] Furtherprotocatechualdehyde (PCA) has been shown to improvethe development of lens opacity (cataract) with a positiveeffect on glycemic control PCA was found to be 80 moreeffective than aminoguanidine in preventing AGE-relatedcomplications of DM [30]

However there are no available reports on the anti-glycation activity of S caryophyllatum leaves and fruits(e results of the present study also demonstrate mod-erate activity for CR extracts for leaf and weak activity forfruits

Dietary antioxidants are essential to strengthen theoxidative defense system of the body particularly underhyperglycemic conditions In this regard the antioxidantactivity of S caryophyllatum fruits and leaves was evaluatedusing four different methods which are having differentmodes of action

Free radical scavenging methods such as DPPH andABTS were used and EC50 values of both assays suggestedpotent radical scavenging activity of S caryophyllatum fruitsand leaf extracts (e DPPH nitrogen centered stable freeradical has been used to determine the free radical scav-enging activity (e reduction of DPPH in methanol leads todiscoloration from violet to yellow by either hydrogen orelectron donation

A previous study on DPPH radical scavenging activity ofthe S caryophyllatum leaves has reported DPPH inhibitionactivity in methanol extract with 8197 followed by EA(7869) and Hex fraction (3434) at the concentration of100 μgmL [14] Annadurai et al [31] have reported DPPHscavenging activity for the EA fraction of S caryophyllatumleaves as 8614 inhibition at a concentration of 400 μgmLSubramanian et al [14] and the present study show similarresults for the EA fraction with a value of 7292 inhibitionat the concentration of 150 μgmL A previous study carriedout by sequential extraction method for leaves and fruits hasreported having IC50 value for DPPH scavenging activity inmethanol as 349plusmn 12 μgmL and 694plusmn 07 μgmL re-spectively which showed high activity compared to thepresent study [15]

(e scavenging activity against cationic radical indicatesthe capability of crude and fractions to act as electron orhydrogen donors Previous studies have reported havingABTS radical scavenging activity with IC50 values of132plusmn 003 μgmL for leaves and 1202plusmn 04 μgmL for fruitpulp in methanol extract [15] (ough previous studies haveused methanolic extracts in this study ethanolwater wasused to prepare the extract as the main focus was on theformulation of a supplement In this regard the Hex fractionof the fruit extract exhibited higher ABTS radical scavenging

activity (EC50 1501plusmn 065 μgmL) compared to the resultsreported in a previous study [15]

(e differences observed among these studies are gen-erally accepted as the samples were from different geo-graphic locations with different degrees of biotic and abioticstress conditions that are associated with biosynthesis andexpression of secondary metabolites [32]

In the FRAP assay the reduction of the ferric-TPTZcomplex to ferrous-TPTZ by accepting electrons from thesample was measured as the total amount of antioxidantsCompounds that exhibit the ferric reducing activity indicatethat their ability to act as electron donors reduces the ox-idized intermediates of the lipid peroxidation process In thisstudy EA fraction of leaves reported the highest valueamong the tested fractions in leaves and fruits which revealsthe best-reducing property

(e ORAC assay is based on hydrogen atom transfermechanism (HAT) and the inhibition of oxidation offluorescein by peroxide radicals generated from 22rsquo-azobis(2-amidino-propane) dihydrochloride (AAPH) with anantioxidant action According to the present study ORACassay results implied that there is no correlation with otherantioxidant assays (is fact still requires more supportingpieces of evidence as there is no available reported data onORAC assay for the S caryophyllatum fruit and leaf extracts

(e Fe2+ chelation assay is considered as an indirectantioxidant activity measurement which indicates the po-tential to inhibit Fenton reaction (e Fe2+ chelation wasestimated quantitatively with ferrozine (e test procedureresults in the formation of a red-colored complex with Fe2+

that reduces its color in the presence of a chelating agent(is is due to the interruption caused by the formedcomplex Extracts and fractions exhibited the metal iron-chelating power where the Hex fraction of fruits exhibitedthe highest iron chelation capacity (is is suggestive of thepresence of bioactive molecules with chelating abilities inaddition to the polyphenolics

