Research Article Phytochemical Content and Pharma...

Research ArticlePhytochemical Content and Pharma-Nutrition Study onEleutherococcus senticosus Fruits Intractum

Daniel ZaBuski1 Marta Olech2 Agnieszka Galanty3 Robert Verpoorte4 RafaB Kufniewski1

Renata Nowak2 and Anna Bogucka-Kocka5

1Department of Pharmacognosy Ludwik Rydygier Collegium Medicum Nicolaus Copernicus University9 Marie Curie-Skłodowska Street 85-094 Bydgoszcz Poland2Department of Pharmaceutical Botany Medical University of Lublin 1 Chodzki Street 20-093 Lublin Poland3Department of Pharmacognosy Collegium Medicum Jagiellonian University 9 Medyczna Street 30-688 Cracow Poland4Natural Products Laboratory Institute of Biology Leiden University 2300 RA Leiden Netherlands5Department of Biology and Genetics Medical University of Lublin 4a Chodzki Street 20-093 Lublin Poland

Correspondence should be addressed to Daniel Załuski daniel zaluskioneteu

Received 12 July 2016 Revised 31 August 2016 Accepted 18 September 2016

Academic Editor Ilaria Peluso

Copyright copy 2016 Daniel Załuski et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In the past two decades public interest in herbal products has increased significantly in Europe especially in the plant-basedproducts from non-European traditions Eleutherococcus senticosus has been used for the treatment of inflammatory diseasesanemia and rheumatoid arthritis The Eleutherococcus senticosus fruits intractum was examined for the content of phenolic acids(LC-ESI-MSMS) minerals (AAS) TPC and TFC (spectrophotometric assay) The antioxidant activity was determined using freeradical scavenging assay and TLC-DB-DPPHlowast dot-blot test An anti-Hyal activity was evaluated by the spectrophotometric assaymethod Cytotoxicity towards HL-60 HL-60MX1 HL-60MX2 CEMC1 and CCRFCEM leukemic cell lines was done usingtrypan blue test Among eight phenolic acids trans-caffeic acid was found in the largest amount (412mggDE) The intractumpresented a high amount ofmacroelements (CaMg K 1750 1300 and 21000mgkg) andmicroelements (FeMn 327 543mgkg)respectively The content of TPC and TFC was 130 and 92mgg DE respectively The intractum showed anti-Hyal activity (216ndash60) and an antioxidant capacity (EC

50 52 120583gmL)The intractummost strongly inhibited the growth of HL-60 HL-60MX1 and

CCRFCEM A better understanding of the intractum health benefits is important in order to increase its utility and enrich dietarysources of health promoting compounds

1 Introduction

Plant-based products have been used to manage variousailments for the centuries in different ethnic communities ofthe world Nowadays such products are used in developedand developing countries separately andor together withsynthetic drugs It is established that about 120 plant derivedcompounds are used in western medicine and about 80 ofthe world population uses medicinal plants in primary healthcare [1]

Eleutherococcus senticosus (Rupr et Maxim) Harmscalled Siberian ginseng is a medicinal plant with a longhistory of use (by the Chinese for over 2000 years) Theplant has been recognized as an adaptogen similarly to Panax

ginseng [C A Meyer (Araliaceae)] Schisandra chinensis[Turcz Baill (Schisandraceae)] orAralia mandshurica [Rupret Maxim (Araliaceae)]

In the Chinese and Russian ethnomedicine its use wasempirical because people used to believe that it was a panaceathat promoted longevity with beneficial effects for the treat-ment of physical fatiguesThe fruits have been used for a longtime as an ingredient of the fermented wine the leaves as atonic as a functional beverage marketed for reducing liverdamage and accelerating alcohol detoxification [2ndash5]

At present in China the ethanol extract of the rootsis a popular health supplement for weakness diseases con-nected with inflammation (rheumatism haemorrhoids) andimpotence It was reported that in the Olympic Games

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 9270691 10 pageshttpdxdoiorg10115520169270691

2 Oxidative Medicine and Cellular Longevity

the players of the Old Soviet Union have increased recordsafter administering the E senticosus roots products [6 7]According to Załuskirsquos previous studies the fruits of thatspecies cultivated in Poland act as antioxidants inductorsof the apoptosis in Jurkat 45 and HL60 leukemic cell linesand inhibitors of MMP-1 MMP-2 MMP-3 and MMP-9[8 9]

