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Research ArticleOptimization of Extraction Conditions of PhytochemicalCompounds and Anti-Gout Activity of Euphorbia hirta L(Ara Tanah) Using Response Surface Methodology and LiquidChromatography-Mass Spectrometry (LC-MS) Analysis

Fazleen Izzany Abu Bakar 1 Mohd Fadzelly Abu Bakar 1 Norazlin Abdullah 1

Susi Endrini2 and Sri Fatmawati3

1Centre of Research for Sustainable Uses of Natural Resources (CoR-SUNR) Faculty of Applied Sciences and TechnologyUniversiti Tun Hussein Onn Malaysia (UTHM) 84600 Muar Johor Malaysia2Faculty of Medicine Universitas Abdurrab Pekanbaru 28292 Riau Indonesia3Department of Chemistry Faculty of Science Institut Teknologi Sepuluh Nopember Surabaya Indonesia

Correspondence should be addressed to Mohd Fadzelly Abu Bakar fadzellyuthmedumy

Received 31 August 2019 Revised 13 December 2019 Accepted 24 December 2019 Published 27 January 2020

Guest Editor Paulo Jorge da Silva Almeida

Copyright copy 2020 Fazleen Izzany Abu Bakar et al is is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in anymedium provided the original work isproperly cited

Gout is a common disease affected most of the people due to the elevation of uric acid in the blood Flavonoid and phenoliccompounds are reported to exert the anti-gout activity of medicinal plants Hence this study aimed at optimizing theextraction conditions of phenolic and flavonoid compounds as well as the anti-gout (xanthine oxidase inhibitory activity) invitro of Euphorbia hirta using response surface methodology (RSM) e plant part used was the whole plant excludingroots e effects of three independent variables (extraction time X1 extraction temperature X2 and solid-to-liquid ratioX3) on three response variables (total flavonoid content Y1 total phenolic content Y2 and xanthine oxidase inhibitoryactivity Y3) were determined using central composite design (CCD) while phytochemical profiling of the extracts wasdetermined by liquid chromatography-mass spectrometry (LC-MS) Quadratic models produced a satisfactory fitting of theexperimental data with regard to total flavonoid content (r2 09407 plt 00001) total phenolic content (r2 09383plt 00001) and xanthine oxidase inhibitory activity (r2 09794 plt 00001) e best extraction conditions observed fortotal flavonoid content total phenolic content and xanthine oxidase inhibitory activity were at a temperature of 7907degC for1742 min with solid-to-liquid ratio of 1 20 gml e optimum values for total flavonoid total phenolic and xanthineoxidase inhibitory activity were 6756 mg REg 15521 mg GAEg and 9142 respectively e main phytochemicalcompounds in the optimized E hirta extract are neochlorogenic acid quercetin-3β-D-glucoside syringic acid caffeic acidellagic acid astragalin afzelin and quercetin As conclusion this study clearly demonstrated the best conditions to obtainhigher xanthine oxidase inhibitory activity and phytochemical compounds which can be further used for the development ofanti-gout agents

1 Introduction

Euphorbia hirta L or locally known as Ara tanah or Gelangsusu in Malaysia is from the family of Euphorbiaceae egenus of Euphorbia is considered to be the largest in theEuphorbiaceae family with about 1600 species and it isnative to tropical and subtropical regions of Asia Africa and

Central as well as South America [1]is plant was found topossess potent antimicrobial [2] antidiarrhoeic [3] anti-hyperglycemic [4] anti-inflammatory [5] and anticancer [6]activities In fact in Malay traditional medicine it has beenused widely to treat various diseases including skin prob-lems gastrointestinal disorders diarrhoea and ulcer [2]However in Ayurvedic treatment this plant has been used

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2020 Article ID 4501261 13 pageshttpsdoiorg10115520204501261

traditionally to treat worm infestations in children dysen-tery jaundice pimples digestive problems female disordersrespiratory ailments (cough coryza bronchitis andasthma) and tumors [1] In addition the decoction or in-fusion of this plant has been used widely by practitioners oftraditional medicine in treating asthma kidney stonesheartburn diarrhea coughs and menstrual problems [7]Interestingly many phytochemical compounds such as sa-ponin sterol terpene alkaloids polyphenols tannins ter-penoids steroids and flavonoids have been found in theaerial parts of this plant [4 7] is plant is also rich inminerals such as zinc potassium calcium and magnesium[4] In 2007 three compounds from the methanolic extractof E hirta namely afzelin quercetin and myricetin whichshowed potent inhibition against malaria have been suc-cessfully isolated [8]

Extraction is considered to be one of the important stepsin discovering potential bioactive compounds from plantmaterials considering their quantity and type of bioactivecompounds as they are widely used in nutraceuticalpharmaceutical and cosmetic products as well as functionalfood ingredients and food additives [9 10] e type andconcentration of solvent temperature time pH solid-liquidratios pressure and the particle size of the plant have beenidentified from the previous studies as important factorsaffecting the extraction efficiency of the plant bioactivecompounds [11ndash13] Traditional method which is known asthe one-factor-at-a-time has been used widely by manyresearchers in optimizing specific parameters in which onlyone factor plays a role as a variable at a time while keeping allother factors constant [14] However this method is con-sidered to be less reliable as it does not include interactiveeffects among factors time-consuming and expensiveMisleading conclusions and false conditions also might beoccurred at the end of this experiment method ignoring thetrue significant factors during the extraction process [9] Toovercome this problem response surface methodology(RSM) has been introduced and widely used nowadays foroptimizing the extraction conditions RSM is considered tobe a powerful mathematical technique used widely in manyindustries for technological operations in order to optimizecertain experimental conditions Moreover it evaluates theeffects between the multiple factors and their interactionstowards one or more response variables simultaneouslyproducing lesser number of experimental measurements[15] It also helps to locate the region where the extraction isoptimized by using clearer visual aid [16] Based on theprevious literature the phenolic and flavonoid contents ofguava puree [17] Curcuma zedoaria leaves [16] apples [18]Feronia limonia fruit [15] and Mangifera pajang [9] havebeen successfully optimized using RSM

Xanthine oxidase inhibitory activity (XO) is an enzymeassay used widely in determining the anti-gout activity of theplant extracts XO is known as the main enzyme causing theaccumulation of uric acid in the blood which eventuallyleads to gout e mechanism involves where it catalyses theoxidation of hypoxanthine into xanthine and then uric acid[19] Optimization of extraction method to obtain highertotal flavonoids and phenolics as well as XO inhibitory

activity from E hirta is essential for future nutraceuticalproduct development especially for the gout treatment Tothe best of our knowledge this is the first report describingoptimization of extraction process of phytochemical com-pounds and XO inhibitory activity from E hirta ereforethis study aimed at optimizing the extraction temperaturetime and solid-to-liquid ratio for the extraction of totalflavonoid content total phenolic content and anti-goutactivity from E hirta whole plant excluding roots usingRSM

2 Materials and Methods

21 Chemicals and Reagents Gallic acid xanthine xanthineoxidase (buttermilk) and potassium dihydrogen phosphate(KH2PO4) were purchased from Sigma-Aldrich Chemicals(St Louis MO USA) FolinndashCiocalteu reagents were ob-tained from Merck (Germany) Allopurinol rutin alumi-num chloride hexahydrate sodium nitrite sodiumhydroxide and dipotassium hydrogen phosphate (K2HPO4)were of the highest purity (95ndash99)

22 PlantMaterials e plant part used was the whole plantexcluding roots E hirta was collected from the Departmentof Agriculture Serdang Selangor Malaysia and authenti-cated by Dr Mohd Firdaus Bin Ismail (Institute of Bio-science Universiti Putra Malaysia) and the voucherspecimen was deposited in the Herbarium of the same in-stitute under the number MFI008419

23 Sample Extraction e whole plant excluding root wassoaked in water washed and oven-dried at 40degC for 72hours en it was ground and stored in a minus 20degC freezerprior to the extraction process In order to mimic the humanconsumption the plant was prepared using hot boiling waterextraction procedure whereby each 10 g of dried plant wasinfused in 10ndash20ml of distilled water at temperature be-tween 50 and 100degC and continuously stirred for 5ndash60min(Table 1) using a magnetic stirrer under 300 rpm e in-fusion was left to cool to 15min en the extract wasfiltered using Whatman No 1 filter paper and subjected tothe xanthine oxidase inhibitory activity assay spectropho-tometrically at 295 nm for determination of total flavonoidand total phenolic contents

24 Experimental Design Central composite design (CCD)was developed in order to obtain optimized extractionconditions for higher total flavonoid content total phenoliccontent and xanthine oxidase inhibitory activity of E hirtaCCD was chosen in this study as it provides more designpoints on each variable ree selected independent vari-ables in this study were extraction time (X1 5ndash60 minutes)extraction temperature (X2 50ndash100degC) and solid-to-liquidratio (X3 1 10ndash1 20 gml) while the dependent variableschosen were total flavonoid content (Y1) total phenoliccontent (Y2) and xanthine oxidase inhibitory activity (Y3)e responses of total flavonoid content and total phenolic

2 Evidence-Based Complementary and Alternative Medicine

content were critical to be studied as the presence of both ofthem in the plants have been reported to be the reasons fortreating the gout disease while xanthine oxidase inhibitoryactivity was the in vitro enzyme assay which has been usedwidely for determining the antigout activity of the plantse complete design was generated by a statistical softwarepackage (Design Expert version 70 Minneapolis USA)which consists of 20 combinations including six replicates atthe central point (Table 1)