Strong antioxidant and antimicrobial potentials of theleaf oil of S caryophyllatum along with GC-MS analysis havebeen reported for Indian plants by Soni et al [33] andNadarajan et al [34] (e major compounds revealed by thepresent study are phytol caryophyllene humulene globuloland α-cadinol (e composition of leaf volatiles of S car-yophyllatum reported in this study relates and correspondsto those reported in the previous study (e previous studyhas reported 55 compounds in the winter season and 129compounds in the summer season with promising anti-bacterial activity [34] (e major identified compound inwinter essential oil was α-cadinol and in summer the es-sential oil was caryophyllene oxide where the phytol was themajor compound in the present study [34] Further fewcommon compounds such as α-cadinol caryophyllene ox-ide globulol and phytol were also found

Mineral deficiencies impose a major burden on publichealth and maintenance of healthy physiology (ereforesupplementation is the best choice in order to fulfil the dailymineral requirement especially when an individual is suf-fering from a health complication such as DM Some dietaryminerals including Cr Mg Ca Mn K Zn and Se play a key

12 Evidence-Based Complementary and Alternative Medicine

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

role in physiological processes related to DM [35] Mag-nesium deficiency is related to the risk of atherosclerosishypertension cardiac arrhythmias stroke alterations inlipid metabolism insulin resistance metabolic syndrometype 2 diabetes mellitus osteoporosis and depression

Zinc is an important microelement with definite roles inmetabolism and growth and it is also essential and vital forthe smooth functioning of over 200 enzymes [36] Zincdeficiency adversely affects the growth of T and B cells andapoptosis growth retardation and cognitive impairment[37] Further Zn is involved in the synthesis and secretionof insulin and it is a structural part of Zn-dependentantioxidant enzymes such as superoxide dismutase [38]Copper is an essential microelement that acts as a cofactorof many redox enzymes and as catalysts for iron ab-sorption [36]

Chromium is an essential microelement for the bio-synthesis of DNA which regulates cell division and growth[39] (e Cr is also necessary for the regulation of normalglucose metabolism and the deficiency of Cr is related toimpaired glucose tolerance [35] Selenium plays a criticalrole in antioxidant defense due to its presence in the activecenter of antioxidative enzymes (glutathione peroxidase(GPX) and thioredoxin reductase (TrxRs)) (is essentialmicroelement impairs lipid peroxidation and protects cellsagainst damage to genetic material [40] A previous studycarried out by Subramanian et al [41] for the leaves ofS caryophyllatum reported having 99275plusmn 0022mgkg17302plusmn0010mgkg 45498plusmn0072mgkg 47344plusmn0002mgkg7634plusmn0050mgkg and 0253plusmn0003mgkg of Fe Zn Cu MnPb and Cd respectively [41] (e content of Na and Kreported was absent and also Zn significantly high Cu andlowMn levels and Cd were detected and a higher amount ofPb was reported compared to the present study [41]

(e results of the present study exhibited that thecontent of the mineral elements differs significantly amongthe parts of the similar plants Furthermore with com-parison to the reported data it reveals that the levels ofmineral elements vary from different geographical locationsdue to the variation in soil types agricultural and industrialactivities local growing conditions plant interactions andweather [8] Nonetheless studies have revealed that themetal-tolerant plants (metallophytes) possess the effects forphytoremediation through exposure to heavy metal stressshowing varying degrees of therapeutically active constitu-ents [42]

(us the risk analysis is required in plants before anypreparation as the wild fruity plants are favored in con-trolling the growth and processing of the environment