The Eleutherococcus senticosus products attract globalattention as a novel medicinal plant and since a few yearshave become popular as dietary supplement in the UnitedStates and European countries Imported products of thisplant have become available in North America with amarketshare of 31 of the $12 billion medicinal herbal industryThe 1994DSHEA (Dietary SupplementHealth and EducationAct) regulation allows a direct commercialization of Esenticosus as a supplement for consumption in the UnitedStates without the regulation of the FDA (Food and DrugAdministration)With the increased awareness of developingcountries that the study of traditional medicines and findingnew leads is important there is a need to avoid importedexpensive eastern medicines and to estimate a new sourceof some eastern herbs in Europe Preparations of the rootsof E senticosus are given in cases of asthenia with weaknessand fatigue for example in convalescence This indicationhas been officially accepted by the Community HerbalMonograph on Eleutherococcus senticosus (Rupr et Maxim)Maxim Radix (EMEAHMPC2445692006) published bythe European Medicines Agency The clinical applicationof E senticosus is generally considered safe however theEuropean Community Herbal Monograph states ldquoarterialhypertensionrdquo as a contraindication [5ndash7]

The roots of E senticosus are source of phenols calledeleutherosides (derivatives of lignans coumarins and phe-nylpropanoids) flavonoids (hyperin rutin afzelin quercetinand kaempferol) phenolic acids triterpenic acids and antho-cyanins (Figure 1) Compounds isolated from the fruitsbelong to eleutherosides (eleutherosides B and E) flavonoidsphenolic acids and essential oil (03 vdw) The driedfruits consumed as food are rich in Ca Mg Mn Zn andCu In the leaves flavonoids (quercetin quercitrin and rutin)have been identified [8ndash11]

Eleutherococcus senticosus is an example of one suchspecies whose activity and chemistry are yet to be studiedin more detail especially the species harvested in differentplaces The Eleutherococcus senticosus products which areavailable in the herbal drugs market are imported fromChina Because of a lack of the assessment of plant materialthere have been many cases of the poor quality of plantssupplied by Chinese traders leading to financial lossesfor some pharmaceutical companies For example 26 ofproducts prepared among others from E senticosus did notmeet label claims with respect to the claimed eleuthero-sides content [12] To avoid that the establishment of thenew source of important medicinal plants in Europe isrequired As a major part of western medicine has beendeveloped from traditional knowledge it makes sense tofirst of all look to our ancestorsrsquo knowledge and study thatwith all the novel concepts and source of the plant mate-rial

The chemical compounds and biological activity of plantsdepend on the geographical zone of the growth This speciesis successfully cultivated at the botanical garden in Rogowwhich lies in the Central Polish Lowlands region with geo-graphic data such as 51∘ 491015840N and 19∘ 531015840EThe average long-term temperature is minus201∘C what classified the garden to the6bth subclimate (according to USDA Frost Hardiness Zones)and to the second zone according to the Kornikrsquos categoryThese plants are grown on the acidic luvic and sandy soils[13]

In view of several ethnopharmacological medicinal andnutritional value of the fruits described in literature and onthe basis of Załuskirsquos previous studies it was decided to exam-ine the fruits intractum for its phytochemicals and bioactivityMany products which are widely available in the health foodor herbal drugs markets are in the form of capsule powderor tea bag So far the intractum is not available on marketAs part of a program to search for bioactive constituentsfrom Eleutherococcus species this study was focused on theestablishment of phenolic compounds (phenolic acids TPCand TFC) minerals content and anti-Hyal anti-DPPHlowast andcytotoxic properties of the intractumprepared from the fruitsharvested in Poland

2 Materials and Methods

21 Standards and Reagents Folin-Ciocalteu reagent DPPHascorbic acid DMSO bovine albumin hyaluronidase frombovine testes type I-S Streptococcus equi hyaluronic acidand sodium phosphate buffer pH 70 were obtained fromSigma-Aldrich DNPH and FeCl