25 Total Flavonoid Content (TFC) Determination TFC ofthe plant extract was determined using aluminium chloridemethod [20] e absorbance of the mixture was measuredspectrophotometrically at 510 nm and the results obtainedwere expressed as milligram rutin equivalent per gram (mgREg) based on the standard curve of various concentrationsof rutin (0ndash100 μgml)

26TotalPhenolicContent (TPC)Determination TPC of theplant extract was determined using FolinndashCiocalteursquoscolorimetric method with slight modification [21] eabsorbance was spectrophotometrically measured at725 nm and the results obtained were expressed as mil-ligram gallic acid equivalent per gram (mg GAEg) basedon the standard curve with various concentrations of gallicacid (0 minus 100 μgml)

27 Xanthine Oxidase (XO) Inhibitory Activity Assay einhibitory effect on XO was measured spectrophotometri-cally at 295 nm under aerobic condition following theexisting method with some modifications [22 23] whereallopurinol (0ndash100 μgml) was used as a positive control edegree of XO inhibitory activity was calculated by using thefollowing equation

XO inhibition 1 minusβα

1113888 1113889 times 100 (1)

where α is the activity of XO without test extract and β is theactivity of XO with test extract

28 Liquid Chromatography-Mass Spectroscopy (LC-MS)Analysis Chromatography was performed according toZakaria et al [24] where 20 μL of sample was injected onto aAgilent C18 reverse-phase column (40times 250mm 18 μm)and held at 50degC with a constant flow rate of 04mLmin andtotal LC run time of 30min Sample elution was performedin a gradient manner using mobile phase comprising ofwater containing 01 acetic acid (solvent A) and HPLCgrade acetonitrile containing 01 acetic acid (solvent B)e mobile phase composition (A B) was gradually in-creased from 595 to 1585 over 25min and returned to theinitial condition (95) for 5min for solvent A and 5 to85 for 25min and then decreased to the initial condition(5 95) over the next 5min For the mass analysis the sourceconditions were as follows nebulizer pressure was 40 psidrying gas flow was set at 12 Lmin and drying gas tem-perature was 350degC and the MSMS acquisitions wereperformed in the negative and positive electrospray ioni-zation mode for the mass range of 150 to 1500mz Dataacquisition was performed by Agilent MassHunter work-station Data Acquisition while data processing was carriedout with MassHunter Qualitative Analysis software Inaddition MSMS experiments were carried out in the au-tomatic and multiple reaction monitoring (MRM) modewhere automatic MSMS low-energy collision dissociation(CID) was performed at 5ndash8 eV collision energy Peakidentification was carried out based on comparison withliterature values and online databases

Table 1 Experimental design for the extraction process from central composite design

Run order X1 (extraction time) (min) X2 (extraction temperature) (degC) X3 (solid-to-liquid ratio) (gml)1a 325 75 152a 325 75 153a 325 75 154 325 50 155a 325 75 156 325 75 207a 325 75 158 5 50 209 325 75 1010 60 50 1011 60 50 2012 60 100 1013 60 75 1514 325 100 1515 5 100 2016 5 75 1517 5 100 1018 5 50 1019 60 100 2020a 325 75 15aCenter point

Evidence-Based Complementary and Alternative Medicine 3

29 Statistical Analysis Design Expertreg Software (version 7Stat-Ease Inc Minneapolis MN) was used in this study Aresponse surface analysis was employed to determine theregression coefficients and statistical significance of themodel terms and to fit the mathematical models of theexperimental data e adequacy of the model was predictedthrough the regression analysis (r2) and the analysis ofvariance (ANOVA) (plt 005)

3 Results and Discussion

31 Fitting the Response Surface Models Fitting the modelsare crucial in interpreting the accuracy of the RSMmathematical models for prediction of the TFC TPC andXO inhibitory activity of E hirta extract In this presentstudy the relationship between the response functions(TFC TPC and XO inhibitory activity) and the inde-pendent variables (extraction time extraction tempera-ture and solid-to-liquid ratio) was successfully identifiedby CCD e results of the responses for the 20 runsfollowing the experimental design are shown in Table 2e yield of the TFC ranged from 5111 to 6555mg REgwhile TPC ranged from 12034 to 15295mg GAEg Interms of XO inhibitory activity the highest activity was9757 whereas the lowest was 7296 As suggested bythe Design Expert software a quadratic polynomial modelwas selected and fitted well for all three independentvariables and responses In terms of coded values thepredicted responses for the TFC TPC and XO inhibitoryactivity could be expressed by the second-order polyno-mial equation via multiple regression analysis

YTFC 6046 + 154X1 + 111X2 + 264X3 minus 228X1X2

+ 128X1X3 + 128X2X3 minus 172X21 minus 573X

22 + 284X

23

(2)

YTPC 14228 + 247X1 + 250X2 + 484X3 minus 572X1X2

+ 308X1X3 + 171X2X3 minus 248X21

minus 1724X22 + 888X

23

(3)

YXO inhibitory activity 9503 minus 330X1 minus 112X2 minus 627X3

minus 063X1X2348X1X3 minus 074X2X3

minus 016X21 minus 291X

22 minus 332X

23

(4)

where X1 X2 and X3 are the coded variables for extractiontime extraction temperature and solid-to-liquid ratio re-spectively A negative sign in each equation represents anantagonistic effect of the variables and a positive signrepresents a synergistic effect of the variables [16]

Positive coefficients for X1 X2 and X3 were observed inequation (2) indicating that TFC was increased with theincrease of extraction time (5ndash60min) extraction temper-ature (50ndash100degC) and solid-to-liquid ratio (1 10ndash120) esame observation was also found for TPC where positive

coefficients for X1 X2 and X3 were observed in equation (3)indicating that TPC was increased with the increase ofextraction time (5ndash60min) extraction temperature(50ndash100degC) and solid-to-liquid ratio (1 10ndash1 20) In con-trast negative coefficients for X1 X2 and X3 were observedin equation (4) indicating that XO inhibitory activity wasdecreased with the increase of extraction time (5ndash60min)extraction temperature (50ndash100degC) and solid-to-liquid ratio(1 10ndash1 20) e RSM model coefficients were validated byanalysis of variance (ANOVA) of the response variables forthe quadratic polynomial model summarized in Supple-mentary Materials (Tables S1 S2 and S3) For the responsesof TFC TPC and XO inhibitory activity the models werehighly significant when the computed F-values were greaterthan the tabulated F-value and the probability values werelow (plt 0001) indicating that the individual terms in eachresponse model were significant on the interaction effect Inaddition the coefficient of determination (r2) of the modelswas 09407 09383 and 09794 respectively indicating that9407 9383 and 9794 match between the values of thepredicted model and the values that were attained in theexperimental data Besides the r2 values were comparable toadjusted r2 demonstrating good statistical model e p

values for the lack of fit were identified as 02462 04015 and05001 highlighting that the lack of fit of the models wassignificant at pgt 005 Hence the results indicate that themodels could predict the TFC TPC and XO inhibitoryactivity of E hirta extract efficiently when independentvariables were within the ranges depicted here

32 Effect of Extraction Parameters on Total FlavonoidContent (TFC) Flavonoids are the most common group ofpolyphenolic compounds in the human diet and are foundubiquitously in plants [25] ey are categorized mainly intoflavone flavanol and flavanone compounds Table S1(Supplementary data) shows that all independent variables(extraction time temperature and solid-to-liquid ratio) had asignificant effect on TFC of E hirta extract Among thesesolid-to-liquid ratio (X3) was found to be the major effect onTFC in comparison with other variables which may be due tothe high F-value of 3399 Interestingly the analysis of theresults also showed that all the interactions between time andtemperature (X1X2) time and solid-to-liquid ratio (X1X3) andtemperature and solid-to-liquid ratio (X2X3) were highlysignificant factors affecting the yield of TFC (plt 005) Basedon the previous studies extraction temperature time andsolid-to-liquid ratio are considered to be the important pa-rameters for the extraction efficiency of flavonoid compounds[26 27] Figures 1(a)ndash1(c) show the three-dimensional re-sponse surface plots for the influences of extraction tem-perature time and solid-to-liquid ratio on TFCe responsesurface plots are very useful to see the interaction effects of thefactors on the responses As in Figure 1(a) the TFC extractionyields significantly increased as the extraction temperatureand time increased from 50 to 75degC and 5 to 60min re-spectively but started to decrease towards 100degC whilekeeping the solid-to-liquid ratio as constant is was sup-ported by Sheng et al [28] where the yield of total flavonoids


in the plant extract rose gradually with the increase oftemperature from 50deg to 90degC e increase of extractiontemperature promotes the solvent extraction process of theplant extract due to multiple effects of temperature on themass transfer process by enhancing both diffusion coefficientsand the solubility of flavonoid compounds as well as pro-moting the degradation of the plant matrix [26 29]

Figures 1(b) and 1(c) display the effect of solid-to-liquidratio-extraction time and solid-to-liquid ratio-extractiontemperature on TFC of E hirta respectively e yield of TFCincreased when the solid-to-liquid ratio and extraction timeincreased (Figure 1(b)) As shown in Figure 1(c) as the ratio ofsolid-to-liquid increased from 1 15 to 1 20 the yield of TFCincreased significantly from 5982mgg to 6406mgg with anincrease in extraction temperature is was supported by theprevious study where the TFC of Gynura medica leaves sig-nificantly increased from 2448 to 3539mg kaempferolg DMas the liquid-to-solid ratio increased from 10 to 40 (vw) [26]e same trendwas also observed by Sheng et al [28] where theTFC increased gradually with the increase of liquid-to-solidratio from 10 1 to 18 1 is might be due to the increase ofthe driving force for the mass transfer of the TFC resulting inthe high TFC obtained at the end of the study [26]