5 Conclusions

(e present study concludes that the leaves and the fruits ofSyzygium caryophyllatum plant possess antioxidant anti-amylase and antiglycation activities and are a rich pool ofessential mineral elements such as ZnMn Cu and Sr whichtogether may act to reduce the risk of associated diabeticcomplications and other noncommunicable diseasesStudies on isolation and characterization of the bioactive

compound in leaves and fruit extracts are important forconfirmation of the pharmacological properties of the re-sponsible active compounds Further it can be concludedthat Syzygium caryophyllatum leaves and fruits are potentnatural sources that possess the potential to be developedinto a natural supplement to aid the management process oftype 2 diabetes mellitus

Data Availability

(e data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

(e authors declare that they have no conflicts of interest

Acknowledgments

(e authors acknowledged Suvini Karunaratne for thelanguage editing of the manuscript

References

[1] World Health Organization ldquoDiabetesrdquo World Health Or-ganization Geneva Switzerland 2016 httpwwwwhointmediacentrefactsheetsfs312en

[2] El Nahas A Meguid and A K Bello ldquoChronic kidneydisease the global challengerdquo Ce Lancet vol 365 no 9456pp 331ndash340 2005

[3] H Kolb and S Martin ldquoEnvironmentallifestyle factors in thepathogenesis and prevention of type 2 diabetesrdquo BMCMedicine vol 15 no 1 p 131 2017

[4] A Phaniendra D B Jestadi and L Periyasamy ldquoFree rad-icals properties sources targets and their implication invarious diseasesrdquo Indian Journal of Clinical Biochemistryvol 30 no 1 pp 11ndash26 2015

[5] M Odjakova E Popova M Al Sharif and R MironovaldquoPlant-derived agents with anti-glycation activityrdquo Glycosyl-ation pp 223ndash256 IntechOpen London UK 2012

[6] R S Tupe N M Sankhe S A Shaikh et al ldquoAqueous extractof some indigenous medicinal plants inhibits glycation atmultiple stages and protects erythrocytes from oxidativedamage-an in vitro studyrdquo Journal of Food Science andTechnology vol 52 no 4 pp 1911ndash1923 2015

[7] H P Gunawardena R Silva R Sivakanesan P Ranasingheand P Katulanda ldquoPoor glycaemic control is associated withincreased lipid peroxidation and glutathione peroxidase ac-tivity in type 2 diabetes patientsrdquo Oxidative Medicine andCellular Longevity vol 2019 Article ID 9471697 10 pages2019

[8] M Sium P Kareru J Keriko B Girmay G Medhanie andS Debretsion ldquoProfile of trace elements in selected medicinalplants used for the treatment of diabetes in Eritreardquo CeScientific World Journal vol 2016 Article ID 27528367 pages 2016

[9] I N Leroux A P S d S Ferreira F P Paniz et al ldquoBrazilianpreschool children attending day care centers show an in-adequate micronutrient intake through 24-h duplicate dietrdquoJournal of Trace Elements in Medicine and Biology vol 54pp 175ndash182 2019

Evidence-Based Complementary and Alternative Medicine 13

[10] E Jarald S B Joshi and D Jain ldquoDiabetes and herbalmedicinesrdquo Iranian Journal of Pharmacology amp Cerapeuticsvol 7 pp 97ndash106 2008

[11] E Idmrsquohand F Msanda and K Cherifi ldquoEthno-pharmacological review of medicinal plants used to managediabetes in Moroccordquo Clinical Phytoscience vol 6 pp 1ndash322020

[12] A Singh and T Marar ldquoInhibitory effect of extracts ofsyzygiumcumini and psidiumguajava on glycosidasesrdquoJournal of Cell and Tissue Research vol 11 no 1 p 2535 2011

[13] K Kala V T Antony M S Sheemole and A Saji ldquoAnalysisof bioactive compounds present in syzygiumcaryophyllatum(L) alston fruitrdquo International Journal of PharmaceuticalSciences Review and Research vol 36 pp 239ndash243 2016