3and ethanol were obtained

from POCH (Lublin Poland) The acetate buffer pH 45was purchased from J T Baker USA The standards ofphenolic acids flavonoids and aescin were obtained fromChromaDex (SantaAna CA) Liquid chromatography- (LC-)grade methanol (MeOH) and acetonitrile (ACN) were pur-chased from Merck (Darmstadt Germany) LC-grade waterwas prepared using a Milli-Q purification system (MilliporeBedford MA USA) All others reagents were of analyticalgrade

22 Plant Material The fruits of E senticosus (Rupr etMaxim) Maxim were collected at the arboretum in Rogow(Poland) in October 2015 (voucher specimen number ES0914F) The sample was deposited at the Department of Phar-macognosy CollegiumMedicum Bydgoszcz Poland Imme-diately after harvesting the intractum was prepared

23 Preparation of Intractum 20 g of fresh fruits was macer-ated in 100mL 40 ethanol for 30 days at room temperatureAfter that the extract was filtered throughWhatman number4 filter paperThe solvent was dried with an evaporator undervacuum conditions at 45∘C and the residue was subjectedto lyophilisation The extraction yield was calculated basedon the dry weight of the extract and was expressed as apercentage calculated according to the formula

Yield = extract weightsample weight

times 100 (1)

Oxidative Medicine and Cellular Longevity 3

O

OO

O

O

O

(+)-Sesamin

OHO

OH

HO OH

O

OO

OH

HH

Eleutheroside B

OHO

OH

HO OH

OO

O

O O

H

H

OO

O

OHO

HO

HOOH

Eleutheroside E

OHO

OH

HO OH

OO

O

O O

H

H

OO

HO

O

O

HO

O

O

Isofraxidin

HO

H

H

OH

OH

Oleanolic acid

HO

HCOOH

H

Ursolic acid

Eleutheroside E1

Figure 1 The chemical structures of main E senticosus compounds [9]

24 Acidic Hydrolysis of Intractum as Sample Preparationbefore in LCMS Analysis 1 g of intractum was hydrolysedin 1mL of H

2SO4in 20mL of 20 aqueous methanol After

refluxing at 80∘C for 2 h the extract was allowed to cooland dissolved in 20mL of dichloromethane (left for 24 hroom temperature) Next the liquid-liquid extraction wasperformed using 5times20mL of ethyl acetate Dichloromethane

and ethyl acetate layers were evaporated and the residueswere used in LCMS analysis

25 Total Phenolic Content (TPC) The total phenolic contentof extracts was determined using themethod of Singleton andRossi [14] Gallic acid was used to calculate the calibrationcurve (20ndash100 120583gmL 119910 = 00026119909 + 0044 1199032 = 0999)


and TPC was expressed as gallic acid equivalents (GAEmL)as well as per typical administered dose (ie per 25mLintractum dose) The experiments were done in triplicate

26 Total Flavonoid Content (TFC) The TFC in investigatedsamples was determined using FeCl

3andDNPHcolorimetric

methods [15] TFC were expressed as means (plusmnSE) mgof quercetin equivalent (QEsmL for FeCl

3method 20ndash

100 120583gmL 119910 = 00041119909 + 0236 1199032 = 0999) and as means(plusmnSE) mg of hesperetin equivalent (HEsmL for DNPHmethod 250ndash1000120583gmL 119910 = 6374119909 minus 0098 1199032 = 0988)as well as per typical administered dose (ie per 25mLintractum dose) The experiments were done in triplicate