33 Effect of Extraction Parameters on Total Phenolic Content(TPC) One of the most important groups of secondarymetabolites produced by all plants is phenolic compoundsPhenolic compounds are secondary metabolites that are de-rivatives of the pentose phosphate shikimate and phenyl-propanoid pathways in plants [30] Phenolics include simplephenols phenolic acids (benzoic and cinnamic acid

derivatives) coumarins flavonoids stilbenes hydrolyzableand condensed tannins lignans and lignins acting mainly asphytoalexins attractants for pollinators contributors to plantpigmentation antioxidants and protective agents againstultraviolet (UV) light [31] Table S2 (Supplementary data)shows that all independent variables (extraction time tem-perature and solid-to-liquid ratio) had the significant effect onTPC of E hirta extract Among these solid-to-liquid ratio (X3)was found to be the major effect on TPC in comparison withother variables which may due to the high F-value of 1942Interestingly the analysis of the results also showed that theinteractions between time and temperature (X1X2) and timeand solid-to-liquid ratio (X1X3) were highly significant factorsaffecting the yield of TFC (plt 005) except for temperatureand solid-to-liquid ratio (X2X3) Figures 2(a)ndash2(c) show theresponse surface plots for the influences of extraction tem-perature extraction time and solid-to-liquid ratio on TPC Asshown in Figure 2(a) TPC was increased as the extractiontemperature increased and reached the maximum at 75degC butstarted to decrease towards 100degC especially at high levels ofextraction time implying that beyond a certain value oftemperature phenolic compounds can be denatured [32 33]Previous study has demonstrated that the yield of phenoliccompounds was increased significantly over the extractiontemperature range (25ndash75degC) where themaximumof TPCwasat 65degC [34] In fact heat has been found to enhance therecovery of phenolic compounds [14 35] It was believed thathigh temperature increased and enhanced the diffusion andsolubility rate of phenolic compounds by softening the planttissue as well as disrupting the interactions between thephenolic compounds and protein or polysaccharides [36] It isinteresting to note that in the present study increasing

Table 2 Response surface central composite design (uncoded) and results for total flavonoid content total phenolic content and xanthineoxidase inhibitory activity

Runorder

X1 (extractiontime) (min)

X2 (extractiontemperature) (degC)

X3 (solid-to-liquid ratio)

(gml)

Y1total flavonoidcontent (mg REg)b

Y2 (total phenoliccontent (mg GAEg)c

Y3 (xanthine oxidaseinhibitory activity) ()

1a 325 75 15 5092 13053 95132a 325 75 15 6062 14076 97663a 325 75 15 6054 14063 97284 325 50 15 5078 12076 90075a 325 75 15 6038 14028 96606 325 75 20 6055 15095 84957a 325 75 15 6044 13066 96448 5 50 20 6034 15087 89759 325 75 10 5027 12049 945710 60 50 10 4098 11038 953111 60 50 20 6099 15097 733212 60 100 10 5011 11069 949413 60 75 15 5044 12043 873914 325 100 15 5089 13045 892615 5 100 20 7041 16092 889316 5 75 15 6026 14030 974417 5 100 10 5017 11081 949518 5 50 10 4096 11034 948319 60 100 20 6075 15042 729620a 325 75 15 6042 14066 9419aCenter point bTotal flavonoid content was expressed as mg rutin equivalents in 1 g of dried sample (mg REg) cTotal phenolic content was expressed as mggallic acid equivalents in 1 g of dried sample (mg GAEg)


extraction temperature had a positive effect to TPC From thisscenario it is believed that the phenolic compounds present inE hirta are thermally stable up to 75degC Figure 2(b) shows thatthe TPC increased significantly as the solid-to-liquid ratioincreased from 1 15 to 1 20 with the increase of extractiontime Figure 2(c) also shows that the TPC increased signifi-cantly as the solid-to-liquid ratio increased from 1 15 to 1 20with the increase of extraction temperature up to 75degC Hencefrom these figures the solid-to-liquid ratio could drasticallyaffect the yield of phenolic compounds during the extractionprocess e similar result was observed from the previousstudy where the yield of phenolic compounds from date seedswater extract increased from 25 to 55 g100 g as solvent ratioincreased to the 60 1 level [37] e same results were re-ported for the milled berries extraction where higher solid-to-liquid ratio resulted in higher yield of phenolic compounds[38] is was in line with the mass transfer principle [39]

34 Effect of Extraction Parameters on XO Inhibitory ActivityXO inhibition assay is a common assay used for determiningthe anti-gout activity of the plant extracts [40] XO catalyses

the oxidation of hypoxanthine to xanthine and then to uricacid which plays a crucial role in gout [41] As reviewed byLing and Bochu [42] phenolic and flavonoid compoundshave been reported to be the potent plant-based XO in-hibitors which make them crucial as anti-gout agents Fewstudies from the previous years have successfully optimizedthe extraction conditions for higher XO inhibitory activity ofthe plants [43ndash45] Figures 3(a)ndash3(c) illustrate the responsesurface plots for the influences of extraction temperatureextraction time and solid-to-liquid ratio on XO inhibitoryactivity Table S3 (Supplementary data) shows that all in-dependent variables (extraction time temperature andsolid-to-liquid ratio) had the significant effect on XO in-hibitory activity of E hirta extract Among these extractiontime (X1) and solid-to-liquid ratio (X3) were found to be themajor effects on XO inhibitory activity in comparison withextraction temperature (X2) which may due to the high F-values of 6627 and 23946 respectively Interestingly theanalysis of the results also showed that the interactionsbetween time and solid-to-liquid ratio (X1X3) was highlysignificant factor affecting the XO inhibitory activity(plt 005) As shown in Figure 3(a) XO inhibitory activity

5001875

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56

60

64

Total flavonoidcontent

6555

5111

A extraction time

B extraction temperature

Tota

l flav

onoi

d co

nten

t

(a)

5001875

32504625

6000

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55

5825

615

6475

68


6555

5111

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(b)

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51

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6555

5111

B extraction temperatureC solid-to-liquid ratio

Tota

l flav

onoi

d co

nten

t

(c)

Figure 1 Response surface plots of E hirta showing the effect of (a) extraction time and extraction temperature (b) solid-to-liquid ratio andextraction time (c) solid-to-liquid ratio and extraction temperature on TFC Color gradients indicate the level of optimization (red highgreen intermediate and blue low)


increased when the temperature increased from 50 to 75degCand started to decrease towards 100degC as the extraction timedecreasedis was in agreement with Edirs et al [46] wherethe antidiabetic activity of Kursi Wufarikun Ziyabit usingprotein tyrosine phosphatase-1B and α-glucosidase inhibi-tion assays increased as the extraction temperature increasedfrom 70 to 75degC and 65 to 70degC respectively is explainsthat an increase in the extraction temperature beyondcertain value will denature or decompose some of thevaluable compounds responsible for inhibiting XO activity[47] In fact the high temperature may cause the enzymes tobe denatured as they are heat sensitive [48]

Meanwhile Figure 3(b) clearly shows that the XO in-hibitory activity decreased when the extraction time andsolvent-to-liquid ratio increased e XO inhibitory activityreached at maximum level (9537) when the extractiontime and solvent to liquid ratio were 325min and 1 15 gmlrespectively is implied that excessive extraction time(more than 325min) was no longer useful to extract moreactive compounds from E hirta which contributed toantigout activity In fact oxidation of the active compoundsduring the prolonged time of extraction process could also

reduce the XO activity However lesser contact time duringthe extraction process of the plants would not allow goodmixing between the samples and solvent resulting in low XOinhibitory activity [43] In addition the interaction effectsbetween the extraction temperature and solid-to-liquid ratioon XO inhibitory activity with a constant value of the ex-traction time at 325min are shown in Figure 3(c) It can beseen that solid-to-liquid ratio gave greater impact on the XOinhibitory activity of E hirta regardless of the temperatureused is was in agreement with Mehmood et al [49] whoalso showed that the solute solvent ratio gave a significanteffect on XO inhibition

35 Optimization of Extraction Conditions In this study thedesirability function approach suggested that the optimalextraction conditions for E hirta water extract in obtainingmaximum XO inhibitory activity (9142) TFC (6756mgREg) and TPC (15521mg GAEg) were at 7907degC with1742min and 1 20 of solid-to-liquid ratio e predictedvalues of all the responses were close to the experimentalvalues with less than 10 error which is relatively desirable

5001875

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6000

50006250

75008750

10000

111

1205

130

1395

149

A extraction time


Tota

l phe

nolic

cont

ent

Total phenoliccontent

15295

12034

(a)

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6000


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2000

137

14275

1485

15425

160

Tota

l phe

nolic

cont

ent


15295

12034

(b)

B extraction temperatureC solid-to-liquid ratio5000

62507500

875010000

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2000

120

12925

1385

14775

157

Tota

l phe

nolic

cont

ent


15295

12034

(c)

Figure 2 Response surface plots of E hirta showing the effect of (a) extraction time and extraction temperature (b) solid-to-liquid ratio andextraction time and (c) solid-to-liquid ratio and extraction temperature on total phenolic contents Color gradients indicate the level ofoptimization (red high green intermediate and blue low)


and all the experimental data were within plusmn95 predictionintervals Besides the accuracy of our model was confirmedas no significant difference was observed (pgt 005) betweenthe experimental values and the predicted values (Table 3)erefore it can be concluded that the model from CCDwasaccurate and reliable enough for predicting the TFC andTPC as well as anti-gout activity of E hirta