[14] R Subramanian P Subbramaniyan and V Raj ldquoPhyto-chemical screening total phenolic contents and antioxidantactivity ofSyzygium caryophyllatumandSyzygium densi-florumrdquo Journal of Biologically Active Products from Naturevol 4 no 3 pp 224ndash235 2014

[15] N Stalin and S P Sudhakar ldquoScreening of phytochemical andpharmacological activities of Syzygiumcaryophyllatum (L)Alstonrdquo Clinical Phytoscience vol 4 no 1 pp 1ndash3 2018

[16] K V Peter ldquoUnderutilized and underexploited cropsrdquo vol 3p 177 New India Publishing Agency New Delhi India 2008

[17] V L Singleton R Orthofer and R M Lamuela-Raventosldquo[14] Analysis of total phenols and other oxidation substratesand antioxidants by means of folin-ciocalteu reagentrdquo Oxi-dants and Antioxidants Part A vol 299 pp 152ndash178 1999

[18] P Ranasinghe G Premakumara C Wijayarathna andW Ratnasooriya ldquoAntioxidant activity ofrdquo Tropical Agri-cultural Research vol 23 no 2 pp 117ndash125 2012

[19] R Re N Pellegrini A Proteggente A Pannala M Yang andC Rice-Evans ldquoAntioxidant activity applying an improvedABTS radical cationdecolourization assayrdquo Free Radical Bi-ology and Medicine vol 26 no 9-10 pp 1231ndash1237 1999

[20] B Ou M Hampsch-Woodill and R L Prior ldquoDevelopmentand validation of an improved oxygen radical absorbancecapacity assay using fluorescein as the fluorescent proberdquoJournal of Agricultural and Food Chemistry vol 49 no 10pp 4619ndash4626 2001

[21] P Carter ldquoSpectrophotometric determination of serum ironat the submicrogram level with a new reagent (ferrozine)rdquoAnalytical Biochemistry vol 40 no 2 pp 450ndash458 1971

[22] G A S Premakumara W K S M AbeysekeraW D Ratnasooriya N V Chandrasekharan andA P Bentota ldquoAntioxidant anti-amylase and anti-glycationpotential of brans of some Sri Lankan traditional and im-proved rice (Oryzasativa L) varietiesrdquo Journal of CerealScience vol 58 no 3 pp 451ndash456 2013

[23] N Matsuura T Aradate C Sasaki et al ldquoScreening systemfor the Maillard reaction inhibitor from natural productextractsrdquo Journal of Health Science vol 48 no 6 pp 520ndash5262002

[24] A B Oyenihi N L Brooks O O Oguntibeju and G AboualdquoAntioxidant-antidiabetic agents and human healthrdquo Olu-wafemiOguntibeju IntechOpen London UK 2014 httpswwwintechopencombooksantioxidant-antidiabetic-agents-and-human-healthantioxidant-rich-natural-products-and-diabetes-mellitus

[25] V P Singh A Bali N Singh and A S Jaggi ldquoAdvancedglycation end products and diabetic complicationsrdquo CeKorean Journal of Physiology amp Pharmacology vol 18 no 1pp 1ndash14 2014

[26] M I Kazeem J O Adamson and I A Ogunwande ldquoModesof inhibition of α-amylase and α-glucosidase by aqueousextract of MorindalucidaBenth leafrdquo BioMed Research In-ternational vol 2013 Article ID 527570 6 pages 2013

[27] C S Rabeque and S Padmavathy ldquoHypoglycaemic effect ofSyzygium caryophyllatum (L) alston on alloxan induced di-abetic albino micerdquo Asian Journal of Pharmaceutical andClinical Research vol 6 no 4 pp 203ndash205 2013

[28] G Keerthana M K Kalaivani and A Sumathy ldquoIn-vitroalpha amylase inhibitory and anti-oxidant activities of etha-nolic leaf extract of Croton bonplandianumrdquo Asian Journal ofPharmaceutical and Clinical Research vol 6 no 4 pp 32ndash362013