27 LC-ESI-MSMS Conditions of Analysis of Phenolic AcidsThe samples were analyzed according to modified methodof Nowacka et al [16] For this purpose an Agilent 1200Series HPLC system (Agilent Technologies USA) equippedwith a binary gradient solvent pump a degasser an autosam-pler and column oven connected to a 3200 QTRAP MassSpectrometer (AB Sciex USA) equippedwith an electrosprayionisation source (ESI) and a triple quadrupole-ion trapmassanalyzer was used The separation of the analytes was carriedout on a Zorbax SB-C18 column (21 times 50mm 18 120583mparticlesize Agilent Technologies USA) maintained at 25∘C using3 120583L injectionsThe solvents used were water containing 01HCOOH(solventA) andmethanol containing 01HCOOH(solvent B) The following gradient elution program at a flowrate of 370 120583Lminminus1was applied 0-1min 5B 2ndash4min 20B 8ndash95min 70 B 115ndash155min 5 B Mass spectrometerwas controlled by the Analyst 15 software ESI worked in thenegative-ion mode with the curtain nebulizer and turbo-gas(all nitrogen) set at 30 60 and 60 psi respectively The ionspray needle voltage was minus4500V and capillary temperature400∘C For each compound the optimum conditions of Mul-tiple Reaction Mode (MRM) were determined in the directinfusion mode Triplicate injections were made for eachstandard solution and sample The analytes were identifiedby comparing retention time and 119898119911 values obtained byMS and MS2 with the mass spectra from correspondingstandards tested under the same conditions The calibrationcurves obtained in MRM mode were used for quantificationof all analytes The identified phenolic acids were quantifiedon the basis of their peak areas and comparison with acalibration curve obtained with the corresponding standardsLinearity ranges for calibration curves were specified Thelimits of detection (LOD) and quantification (LOQ) forphenolic compounds were determined at a signal-to-noiseratio of 3 1 and 10 1 respectively by injecting a series ofdilute solutions with known concentrations Detailed LC-ESI-MSMSmethods parameters are given in SupplementaryMaterial (see Supplementary Material available online athttpdxdoiorg10115520169270691)

28 AAS of Minerals 05 g of the lyophilised intractum wasput into a burning cup and 2mL of pure HNO

3was added

The samples were incinerated in a MARS 5 microwave oven(Manufactured by CEM corporation USA) at a temperatureof 90∘C for 15min next 120∘C for 10min and 210∘C for

30min and the solution was diluted to 100mL with waterConcentrations were determined with an Varian SpektrAA280FS +Autosampler SPS 3 SpectrometerMinerals and traceelements were determined using the instrumental conditionsrecommended for eachmineral and were calculated based onthe respective standard curve

29 Antihyaluronidase Studies The ability of the extractsto inhibit Hyal was determined by the spectrophotometricmethod of Yahaya et al [17] The intractum concentrationwas 10mgmL in 10 water ethanol solution The finalconcentration in the reactionrsquos mixture was 22120583g0161mLThe activity was determine on the basis of precipitation ofundigested HA with albumin 50120583L of enzyme in acetatebuffer pH 45 50 120583L of sodium phosphate buffer (50mM pH70 with 77mM NaCl and 1mgmL of albumin) and 22 120583Lof the analyzed samples were combined All the reactionmixtures were incubated at 37∘C for 10min Next 50120583Lof HA (03mgmL of acetate buffer pH 45) was addedand incubated at 37∘C for 45min The undigested HA wasprecipitated with 1mL acid albumin solution made up 01bovine serum albumin in 24mM sodium acetate and 85mMacetic acid The mixture was kept at room temperature for10min the absorbance of the reactionmixture wasmeasuredat 600 nm (Multidetection Microplate Reader SynergyTMHT BioTek) Aescin was used as the positive control atthe following concentrations 005 01 02 04 06 and08mg0161mL the absorbance in the absence of enzymewas used as the blind control All assays were done intriplicate The percentage of inhibition was calculated as

inhibition = [(AB minus AE)(AS minus AE) ] times 100 (2)

where AB is absorbance of the enzyme + substrate + extractAE is absorbance of the enzyme + substrate sample AS isabsorbance of the enzyme + substance sample

210 Cytotoxic Activity Leukemic cells HL-60-human Cau-casian promyelocytic leukemia from American Type Cul-ture Collection (ATCC CCL-240) HL-60MX1-humanCaucasian acute promyelocytic leukemia from AmericanType Culture Collection (ATCC CRL-2258) and HL60MX2-human Caucasian acute promyelocytic leukemia fromAmerican Type Culture Collection (ATCC CRL-2257)CEMC1-human Caucasian acute lymphoblastic leukemiafrom American Type Culture Collection (ATCC CRL-2265) and CCRFCEM-human Caucasian acute lym-phoblastic leukemia from American Type Culture Collection(ATCC CCL-119) were incubated at the concentration of5 times 105 cellsmL in 5 CO

2atmosphere for 24 h at 37∘C

An RPMI 1640 medium (Sigma St Luis USA) with 15fetal bovine serum (Sigma) 2mM L-glutamine and antibi-otics [100UmL penicillin 100 120583MmL streptomycin and25 120583gmL amphotericin B (Gibco Carlsbad USA)] served asa growing medium