36 LiquidChromatography-Mass Spectrometry (LC-MSMS)Metabolite Profile of E hirta Extract e E hirta optimizedextracts were analyzed and profiled by LC-MSMS analysis inpositive and negative ionization modes in order to perform aqualitative characterization of chemical constituents in Ehirta To our knowledge this is the first validated method onthe detection of active compounds in E hirta whole plantexcluding roots using LC-MSMS analysis e base peakchromatogram is depicted in Figures 4 (negative ionizationmode) and 5 (positive ionization mode) Compounds werecharacterized for their retention times the absorption spec-trum in the UV-vis region the mass spectrum obtained by

MS-ESI and the fragmentation profile as well as comparedwith the previous literature reports [50ndash53] e results ob-tained from the LC-MSMS analysis allowed the tentativeassignment of nine chemical constituents using negativeionization mode comprising four phenolic acids neo-chlorogenic acid ([M minus H]minus ion at mz 353) caffeic acid([M minus H]minus ion at mz 179034) syringic acid ([M minus H]minus ion atmz 197045) and ellagic acid ([M minus H]minus ion at mz 300999)and four flavonoids quercetin-3β-D-glucoside ([M minus H]minus ionat mz 463087) astragalin ([M minus H]minus ion at mz 447094)afzelin ([M+FA minus H]minus ion at mz 477104) quercetin([M minus H]minus ion at mz 301035) as well as one fatty acidcorchorifatty acid F ([M minus H]minus ion at mz 346081) In ad-dition LC-MSMS analysis also allowed the tentative as-signment of two chemical constituents using positiveionization mode 2-undecanone 24-dinitrophenylhydrazone([M+H]+ ion at mz 3514) and demexiptiline ([M+H]+ ionat mz 2793) In this study the negative mode ESI was moresensitive for the detection of the phenolics and flavonoids inthe extract e same observations were also shown byZakaria et al [24] and Keskes et al [54]

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8925

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9575

99

A extraction time


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

Xanthine oxidaseinhibitory activity

9757

7296

(a)

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78

8325

885

9375

99

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(b)


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

50006250

75008750

10000

10001250

15001750

2000

80

8475

895

9425

99


9757

7296

(c)

Figure 3 Response surface plots of E hirta showing the effect of (a) extraction time and extraction temperature (b) solid-to-liquid ratio andextraction time and (c) solid-to-liquid ratio and extraction temperature on xanthine oxidase inhibitory activity Color gradients indicate thelevel of optimization (red high green intermediate and blue low)


0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

853

069

689112

220

1506

487346728454 509

548 993790647 212916431280259 15371010934 1490 16551125 1858 287114611171 28511084 1703 1969 268525371989 23462189

1

2

3

4

5

6

7

9

8

Figure 4 LC-MS chromatogram acquired in the negative ion mode from an extract of E hirta where peak labelling represents thecompounds identified and listed in Table 4

Table 3 Differences between the predicted value and the experimental value

Parameters Predicted value Experimental value Percentage of error ()Total flavonoid content (mg REg) 6431plusmn 000a 6756plusmn 083a 481Total phenolic content (mg GAEg) 15295plusmn 000b 15521plusmn 067b 146Xanthine oxidase inhibitory activity () 8878plusmn 000c 9142plusmn 074c 289Values were presented as meanplusmn standard deviation Same superscripts within the row indicate no significant difference (pgt 005) between predicted andexperimental values

Table 4 Tentative identification of chemical constituents in the E hirta water extract

No Tentative assignment RT(min)

Molecularweight Precursor type Precursor

mzMolecularformula

MSMSfragmentions

References

1 Neochlorogenic acid 22 35409 [M minus H]minus 353 C16 H18 O9 191 179 Massbank PM013904

2 Caffeic acid 346 18004 [M minus H]minus 1790341 C9 H8 O4135 179

134Massbank

FiehnHILIC001102

3 Syringic acid 548 198052 [M minus H]minus 197045 C9 H10 O5182 166

197Massbank

FiehnHILIC001522

4 Ellagic acid 647 302006 [M minus H]minus 300999 C14 H6 O8 303 300 MassbankFiehnHILIC001170

5 Quercetin-3β-D-glucoside 689 46409 [M minus H]minus 4630879 C21 H20 O12300 301

271 Pubchem (CID5280804)

6 Astragalin 853 4481 [M minus H]minus 447094 C21 H20 O11447 285

284Massbank

CCMSLIB00000845312

7 Afzelin 934 4321 [M+FA minus H]minus 477104 C21 H20 O10285 431

284Massbank

CCMSLIB00000845703

8 Quercetin 1125 30204 [M minus H]minus 3010359 C15 H10 O7

107 121151 178

301

MassbankFiehnHILIC002955

9 Corchorifatty acid F 1506 328224 [M minus H]minus 346081 C18 H32 O5235 311

219Pubchem

(CID44559173)

10 2-Undecanone 24-dinitrophenylhydrazone 1508 3504 [M+H]+ 3514 C17 H26 N4

O4351 352 Pubchem (CID 5717665)

11 Demexiptiline 2716 2783 [M+H]+ 2793 C18 H18 N2O

204 203149 Massbank WA002119


Previous studies have demonstrated that flavonoids andphenolic acids possess high biological and pharmacologicalactivities [55] In the LC chromatogram the peak at re-tention time (RT) 220min was assigned to the presence ofneochlorogenic acid which was also reported by the previousstudies [56 57] In fact neochlorogenic acid showed potentanti-inflammatory activity [58] In this study astragalin(kaempferol-3-O-glucoside) a natural flavonoid was themain compound obtained in E hirta water extract Previousstudy also reported the presence of astragalin in other Eu-phorbia species [59] As reviewed by Riaz et al [60]astragalin showed potent anti-inflammatory activity byinhibiting the production of inflammatorymediators such asinterleukin-1 beta (IL-1β) interleukin-6 (IL-6) and tumornecrosis factor-alpha (TNF-α) in macrophages [61 62]Moreover Jia et al [63] demonstrated for the first time thatastragalin effectively inhibited the worsening of synovialinflammation and joint destruction in the animal model Infact flavonoid compounds from Malaysian medicinal plantsshowed potent anti-gout and anti-inflammatory activitiesfrom the previous studies [64 65] Hence in this presentstudy we speculated that the anti-gout effects of E hirtamight be due to the presence of high concentration ofastragalin in the extract

4 Conclusion

RSM was successfully employed to optimize the phyto-chemical compounds and anti-gout activity of E hirtawhereCCD proved to be an efficient tool for the optimization ofthese parameters e second-order polynomial modelsfor predicting responses were obtained and the best

combination of extraction temperature time and solid-to-liquid ratio were found to be 7907degC with 1742min and 1 20 respectively Further investigation on the secondarymetabolites present in the optimized extract of E hirta usingLC-MS analysis revealed the presence of neochlorogenicacid quercetin-3β-D-glucoside syringic acid caffeic acidellagic acid astragalin afzelin quercetin corchorifatty acidF 2-undecanone 24-dinitrophenylhydrazone and demex-iptiline Hence E hirta could be a natural source of poly-phenols compounds which serve as ingredients of functionalfoods and be used in pharmaceuticals for the treatment ofgout disease

Abbreviations

CCD Central composite designLC-MS Liquid chromatography-mass spectrometryRSM Response surface methodologyTFC Total flavonoid contentTPC Total phenolic contentXO Xanthine oxidase

Data Availability

e datasets generated andor analyzed during the currentstudy are available from the first author on reasonablerequest

Conflicts of Interest

e authors do not have any conflicts of interest regardingthe content of the present work

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

852

688

150 344092336

591 710073

1282 26891508780222 991 2716679486 2131238 1979 2935507 28411051 1434364 1857935 1126 1642 277816731319 2276202019651221 2351 2428 2677

1011

Figure 5 LC-MS chromatogram acquired in the positive ion mode from an extract of E hirta where peak labelling represents thecompounds identified and listed in Table 4


Acknowledgments

is research was financially supported by the MalaysiaMinistry of Education under Fundamental Research GrantScheme (FRGS) (Vot No K099) as well as Universiti TunHussein Onn Malaysia under GPPS scheme (UTHM) (VotNo U758)

Supplementary Materials

Table S1 the regression coefficients and results of ANOVAfor response surface quadratic model of total flavonoidcontent Table S2 the regression coefficients and results ofANOVA for response surface quadratic model of totalphenolic content Table S3 the regression coefficients andresults of ANOVA for xanthine oxidase inhibitory activity(Supplementary Materials)

References

[1] S Kumar R Malhotra and D Kumar ldquoEuphorbia hirta itschemistry traditional and medicinal uses and pharmaco-logical activitiesrdquo Pharmacognosy Reviews vol 4 no 7 p 582010

[2] S Perumal R Mahmud S Pillai W C Lee andS Ramanathan ldquoAntimicrobial activity and cytotoxicityevaluation of Euphorbia hirta (L) extracts from MalaysiardquoAPCBEE Procedia vol 2 pp 80ndash85 2012

[3] J Galvez A Zarzuelo M Crespo M Lorente M Ocete andJ Jimenez ldquoAntidiarrhoeic activity of Euphorbia hirta extractand isolation of an active flavonoid constituentrdquo PlantaMedica vol 59 no 4 pp 333ndash336 1993