[29] J S Ramkissoon F M Mahomoodally A Ahmed andH A Subratty ldquoNatural inhibitors of advanced glycation end-productsrdquo Nutrition amp Food Science vol 42 no 6pp 397ndash404 2012

[30] G Abbas A S Al-Harrasi H Hussain J Hussain R Rashidand M I Choudhary ldquoAnti-glycation therapy discovery ofpromising anti-glycation agents for the management of di-abetic complicationsrdquo Pharmaceutical Biology vol 54 no 2pp 198ndash206 2016

[31] G Annadurai B R P Masilla S JothiramshekarE Palanisami S Puthiyapurayil and A K Parida ldquoAnti-microbial antioxidant anticancer activities of Syzygiumcar-yophyllatum (L) Alstonrdquo International Journal of GreenPharmacy (IJGP) vol 6 no 4 pp 285ndash288 2012

[32] C M Andre M Oufir L Hoffmann et al ldquoInfluence ofenvironment and genotype on polyphenol compounds and invitro antioxidant capacity of native Andean potatoes (Sol-anumtuberosum L)rdquo Journal of Food Composition andAnalysis vol 22 no 6 pp 517ndash524 2009

[33] A M R I T A Soni and P R A V E E N DahiyaldquoPhytochemical analysis antioxidant and antimicrobial ac-tivity of Syzygiumcaryophyllatum essential oilrdquo Asian Journalof Pharmaceutical and Clinical Research vol 7 no 2pp 202ndash205 2014

[34] S Nadarajan and S S Pujari ldquoLeaf essential oil compositionand biochemical activity of an endangered medicinal treesyzygiumcaryophyllatum (L) alston (wild black plum)rdquoJournal of Essential Oil Bearing Plants vol 17 no 3pp 371ndash379 2014

[35] M N Piero J M Njagi C M Kibiti J J N NgeranwaE N M Njagi and P M Miriti ldquo(e role of vitamins andmineral elements in management of type 2 diabetes mellitus areviewrdquo South Asian Journal of Biological Science vol 2pp 107ndash115 2012

[36] N K Singh C B Devi T S Singh and N R Singh ldquoTraceelements of some selected medicinal plants of ManipurrdquoIndian Journal of Natural Products and Resources vol 1 no 2pp 227ndash231 2010

[37] A P Prasad ldquoImpact of the discovery of human zinc defi-ciency on healthrdquo Journal of the American College of Nutri-tion vol 28 no 3 pp 257ndash265 2009

[38] R Jayawardena P Ranasinghe P GalappatthyR L D K Malkanthi G R Constantine and P KatulandaldquoEffects of zinc supplementation on diabetes mellitus asystematic review and meta-analysisrdquo Diabetology amp Meta-bolic Syndrome vol 4 no 1 p 13 2012

[39] K O Soetan C O Olaiya and O E Oyewole ldquo(e im-portance of mineral elements for humans domestic animalsand plants-A reviewrdquo African Journal of Food Science vol 4no 5 pp 200ndash222 2010

14 Evidence-Based Complementary and Alternative Medicine

[40] J Pieczy P Sylwia K Orywal B Mroczko and H Grajeta ldquoIsmaternal dietary selenium intake related to antioxidant statusand the occurrence of pregnancy complicationsrdquo Journal ofTrace Elements in Medicine and Biology vol 54 pp 110ndash1172019

[41] R Subramanian P Subbramaniyan and V Raj ldquoDetermi-nation of some minerals and trace elements in two tropicalmedicinal plantsrdquo Asian Pacific Journal of Tropical Biomed-icine vol 2 no 2 pp 555ndash558 2012

[42] R A Street ldquoHeavy metals in medicinal plant productsmdashanAfrican perspectiverdquo South African Journal of Botany vol 82pp 67ndash74 2012

Evidence-Based Complementary and Alternative Medicine 15