The in vitro cytotoxicity assay was carried out usingtrypan blue assay The cell lines at concentration 5 times 105cellsmL were treated with different concentrations of testingintractum and incubated for 24 h at 37∘C in air atmosphere


humidified by 5 CO2 At the end of this period the

mediumwas removed fromeach plate by aspirationNext thecells were washed with PBS and centrifuged at 800 rpm for10min and then PBS was removed by aspiration Then 10120583Lsuspension cells were incubated for 5minwith the 10 120583L 04trypan blue solution (Bio-Rad Laboratories Inc HerculesCA) The samples were analyzed using an Olympus BX41light microscope The cells were stimulated with the ethanolextracts from the roots and leaves dissolved in DMSO at thefinal concentration from 10 to 100120583gmL of cell culture Thefinal concentration of DMSO in incubating mixture was 1The experiments were done in triplicate As a positive controlpodophyllotoxin was used

211 DPPH Assay The ability to scavenge DPPHlowast free rad-icals was determined by the modified method of Brand-Williams et al [18] The methanol solutions of the extractsat the following concentrations 01 05 10 15mgmL wereused Ascorbic acid and tocopherol were used as positivecontrol (125 25 50 75120583gmL) Absorbance was measuredon a Multidetection Microplate Reader SynergyTM HTBioTek The experiments were done in triplicate The sampleconcentrations providing 50 of scavenging (EC

50) were

calculated from the graph plotted between the percentages ofscavenging and the sample concentrations

212 TLC-DB-DPPH Dot-Blot Test (TLC-Direct Bioautogra-phy Dot-Blot Test) The TLC-DPPH dot-blot test was used1 120583L of extracts (10 120583g120583L) was applied on silica gel plates andthe plate was immersed for 5 s in freshly prepared 02mmolmethanolic DPPHlowast solution After removing DPPHlowast excessthe decolourization of DPPHlowast was observed after 1 5 10 and30min

213 Statistical Analysis All determination was done intriplicateThe obtained data were subjected to statistical anal-ysis using Statistica 70 (StatSoft Cracow) The evaluationswere analyzed for one-factor variance analysis Statisticaldifferences between the treatment groups were estimated bySpearmanrsquos (119877) and Personrsquos (119903) test All statistical tests werecarried out at significance level of 120572 = 005

3 Results and Discussion

31 TPC and TFC The literature regarding the pharma-ceutical and dietary products of Eleutherococcus senticosusis scare and this study may contribute to confirming theethnopharmacological use of this plantThe extraction of thefruits resulted in 451 dry extract yield After lyophilisationthe intractumwas a dark red paste with a characteristic smelland with a very sweet taste Taking into consideration variouspreparations in liquid or solid dosage forms for oral use wecalculated the amount of TPC and TFC as mgmL mgperserving and mgg exc The results revealed a significantlyhigh amount of TPC andTFC (Table 1) It is worth noting thatthe intractumcontainsmore flavanones and dihydroflavonols(735mgg DE) the compounds whose occurrence in natureis limited than flavoneflavonols (185mgg DE)

Table 1 TPC and TFC in intractum from the fruits of E senticosus(mg GAEmL QEsmL HEsmL mgper serving and mgg dryextract) One serving = 25mL for intractum

TPC Flavonoid content TFCFeCl3

DNPHmgmLmgperservingmgg excplusmnSD

mgmLmgperservingmgg excplusmnSD



26 plusmn 011 037 plusmn 012 147 plusmn 012 184 plusmn 01265 plusmn 012 925 plusmn 012 3675 plusmn 012 46 plusmn 012130 plusmn 055 185 plusmn 023 735 plusmn 034 92 plusmn 036The data are represented as the mean (plusmnSD) of three independent measure-ments

The TPC and TFC were higher than those previouslyreported for the various Eleutherococcus species cultivatedin Poland Załuski et al [8] reported on the TPC in the 75ethanolic extracts from the spring leaves (203 to 372mgg)fresh fruits (61ndash197mgg) and roots (69ndash106mgg) Inaddition to this the content of TPC and TFC in the fruits isnot changed during a storage at room temperature After 1-year storage the amount did not change significantly and wasbetween 385 and 413 g100 g for E senticosus and E henryicompared to the freshly dried fruits (411 to 435 g100 g)[19] Heo et al [20] studied the ethanol methanol and waterextracts of the E senticosus fruits growing in Korea butreported a lower concentration of polyphenols and flavonoidsthan that now estimated (03 06 06 and 020 023 and03 resp) which is also in agreement with the studies ofShohael et al [21] According to Jang et al [22] the fruits ofE senticosus collected in Korea have contained the TPC andTFC in the range of 1979ndash3343mgg and 412ndash2037mgg