[4] N G B Y Fofie R Sanogo K Coulibaly and K B DienebaldquoMinerals salt composition and secondary metabolites ofEuphorbia hirta Linn an antihyperglycemic plantrdquo Phar-macognosy Research vol 7 no 1 p 7 2015

[5] G D Singh P Kaiser M S Youssouf et al ldquoInhibition ofearly and late phase allergic reactions by Euphorbia hirta LrdquoPhytotherapy Research vol 20 no 4 pp 316ndash321 2006

[6] R A Mothana U Lindequist R Gruenert andP J Bednarski ldquoStudies of the in vitro anticancer antimi-crobial and antioxidant potentials of selected Yemeni me-dicinal plants from the island Soqotrardquo BMC Complementaryand Alternative Medicine vol 9 no 1 p 7 2009

[7] A A Basma Z Zakaria L Y Latha and S SasidharanldquoAntioxidant activity and phytochemical screening of themethanol extracts of Euphorbia hirta Lrdquo Asian Pacific Journalof Tropical Medicine vol 4 no 5 pp 386ndash390 2011

[8] Y Liu N Murakami H Ji P Abreu and S Zhang ldquoAnti-malarial flavonol glycosides from Euphorbia hirtardquo Phar-maceutical Biology vol 45 no 4 pp 278ndash281 2007

[9] K N Prasad K W Kong R N Ramanan A Azlan andA Ismail ldquoSelection of experimental domain using two-levelfactorial design to determine extract yield antioxidant ca-pacity phenolics and flavonoids from Mangifera pajangKostermrdquo Separation Science and Technology vol 47 no 16pp 2417ndash2423 2012

[10] L Gao and G Mazza ldquoExtraction of anthocyanin pigmentsfrom purple sunflower hullsrdquo Journal of Food Science vol 61no 3 pp 600ndash603 1996

[11] O Aybastıer E Isık S Sahin and C Demir ldquoOptimization ofultrasonic-assisted extraction of antioxidant compounds from

blackberry leaves using response surface methodologyrdquo In-dustrial Crops and Products vol 44 pp 558ndash565 2013

[12] K N Prasad E Yang C Yi M Zhao and Y Jiang ldquoEffects ofhigh pressure extraction on the extraction yield total phenoliccontent and antioxidant activity of longan fruit pericarprdquoInnovative Food Science amp Emerging Technologies vol 10no 2 pp 155ndash159 2009

[13] T Juntachote E Berghofer S Siebenhandl and F Bauerldquoe antioxidative properties of Holy basil and Galangal incooked ground porkrdquo Meat Science vol 72 no 3 pp 446ndash456 2006

[14] E Silva H Rogez and Y Larondelle ldquoOptimization of ex-traction of phenolics from Inga edulis leaves using responsesurface methodologyrdquo Separation and Purification Technol-ogy vol 55 no 3 pp 381ndash387 2007

[15] N Ilaiyaraja K R Likhith G R Sharath Babu andF Khanum ldquoOptimisation of extraction of bioactive com-pounds from Feronia limonia (wood apple) fruit using re-sponse surface methodology (RSM)rdquo Food Chemistryvol 173 pp 348ndash354 2015

[16] N F Azahar S S A Gani and N F M Mokhtar ldquoOpti-mization of phenolics and flavonoids extraction conditions ofCurcuma Zedoaria leaves using response surface methodol-ogyrdquo Chemistry Central Journal vol 11 no 1 p 96 2017

[17] K-W Kong A R Ismail S-T Tan K M Nagendra Prasadand A Ismail ldquoResponse surface optimisation for the ex-traction of phenolics and flavonoids from a pink guava pureeindustrial by-productrdquo International Journal of Food Scienceamp Technology vol 45 no 8 pp 1739ndash1745 2010

[18] A Alberti A A F Zielinski D M Zardo I M DemiateA Nogueira and L I Mafra ldquoOptimisation of the extractionof phenolic compounds from apples using response surfacemethodologyrdquo Food Chemistry vol 149 pp 151ndash158 2014

[19] N Kapoor and S Saxena ldquoPotential xanthine oxidase in-hibitory activity of endophytic Lasiodiplodia pseudotheo-bromaerdquo Applied Biochemistry and Biotechnology vol 173no 6 pp 1360ndash1374 2014

[20] S H Ali Hassan and M F Abu Bakar ldquoAntioxidative andanticholinesterase activity of Cyphomandra betacea fruitrdquoGeScientific World Journal vol 2013 Article ID 278071 7 pages2013

[21] M F Abu Bakar F Abdul Karim and E Perisamy ldquoCom-parison of phytochemicals and antioxidant properties ofdifferent fruit parts of selected Artocarpus species from SabahMalaysiardquo Sains Malaysiana vol 44 no 3 pp 355ndash363 2015

[22] M Umamaheswari K AsokKumar A SomasundaramT Sivashanmugam V Subhadradevi and T K RavildquoXanthine oxidase inhibitory activity of some Indian medicalplantsrdquo Journal of Ethnopharmacology vol 109 no 3pp 547ndash551 2007

[23] T Unno A Sugimoto and T Kakuda ldquoXanthine oxidaseinhibitors from the leaves of Lagerstroemia speciosa (L)rdquoJournal of Ethnopharmacology vol 93 no 2-3 pp 391ndash3952004

[24] F Zakaria W N W Ibrahim I S Ismail et al ldquoLCMSMSmetabolite profiling and analysis of acute toxicity effect of theethanolic extract of Centella asiatica on zebrafish modelrdquoPertanika Journal of Science amp Technology vol 27 no 2pp 985ndash1003 2019

[25] S Kumar and A K Pandey ldquoChemistry and biological ac-tivities of flavonoids an overviewrdquo Ge Scientific WorldJournal vol 2013 Article ID 162750 16 pages 2013

[26] W Liu Y Yu R Yang C Wan B Xu and S Cao ldquoOpti-mization of total flavonoid compound extraction from


Gynura medica leaf using response surface methodology andchemical composition analysisrdquo International Journal ofMolecular Sciences vol 11 no 11 pp 4750ndash4763 2010

[27] M Bimakr R A Rahman F S Taip et al ldquoComparison ofdifferent extraction methods for the extraction of majorbioactive flavonoid compounds from spearmint (Menthaspicata L) leavesrdquo Food and Bioproducts Processing vol 89no 1 pp 67ndash72 2011

[28] Z-L Sheng P-F Wan C-L Dong and Y-H Li ldquoOptimi-zation of total flavonoids content extracted from Flos Populiusing response surface methodologyrdquo Industrial Crops andProducts vol 43 pp 778ndash786 2013

[29] R-E Ghitescu I Volf C Carausu et al ldquoOptimization ofultrasound-assisted extraction of polyphenols from sprucewood barkrdquo Ultrasonics Sonochemistry vol 22 pp 535ndash5412015

[30] R Randhir Y T Lin and K Shetty ldquoPhenolics their anti-oxidant and antimicrobial activity in dark germinated fenu-greek sprouts in response to peptide and phytochemicalelicitorsrdquo Asia Pacific Journal of Clinical Nutrition vol 13no 3 pp 295ndash307 2004

[31] O R Gottlieb and M R D M Borin ldquoMedicinal productsregulation of biosynthesis in space and timerdquo Memorias doInstituto Oswaldo Cruz vol 95 no 1 pp 115ndash120 2000

[32] G Spigno L Tramelli and D M De Faveri ldquoEffects of ex-traction time temperature and solvent on concentration andantioxidant activity of grape marc phenolicsrdquo Journal of FoodEngineering vol 81 no 1 pp 200ndash208 2007

[33] D R Pompeu E M Silva and H Rogez ldquoOptimisation of thesolvent extraction of phenolic antioxidants from fruits ofEuterpe oleracea using response surface methodologyrdquo Bio-resource Technology vol 100 no 23 pp 6076ndash6082 2009

[34] J Wang B Sun Y Cao Y Tian and X Li ldquoOptimisation ofultrasound-assisted extraction of phenolic compounds fromwheat branrdquo Food Chemistry vol 106 no 2 pp 804ndash8102008

[35] N Durling O Catchpole J Grey et al ldquoExtraction of phe-nolics and essential oil from dried sage (Salvia officinalis)using ethanol-water mixturesrdquo Food Chemistry vol 101no 4 pp 1417ndash1424 2007

[36] J Shi J Yu J Pohorly J C Young M Bryan and Y WuldquoOptimization of the extraction of polyphenols from grapeseedmeal by aqueous ethanol solutionrdquo Food Agriculture andEnvironment vol 1 no 2 pp 42ndash47 2003

[37] M A Al-Farsi and C Y Lee ldquoOptimization of phenolics anddietary fibre extraction from date seedsrdquo Food Chemistryvol 108 no 3 pp 977ndash985 2008

[38] J E Cacace and G Mazza ldquoMass transfer process duringextraction of phenolic compounds from milled berriesrdquoJournal of Food Engineering vol 59 no 4 pp 379ndash389 2003

[39] D Campos R Chirinos O Barreto G Noratto andR Pedreschi ldquoOptimized methodology for the simultaneousextraction of glucosinolates phenolic compounds and anti-oxidant capacity from maca (Lepidium meyenii)rdquo IndustrialCrops and Products vol 49 pp 747ndash754 2013

[40] M T T Nguyen S Awale Y Tezuka Q L TranH Watanabe and S Kadota ldquoXanthine oxidase inhibitoryactivity of Vietnamese medicinal plantsrdquo Biological ampPharmaceutical Bulletin vol 27 no 9 pp 1414ndash1421 2004