Our findings revealed that the intractum contains morethe TPC than the blueberries or rose fruits which in Polandor other European countries are very widely used in foodproducts for medical purposes and are recognized as arich source of polyphenols According to Grace et al [23]blueberries contained from 227 to 393mgg extract ofpolyphenols Nowak et al [24] found that tincture of therosehip contains 118mgmL

The high content of polyphenols and flavonoids mayresult from the polarity of ethanol that can penetrate thecellular membrane and dissolve the intracellular constituentsin the plant cells Another factormay be time of the intractumpreparing 30 days of storage

32 LC-ESI-MSMS of Phenolic Acids Content The differ-ent types of compounds present in the intractum and theobtained chromatogram are presented in Figure 2 Com-pounds were identified by comparison of retention times andmass fragmentation pattern with data obtained for commer-cial standards and molecular masses were clearly recognizedfrom the negative ESI-MS spectra The obtained results arepresented in Supplementary Material (Tables S1 and S2)


Time (min)

1

2

3 4

5

6

00

50000

10e4

15e4

20e4

25e4

30e4

35e4

40e4

45e4

50e4

55e4

60e4

65e4

857565554535251505

017027

088118

162 244 282

326

348382

402437 490

500512

575

588

629

652 688714 754783

823843 891

448

40 50

Time (min)

XIC of -MRM (27 pairs) 1368929 Da ID sample 1

Max 41e4 cps

Figure 2 Chromatogram of phenolic acids (acquired in MRMmode) found in the intractum Monitored MRM transitions aregiven in brackets 1 protocatechuic acid (119898119911 1529rarr1078) 24-hydroxybenzoic acid (119898119911 1368rarr929) 3 vanillic acid (1198981199111668rarr1079) 4 trans-caffeic acid (119898119911 1787rarr1349) 5 trans-p-coumaric acid (119898119911 1627rarr119) and 6 trans-ferulic acid (1198981199111928rarr1779) Unnumbered peaks represent uncharacterized con-stituents Please refer to Supplementary Material for details

Among eighteen phenolic acids (gallic protocatechuic gen-tisic 4-OH-benzoic 3-OH-benzoic vanillic trans-caffeiccis-caffeic syringic trans-p-coumaric trans-ferulic veratricsalicylic 3-OH-cinnamic trans-sinapic and cis-sinapic) justeight were found and quantitatively determined The con-centrations of individual compounds which were quantifiedby comparison of peak areas with the calibration curvesobtained for the corresponding standards are reported inTable 2 To the best of our knowledge there are no studiesinvestigating the profiles of phenolic acids in the intractumThe findings revealed the presence of benzoic and cinnamicacid derivatives among them a majority is present in aglycosidic form The raw intractum was found to be rich invanillic and trans-caffeic acids (42 and 412mgg DE resp)It also contained significant amount of trans-ferulic trans-p-coumaric and 4-OH-benzoic acid Analysis of hydrolysatesprovided additional information about composition of fruitintractum Vanillic acid was the most abundant one indichloromethane and ethyl acetate layer Moreover largequantities of 4-OH-benzoic acid (70 and 74mgg DE resp)were found

Data in the literature indicated that Kurkin et al [25]identified free phenolic acids (syringic p-coumaric vanillicp-hydroxybenzoic caffeic and ferulic acids) and depside(chlorogenic acid) in the roots of E senticosus growingin Russia In turn Li et al [26] identified protocatechuicchlorogenic and caffeic acids in the roots of Chinese sampleBączek identified rosmarinic chlorogenic ferulic and caffeicacids in the roots fruits and stem barks of six species [27 28]It is worth noting that the intractum contains trans-caffeicacid whose activity is beneficial for healthThat compound is

Table 2 Concentrations of phenolic acids in the raw intractum andafter acidic hydrolysis (mgg DE)

Compound Rawintractum

Ethyl acetatelayer

Dichloromethanelayer

Protocatechuic 045 plusmn 21 010 plusmn 42 01 plusmn 21Gentisic mdash 0004 plusmn 002 001 plusmn 074-OH-benzoic 2 plusmn 14 70 plusmn 03 73 plusmn 07Vanillic 42 plusmn 02 211 plusmn 63 160 plusmn 47trans-Caffeic 412 plusmn 28 mdash mdashSyringic mdash 08 plusmn 04 mdashtrans-p-Coumaric 25 plusmn 21 mdash mdashtrans-Ferulic 36 plusmn 09 mdash mdash

Table 3 Mineral compositions of fruits [mgkg of dry weight]

Ca Mg K Na Fe Cu Zn Mn1750 1300 21000 218 327 435 235 543

present in other plant species used in traditional medicine orhuman diet A content of trans-caffeic acid in Allium cepa Lwas dependent on a type of rawmaterialThe highest contentwas determined in a dried material (224 120583gg) contrary toa fresh material (017 120583gg) [29] Caffeic acid occurred insmaller amount in rose petals (014 120583ggDE) [24]33 Mineral Composition The diet of the European popula-tion is mainly composed of starchy foods fruits vegetablesmeat eggs and milk Because starchy foods are presentin a majority of meals many people suffer from nutrientrsquosdeficiencies [30ndash32] Therefore there is nowadays a growinginterest in products with high nutripharmacological valueFor this reason the minerals content of the intractum wasevaluated The concentrations [mgkg of dry weight] of themineral components of the intractum according to the min-eralisation and identificationmethods are reported in Table 3The intractum presented a high amount of macroelements(Ca Mg and K 1750 1300 and 21000mgkg resp) andmicroelements (Fe Mn 327 543mgkg resp)

Heo et al [20] reported on mineral composition of theE senticosus fruits growing in Korea That species contains465 1433 199 and 13mgkg dry weight of Ca Mg Mnand Zn respectively Compared with species cultivated inPoland there is a wide variation in the level of Ca andMn The Polish one contains higher level of Ca and Znbut lower level of Mg and Mn A major finding is that thespecies native to Asia does not contain Fe the factor thatexcludes these fruits as an ingredient of antianemic diet Itis worth noting that the investigated samples have higher Feconcentration (327mgkg) than the Rosa canina L and Rosadamascena Mill fruits (270 and 110mgkg resp) whichare very popular in the diet of the Europeans Accordingto a WHO report over 2000 million people in developingcountries have iron deficiency anemia [33] In that case theintractum should be considered a new dietary ingredient thatmay be included in the diet Moreover the results obtained


Table 4 Inhibition ofHyal () for a different activity unit of enzyme(mIUmg protein)

Sample mIUmg protein30 40 53 90 109

Intractum 60 plusmn 23 314 plusmn 13 51 plusmn 03 21 plusmn 04 nsAescin 50 plusmn 18 293 plusmn 09 135 plusmn 15 ns nsNaringenin 389 plusmn 29 154 plusmn 21 ns ns nsns not showed

in the present study indicated higher content of Mn Zn andCu than that in the Rosa canina L and Rosa damascenaMillfruits [34] It should be mentioned that the intractum alsocontains more Zn and Mn than walnut of kernels (from 179to 206 and 175 to 222mgkg)

34 AntihyaluronidaseActivity Hyaluronidases belong to thehydrolases and digestive hyaluronic acid Many investigatorshave reported a positive correlation between the expression ofHyal and the development of inflammation and tumor inva-siveness It was found that its activity is growing up in sev-eral diseases such as the borderline tumors (1089mIUmgprotein) the benign epithelial tumors (892mIUmgprotein)the functional cysts (534mIUmg protein) and malignantepithelial tumors (487mIUmg protein) in contrary to thenormal ovaries (385mIUmg protein) Therefore identi-fication and characterization of Hyal inhibitors would bevaluable for developing antitumor and anti-inflammatoryagents Recently new hyaluronidase inhibitors are urgentlyrequired in the clinic and there are none In this studythe impact of the intractum at the inhibitor concentration22120583g016mL of the reaction mixture was measured Inorder to compare the antienzymatic activity of the intractumanalyzed aescin was used as the standard compound becauseof its well-recognized activity Aescin inhibited Hyal at thelevel of 100 at concentration 800 120583g016mL of the reactionmixture (Figure 3)