[41] L D Kong Y Cai WW Huang C H Cheng and R X TanldquoInhibition of xanthine oxidase by some Chinese medicinalplants used to treat goutrdquo Journal of Ethnopharmacologyvol 73 no 1-2 pp 199ndash207 2000

[42] X Ling and W Bochu ldquoA review of phytotherapy of goutperspective of new pharmacological treatmentsrdquo Die Phar-mazie-An International Journal of Pharmaceutical Sciencesvol 69 no 4 pp 243ndash256 2014

[43] P Jamal S M N Azmi A Amid H M Salleh andY Z H Y Hashim ldquoDevelopment and improvement of anti-gout property from aqueous-methanol extract of Morindaelliptica using central composite designrdquo Advances in Envi-ronmental Biology vol 8 no 3 pp 734ndash744 2014

[44] M Sang G Du J Hao et al ldquoModeling and optimizinginhibitory activities of Nelumbinis folium extract on xanthineoxidase using response surface methodologyrdquo Journal ofPharmaceutical and Biomedical Analysis vol 139 pp 37ndash432017

[45] B Deng Z Liu and Z Zou ldquoOptimization of microwave-assisted extraction saponins from Sapindus mukorossi peri-carps and an evaluation of their inhibitory activity on xan-thine oxidaserdquo Journal of Chemistry vol 2019 Article ID5204534 11 pages 2019

[46] S Edirs A Turak S Numonov X Xin and H A AisaldquoOptimization of extraction process for antidiabetic andantioxidant activities of Kursi Wufarikun Ziyabit using re-sponse surface methodology and quantitative analysis of maincomponentsrdquo Evidence-Based Complementary and Alterna-tive Medicine vol 2017 Article ID 6761719 14 pages 2017

[47] G Spigno and D M De Faveri ldquoAntioxidants from grapestalks and marc influence of extraction procedure on yieldpurity and antioxidant power of the extractsrdquo Journal of FoodEngineering vol 78 no 3 pp 793ndash801 2007

[48] M Amid A Manap M Yazid and N Zohdi ldquoOptimizationof processing parameters for extraction of amylase enzymefrom dragon (Hylocereus polyrhizus) peel using responsesurface methodologyrdquoGe Scientific World Journal vol 2014Article ID 640949 12 pages 2014

[49] A Mehmood L Zhao M Ishaq B Safdar C Wang andM Nadeem ldquoOptimization of total phenolic contents anti-oxidant and in-vitro xanthine oxidase inhibitory activity ofsunflower headrdquo CyTAmdashJournal of Food vol 16 no 1pp 957ndash964 2018

[50] A R M Costa L A P Freitas J Mendiola and E IbantildeezldquoCopaifera langsdorffii supercritical fluid extraction chemicaland functional characterization by LCMS and in vitro as-saysrdquo Ge Journal of Supercritical Fluids vol 100 pp 86ndash962015

[51] Y Lin W Xu M Huang et al ldquoQualitative and quantitativeanalysis of phenolic acids flavonoids and iridoid glycosides inYinhua Kanggan tablet by UPLC-QqQ-MSMSrdquo Moleculesvol 20 no 7 pp 12209ndash12228 2015

[52] M Wang A Cao C Ouyang Y Li and Y Wei ldquoRapidscreening and identification of non-target flavonoid com-ponents in invasive weeds by LCMS-IT-TOFrdquo AnalyticalMethods vol 7 no 24 pp 10207ndash10216 2015

[53] M Zehl C Braunberger J Conrad et al ldquoIdentification andquantification of flavonoids and ellagic acid derivatives intherapeutically important Drosera species by LC-DAD LC-NMR NMR and LC-MSrdquo Analytical and BioanalyticalChemistry vol 400 no 8 pp 2565ndash2576 2011

[54] H Keskes S Belhadj L Jlail et al ldquoLC-MS-MS and GC-MSanalyses of biologically active extracts and fractions fromTunisianJuniperus phoeniceleavesrdquo Pharmaceutical Biologyvol 55 no 1 pp 88ndash95 2017

[55] U P Santos J F Campos H F V Torquato et al ldquoAnti-oxidant antimicrobial and cytotoxic properties as well as the


phenolic content of the extract from Hancornia speciosaGomesrdquo PLoS One vol 11 no 12 Article ID e0167531 2016

[56] T Yoshida L Chen T Shingu and T Okuda ldquoTannins andrelated polyphenols of Euphorbiaceous plants IV Euphor-bins A and B novel dimeric dehydroellagitannins from Eu-phorbia hirta Lrdquo Chemical and Pharmaceutical Bulletinvol 36 no 8 pp 2940ndash2949 1998

[57] M A B Nyeem M S Haque M Akramuzzaman R SiddikaS Sultana and B R Islam ldquoEuphorbia hirta Linn A won-derful miracle plant of mediterranean region a reviewrdquoJournal of Medicinal Plants Studies vol 5 no 3 pp 170ndash1752017

[58] J Zhen T S Villani Y Guo et al ldquoPhytochemistry anti-oxidant capacity total phenolic content and anti-inflamma-tory activity of Hibiscus sabdariffa leavesrdquo Food Chemistryvol 190 pp 673ndash680 2016

[59] I Agata T Hatano Y Nakaya et al ldquoTannins and relatedpolyphenols of euphorbiaceous plants VIII Eumaculin A andeusupinin A and accomapanying polyphenols from Eu-phorbia maculata L and E supina Rafinrdquo Chemical ampPharmaceutical Bulletin vol 39 no 4 pp 881ndash883 1991

[60] A Riaz A Rasul G Hussain et al ldquoAstragalin a bioactivephytochemical with potential therapeutic activitiesrdquoAdvancesin Pharmacological Sciences vol 2018 Article ID 979462515 pages 2018

[61] L W Soromou N Chen L Jiang et al ldquoAstragalin attenuateslipopolysaccharide-induced inflammatory responses bydown-regulating NF-κB signaling pathwayrdquo Biochemical andBiophysical Research Communications vol 419 no 2pp 256ndash261 2012

[62] F Li D Liang Z Yang et al ldquoAstragalin suppresses in-flammatory responses via down-regulation of NF-κB sig-naling pathway in lipopolysaccharide-induced mastitis in amurine modelrdquo International Immunopharmacology vol 17no 2 pp 478ndash482 2013

[63] Q Jia T Wang X Wang et al ldquoAstragalin suppresses in-flammatory responses and bone destruction in mice withcollagen-induced arthritis and in human fibroblast-likesynoviocytesrdquo Frontiers in Pharmacology vol 10 no 94 2019

[64] F I Abu Bakar M F Abu Bakar A Rahmat N AbdullahS F Sabran and S Endrini ldquoAnti-gout potential of Malaysianmedicinal plantsrdquo Frontiers in Pharmacology vol 9 no 2612018

[65] F I Abu Bakar M F Abu Bakar N Abdullah S Endrini andA Rahmat ldquoA review of Malaysian medicinal plants withpotential anti-inflammatory activityrdquo Advances in Pharma-cological Sciences vol 2018 Article ID 8603602 13 pages2018


traditionally to treat worm infestations in children dysen-tery jaundice pimples digestive problems female disordersrespiratory ailments (cough coryza bronchitis andasthma) and tumors [1] In addition the decoction or in-fusion of this plant has been used widely by practitioners oftraditional medicine in treating asthma kidney stonesheartburn diarrhea coughs and menstrual problems [7]Interestingly many phytochemical compounds such as sa-ponin sterol terpene alkaloids polyphenols tannins ter-penoids steroids and flavonoids have been found in theaerial parts of this plant [4 7] is plant is also rich inminerals such as zinc potassium calcium and magnesium[4] In 2007 three compounds from the methanolic extractof E hirta namely afzelin quercetin and myricetin whichshowed potent inhibition against malaria have been suc-cessfully isolated [8]

Extraction is considered to be one of the important stepsin discovering potential bioactive compounds from plantmaterials considering their quantity and type of bioactivecompounds as they are widely used in nutraceuticalpharmaceutical and cosmetic products as well as functionalfood ingredients and food additives [9 10] e type andconcentration of solvent temperature time pH solid-liquidratios pressure and the particle size of the plant have beenidentified from the previous studies as important factorsaffecting the extraction efficiency of the plant bioactivecompounds [11ndash13] Traditional method which is known asthe one-factor-at-a-time has been used widely by manyresearchers in optimizing specific parameters in which onlyone factor plays a role as a variable at a time while keeping allother factors constant [14] However this method is con-sidered to be less reliable as it does not include interactiveeffects among factors time-consuming and expensiveMisleading conclusions and false conditions also might beoccurred at the end of this experiment method ignoring thetrue significant factors during the extraction process [9] Toovercome this problem response surface methodology(RSM) has been introduced and widely used nowadays foroptimizing the extraction conditions RSM is considered tobe a powerful mathematical technique used widely in manyindustries for technological operations in order to optimizecertain experimental conditions Moreover it evaluates theeffects between the multiple factors and their interactionstowards one or more response variables simultaneouslyproducing lesser number of experimental measurements[15] It also helps to locate the region where the extraction isoptimized by using clearer visual aid [16] Based on theprevious literature the phenolic and flavonoid contents ofguava puree [17] Curcuma zedoaria leaves [16] apples [18]Feronia limonia fruit [15] and Mangifera pajang [9] havebeen successfully optimized using RSM

Xanthine oxidase inhibitory activity (XO) is an enzymeassay used widely in determining the anti-gout activity of theplant extracts XO is known as the main enzyme causing theaccumulation of uric acid in the blood which eventuallyleads to gout e mechanism involves where it catalyses theoxidation of hypoxanthine into xanthine and then uric acid[19] Optimization of extraction method to obtain highertotal flavonoids and phenolics as well as XO inhibitory

activity from E hirta is essential for future nutraceuticalproduct development especially for the gout treatment Tothe best of our knowledge this is the first report describingoptimization of extraction process of phytochemical com-pounds and XO inhibitory activity from E hirta ereforethis study aimed at optimizing the extraction temperaturetime and solid-to-liquid ratio for the extraction of totalflavonoid content total phenolic content and anti-goutactivity from E hirta whole plant excluding roots usingRSM

2 Materials and Methods

21 Chemicals and Reagents Gallic acid xanthine xanthineoxidase (buttermilk) and potassium dihydrogen phosphate(KH2PO4) were purchased from Sigma-Aldrich Chemicals(St Louis MO USA) FolinndashCiocalteu reagents were ob-tained from Merck (Germany) Allopurinol rutin alumi-num chloride hexahydrate sodium nitrite sodiumhydroxide and dipotassium hydrogen phosphate (K2HPO4)were of the highest purity (95ndash99)

22 PlantMaterials e plant part used was the whole plantexcluding roots E hirta was collected from the Departmentof Agriculture Serdang Selangor Malaysia and authenti-cated by Dr Mohd Firdaus Bin Ismail (Institute of Bio-science Universiti Putra Malaysia) and the voucherspecimen was deposited in the Herbarium of the same in-stitute under the number MFI008419

23 Sample Extraction e whole plant excluding root wassoaked in water washed and oven-dried at 40degC for 72hours en it was ground and stored in a minus 20degC freezerprior to the extraction process In order to mimic the humanconsumption the plant was prepared using hot boiling waterextraction procedure whereby each 10 g of dried plant wasinfused in 10ndash20ml of distilled water at temperature be-tween 50 and 100degC and continuously stirred for 5ndash60min(Table 1) using a magnetic stirrer under 300 rpm e in-fusion was left to cool to 15min en the extract wasfiltered using Whatman No 1 filter paper and subjected tothe xanthine oxidase inhibitory activity assay spectropho-tometrically at 295 nm for determination of total flavonoidand total phenolic contents

24 Experimental Design Central composite design (CCD)was developed in order to obtain optimized extractionconditions for higher total flavonoid content total phenoliccontent and xanthine oxidase inhibitory activity of E hirtaCCD was chosen in this study as it provides more designpoints on each variable ree selected independent vari-ables in this study were extraction time (X1 5ndash60 minutes)extraction temperature (X2 50ndash100degC) and solid-to-liquidratio (X3 1 10ndash1 20 gml) while the dependent variableschosen were total flavonoid content (Y1) total phenoliccontent (Y2) and xanthine oxidase inhibitory activity (Y3)e responses of total flavonoid content and total phenolic







1113888 1113889 times 100 (1)









YTFC 6046 + 154X1 + 111X2 + 264X3 minus 228X1X2


22 + 284X

23

(2)

YTPC 14228 + 247X1 + 250X2 + 484X3 minus 572X1X2

+ 308X1X3 + 171X2X3 minus 248X21

minus 1724X22 + 888X

23

(3)




22 minus 332X

23

(4)












Runorder




(gml)









5001875

32504625

6000

50006250

75008750

10000

48

52

56

60

64


6555

5111

A extraction time


Tota

l flav

onoi

d co

nten

t

(a)

5001875

32504625

6000

10001250

15001750

2000

55

5825

615

6475

68


6555

5111


Tota

l flav

onoi

d co

nten

t

(b)

50006250

75008750

10000

10001250

15001750

2000

51

55

59

63

67


6555

5111


Tota

l flav

onoi

d co

nten

t

(c)







5001875

32504625

6000

50006250

75008750

10000

111

1205

130

1395

149

A extraction time


Tota

l phe

nolic

cont

ent


15295

12034

(a)

5001875

32504625

6000


10001250

15001750

2000

137

14275

1485

15425

160

Tota

l phe

nolic

cont

ent


15295

12034

(b)


62507500

875010000

10001250

15001750

2000

120

12925

1385

14775

157

Tota

l phe

nolic

cont

ent


15295

12034

(c)






5001875

32504625

6000

50006250

75008750

10000

86

8925

925

9575

99

A extraction time


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(a)

5001875

32504625

6000


10001250

15001750

2000

78

8325

885

9375

99

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(b)


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

50006250

75008750

10000

10001250

15001750

2000

80

8475

895

9425

99


9757

7296

(c)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

853

069

689112

220

1506

487346728454 509

548 993790647 212916431280259 15371010934 1490 16551125 1858 287114611171 28511084 1703 1969 268525371989 23462189

1

2

3

4

5

6

7

9

8







mzMolecularformula

MSMSfragmentions

References



134Massbank

FiehnHILIC001102


197Massbank

FiehnHILIC001522





284Massbank

CCMSLIB00000845312


284Massbank

CCMSLIB00000845703


107 121151 178

301



219Pubchem

(CID44559173)


O4351 352 Pubchem (CID 5717665)


204 203149 Massbank WA002119



4 Conclusion



Abbreviations


Data Availability




0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

852

688

150 344092336

591 710073

1282 26891508780222 991 2716679486 2131238 1979 2935507 28411051 1434364 1857935 1126 1642 277816731319 2276202019651221 2351 2428 2677

1011



Acknowledgments




References













































































1113888 1113889 times 100 (1)









YTFC 6046 + 154X1 + 111X2 + 264X3 minus 228X1X2


22 + 284X

23

(2)

YTPC 14228 + 247X1 + 250X2 + 484X3 minus 572X1X2

+ 308X1X3 + 171X2X3 minus 248X21

minus 1724X22 + 888X

23

(3)




22 minus 332X

23

(4)












Runorder




(gml)









5001875

32504625

6000

50006250

75008750

10000

48

52

56

60

64


6555

5111

A extraction time


Tota

l flav

onoi

d co

nten

t

(a)

5001875

32504625

6000

10001250

15001750

2000

55

5825

615

6475

68


6555

5111


Tota

l flav

onoi

d co

nten

t

(b)

50006250

75008750

10000

10001250

15001750

2000

51

55

59

63

67


6555

5111


Tota

l flav

onoi

d co

nten

t

(c)







5001875

32504625

6000

50006250

75008750

10000

111

1205

130

1395

149

A extraction time


Tota

l phe

nolic

cont

ent


15295

12034

(a)

5001875

32504625

6000


10001250

15001750

2000

137

14275

1485

15425

160

Tota

l phe

nolic

cont

ent


15295

12034

(b)


62507500

875010000

10001250

15001750

2000

120

12925

1385

14775

157

Tota

l phe

nolic

cont

ent


15295

12034

(c)






5001875

32504625

6000

50006250

75008750

10000

86

8925

925

9575

99

A extraction time


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(a)

5001875

32504625

6000


10001250

15001750

2000

78

8325

885

9375

99

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(b)


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

50006250

75008750

10000

10001250

15001750

2000

80

8475

895

9425

99


9757

7296

(c)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

853

069

689112

220

1506

487346728454 509

548 993790647 212916431280259 15371010934 1490 16551125 1858 287114611171 28511084 1703 1969 268525371989 23462189

1

2

3

4

5

6

7

9

8







mzMolecularformula

MSMSfragmentions

References



134Massbank

FiehnHILIC001102


197Massbank

FiehnHILIC001522





284Massbank

CCMSLIB00000845312


284Massbank

CCMSLIB00000845703


107 121151 178

301



219Pubchem

(CID44559173)


O4351 352 Pubchem (CID 5717665)


204 203149 Massbank WA002119



4 Conclusion



Abbreviations


Data Availability




0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

852

688

150 344092336

591 710073

1282 26891508780222 991 2716679486 2131238 1979 2935507 28411051 1434364 1857935 1126 1642 277816731319 2276202019651221 2351 2428 2677

1011



Acknowledgments




References











































































YTFC 6046 + 154X1 + 111X2 + 264X3 minus 228X1X2


22 + 284X

23

(2)

YTPC 14228 + 247X1 + 250X2 + 484X3 minus 572X1X2

+ 308X1X3 + 171X2X3 minus 248X21

minus 1724X22 + 888X

23

(3)




22 minus 332X

23

(4)












Runorder




(gml)









5001875

32504625

6000

50006250

75008750

10000

48

52

56

60

64


6555

5111

A extraction time


Tota

l flav

onoi

d co

nten

t

(a)

5001875

32504625

6000

10001250

15001750

2000

55

5825

615

6475

68


6555

5111


Tota

l flav

onoi

d co

nten

t

(b)

50006250

75008750

10000

10001250

15001750

2000

51

55

59

63

67


6555

5111


Tota

l flav

onoi

d co

nten

t

(c)







5001875

32504625

6000

50006250

75008750

10000

111

1205

130

1395

149

A extraction time


Tota

l phe

nolic

cont

ent


15295

12034

(a)

5001875

32504625

6000


10001250

15001750

2000

137

14275

1485

15425

160

Tota

l phe

nolic

cont

ent


15295

12034

(b)


62507500

875010000

10001250

15001750

2000

120

12925

1385

14775

157

Tota

l phe

nolic

cont

ent


15295

12034

(c)






5001875

32504625

6000

50006250

75008750

10000

86

8925

925

9575

99

A extraction time


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(a)

5001875

32504625

6000


10001250

15001750

2000

78

8325

885

9375

99

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(b)


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

50006250

75008750

10000

10001250

15001750

2000

80

8475

895

9425

99


9757

7296

(c)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

853

069

689112

220

1506

487346728454 509

548 993790647 212916431280259 15371010934 1490 16551125 1858 287114611171 28511084 1703 1969 268525371989 23462189

1

2

3

4

5

6

7

9

8







mzMolecularformula

MSMSfragmentions

References



134Massbank

FiehnHILIC001102


197Massbank

FiehnHILIC001522





284Massbank

CCMSLIB00000845312


284Massbank

CCMSLIB00000845703


107 121151 178

301



219Pubchem

(CID44559173)


O4351 352 Pubchem (CID 5717665)


204 203149 Massbank WA002119



4 Conclusion



Abbreviations


Data Availability




0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

852

688

150 344092336

591 710073

1282 26891508780222 991 2716679486 2131238 1979 2935507 28411051 1434364 1857935 1126 1642 277816731319 2276202019651221 2351 2428 2677

1011



Acknowledgments




References













































































Runorder




(gml)









5001875

32504625

6000

50006250

75008750

10000

48

52

56

60

64


6555

5111

A extraction time


Tota

l flav

onoi

d co

nten

t

(a)

5001875

32504625

6000

10001250

15001750

2000

55

5825

615

6475

68


6555

5111


Tota

l flav

onoi

d co

nten

t

(b)

50006250

75008750

10000

10001250

15001750

2000

51

55

59

63

67


6555

5111


Tota

l flav

onoi

d co

nten

t

(c)







5001875

32504625

6000

50006250

75008750

10000

111

1205

130

1395

149

A extraction time


Tota

l phe

nolic

cont

ent


15295

12034

(a)

5001875

32504625

6000


10001250

15001750

2000

137

14275

1485

15425

160

Tota

l phe

nolic

cont

ent


15295

12034

(b)


62507500

875010000

10001250

15001750

2000

120

12925

1385

14775

157

Tota

l phe

nolic

cont

ent


15295

12034

(c)






5001875

32504625

6000

50006250

75008750

10000

86

8925

925

9575

99

A extraction time


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(a)

5001875

32504625

6000


10001250

15001750

2000

78

8325

885

9375

99

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(b)


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

50006250

75008750

10000

10001250

15001750

2000

80

8475

895

9425

99


9757

7296

(c)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

853

069

689112

220

1506

487346728454 509

548 993790647 212916431280259 15371010934 1490 16551125 1858 287114611171 28511084 1703 1969 268525371989 23462189

1

2

3

4

5

6

7

9

8







mzMolecularformula

MSMSfragmentions

References



134Massbank

FiehnHILIC001102


197Massbank

FiehnHILIC001522





284Massbank

CCMSLIB00000845312


284Massbank

CCMSLIB00000845703


107 121151 178

301



219Pubchem

(CID44559173)


O4351 352 Pubchem (CID 5717665)


204 203149 Massbank WA002119



4 Conclusion



Abbreviations


Data Availability




0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

852

688

150 344092336

591 710073

1282 26891508780222 991 2716679486 2131238 1979 2935507 28411051 1434364 1857935 1126 1642 277816731319 2276202019651221 2351 2428 2677

1011



Acknowledgments




References











































































5001875

32504625

6000

50006250

75008750

10000

48

52

56

60

64


6555

5111

A extraction time


Tota

l flav

onoi

d co

nten

t

(a)

5001875

32504625

6000

10001250

15001750

2000

55

5825

615

6475

68


6555

5111


Tota

l flav

onoi

d co

nten

t

(b)

50006250

75008750

10000

10001250

15001750

2000

51

55

59

63

67


6555

5111


Tota

l flav

onoi

d co

nten

t

(c)







5001875

32504625

6000

50006250

75008750

10000

111

1205

130

1395

149

A extraction time


Tota

l phe

nolic

cont

ent


15295

12034

(a)

5001875

32504625

6000


10001250

15001750

2000

137

14275

1485

15425

160

Tota

l phe

nolic

cont

ent


15295

12034

(b)


62507500

875010000

10001250

15001750

2000

120

12925

1385

14775

157

Tota

l phe

nolic

cont

ent


15295

12034

(c)






5001875

32504625

6000

50006250

75008750

10000

86

8925

925

9575

99

A extraction time


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(a)

5001875

32504625

6000


10001250

15001750

2000

78

8325

885

9375

99

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(b)


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

50006250

75008750

10000

10001250

15001750

2000

80

8475

895

9425

99


9757

7296

(c)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

853

069

689112

220

1506

487346728454 509

548 993790647 212916431280259 15371010934 1490 16551125 1858 287114611171 28511084 1703 1969 268525371989 23462189

1

2

3

4

5

6

7

9

8







mzMolecularformula

MSMSfragmentions

References



134Massbank

FiehnHILIC001102


197Massbank

FiehnHILIC001522





284Massbank

CCMSLIB00000845312


284Massbank

CCMSLIB00000845703


107 121151 178

301



219Pubchem

(CID44559173)


O4351 352 Pubchem (CID 5717665)


204 203149 Massbank WA002119



4 Conclusion



Abbreviations


Data Availability




0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

852

688

150 344092336

591 710073

1282 26891508780222 991 2716679486 2131238 1979 2935507 28411051 1434364 1857935 1126 1642 277816731319 2276202019651221 2351 2428 2677

1011



Acknowledgments




References












































































5001875

32504625

6000

50006250

75008750

10000

111

1205

130

1395

149

A extraction time


Tota

l phe

nolic

cont

ent


15295

12034

(a)

5001875

32504625

6000


10001250

15001750

2000

137

14275

1485

15425

160

Tota

l phe

nolic

cont

ent


15295

12034

(b)


62507500

875010000

10001250

15001750

2000

120

12925

1385

14775

157

Tota

l phe

nolic

cont

ent


15295

12034

(c)






5001875

32504625

6000

50006250

75008750

10000

86

8925

925

9575

99

A extraction time


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(a)

5001875

32504625

6000


10001250

15001750

2000

78

8325

885

9375

99

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(b)


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

50006250

75008750

10000

10001250

15001750

2000

80

8475

895

9425

99


9757

7296

(c)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

853

069

689112

220

1506

487346728454 509

548 993790647 212916431280259 15371010934 1490 16551125 1858 287114611171 28511084 1703 1969 268525371989 23462189

1

2

3

4

5

6

7

9

8







mzMolecularformula

MSMSfragmentions

References



134Massbank

FiehnHILIC001102


197Massbank

FiehnHILIC001522





284Massbank

CCMSLIB00000845312


284Massbank

CCMSLIB00000845703


107 121151 178

301



219Pubchem

(CID44559173)


O4351 352 Pubchem (CID 5717665)


204 203149 Massbank WA002119



4 Conclusion



Abbreviations


Data Availability




0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

852

688

150 344092336

591 710073

1282 26891508780222 991 2716679486 2131238 1979 2935507 28411051 1434364 1857935 1126 1642 277816731319 2276202019651221 2351 2428 2677

1011



Acknowledgments




References











































































5001875

32504625

6000

50006250

75008750

10000

86

8925

925

9575

99

A extraction time


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(a)

5001875

32504625

6000


10001250

15001750

2000

78

8325

885

9375

99

Xant

hine

oxi

dase

inhi

bito

ry ac

tivity


9757

7296

(b)


Xant

hine

oxi

dase

inhi

bito

ry ac

tivity

50006250

75008750

10000

10001250

15001750

2000

80

8475

895

9425

99


9757

7296

(c)



0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

853

069

689112

220

1506

487346728454 509

548 993790647 212916431280259 15371010934 1490 16551125 1858 287114611171 28511084 1703 1969 268525371989 23462189

1

2

3

4

5

6

7

9

8







mzMolecularformula

MSMSfragmentions

References



134Massbank

FiehnHILIC001102


197Massbank

FiehnHILIC001522





284Massbank

CCMSLIB00000845312


284Massbank

CCMSLIB00000845703


107 121151 178

301



219Pubchem

(CID44559173)


O4351 352 Pubchem (CID 5717665)


204 203149 Massbank WA002119



4 Conclusion



Abbreviations


Data Availability




0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

852

688

150 344092336

591 710073

1282 26891508780222 991 2716679486 2131238 1979 2935507 28411051 1434364 1857935 1126 1642 277816731319 2276202019651221 2351 2428 2677

1011



Acknowledgments




References








































































0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

853

069

689112

220

1506

487346728454 509

548 993790647 212916431280259 15371010934 1490 16551125 1858 287114611171 28511084 1703 1969 268525371989 23462189

1

2

3

4

5

6

7

9

8







mzMolecularformula

MSMSfragmentions

References



134Massbank

FiehnHILIC001102


197Massbank

FiehnHILIC001522





284Massbank

CCMSLIB00000845312


284Massbank

CCMSLIB00000845703


107 121151 178

301



219Pubchem

(CID44559173)


O4351 352 Pubchem (CID 5717665)


204 203149 Massbank WA002119



4 Conclusion



Abbreviations


Data Availability




0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

852

688

150 344092336

591 710073

1282 26891508780222 991 2716679486 2131238 1979 2935507 28411051 1434364 1857935 1126 1642 277816731319 2276202019651221 2351 2428 2677

1011



Acknowledgments




References









































































4 Conclusion



Abbreviations


Data Availability




0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Rela

tive a

bund

ance

852

688

150 344092336

591 710073

1282 26891508780222 991 2716679486 2131238 1979 2935507 28411051 1434364 1857935 1126 1642 277816731319 2276202019651221 2351 2428 2677

1011



Acknowledgments




References








































































Acknowledgments




References








































































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