As it is seen in Table 4 the intractum showed the highestinhibition in comparison to aescin and naringenin Thedecrease in the inhibitory activity of compounds correlatedwith the increase of enzymersquos unit No inhibition was foundin case 109mIUThe high inhibition by intractummay resultfrom the nature of chemical constituents of the intractum asa mixture It is known that the activity of Hyal is dependenton the presence of Ca2+ [19] in turn the intractum containsa high amount of polyphenols acting as chelators

Because there are no general procedures for anti-Hyalassay it is hard to compare the results to others The majorfactor is different enzymersquos unit taken into assays whichexcludes a reliable comparison Therefore in our work wedecided to take the enzyme activity like in the above diseasesDespite a lack of a large amount of scientific literature wetried to compare our results to the others Bralley et al [35]reported on anti-Hyal activity of the ethanol extracts of theVitis rotundifolia Michx peels and seeds of Early Fry (bronze)and Ison (purple) Vitis rotundifolia varieties The obtainedresults indicated lower activity of the peel extracts contraryto the seed ones (IC

5010 10 for the Ison and Early Fry

0

20

40

60

80

100

120

Inhi

bitio

n (

)

200 400 600 800 10000Aescin 120583g161120583L of the reaction mixture

Figure 3 Inhibition of Hyal by aescin as the standard compounds()

peels and 03 06 for the Ison and Early Fry seeds mgmL)Considering isolates from the plant extracts inhibitors ofHyal have been mainly found in flavonoids and triterpenesAmong flavonoids 7-O-butyl naringenin had a high valuewith 4484 inhibition at 200120583M concentration Takinginto account the chemical structure of flavonoids theirinhibitory activity towards Hyal increases with the number ofhydroxyl groups especially in 231015840 position (quercetin) and 51015840(myricetin) Some authors state that the inhibition is depen-dent on the number of free available hydroxyl groups andextension of side-chains It was noticed that the inhibitoryactivity was decreased after glycosylation or substitution ofhydroxyl groups In turn another compound chlorogenicacid inhibitedHyal with IC

50225mMApromising source of

Hyal inhibitors may be some compounds present in essentialoil such as120573-caryophyllene (IC

50416 120583gmL) and 18-cineole

(IC50

117mgmL) The mechanism of action may be relatedto the formation of complexes of polyphenols present in theextracts with ions present in the reaction medium [19 36]

35 Analysis of Cell Viability During the last few years anincrease of leukemia evidences has been noticed Leukemiais one of the most frequently occurring diseases amongyoung people up to 30 years of age Because of growingresistance to drugs the treatment of leukemia is very difficulttherefore newdrugswith antileukemic or strengthening bodyproperties are still being searched for In addition to thisit was confirmed that a higher activity of Hyal is in closecorrelation with metastasis and inflammation Therefore theinhibition of Hyal and the cytotoxicity of intractum wasevaluated

Five leukemic cell lines HL-60 HL-60MX1 HL-60MX2 CEMC1 and CCRFCEM were used These cell linesrepresent different types of leukemia namely acute leukemiaand myeloma As a positive control podophyllotoxin wasused the results were published previously by Och et al [37]

As presented in Table 5 the highest mortality of cells wasobserved inHL-60 cell line with the IC

50value of 104 120583gmL

Previous studies on cytotoxic activity of E senticosushave shown a significant cytotoxicity towards HL-60 andJurkat 45 leukemia cell lines The ethanol extracts from rootsand spring and autumn leaves revealed the IC

50values at


Table 5 IC50(120583gmL) of the intractum and podophyllotoxin

Cell line CEMC1 CCRFCEM HL-60 HL-60MX1 HL-60MX2Intractum 304 181 104 157 50Podophyllotoxin 00286 00064 00085 00078 00106

concentrations 208 312 and 299 120583gmL towards HL-60 Inturn the chloroform extract from the roots (IC

5028120583gmL)

the ethanol extracts from the roots (1347 120583gmL) the springleaves (1997120583gmL) the fresh fruits (3312 120583gmL) and thedried fruits (2744 120583gmL) affected Jurkat 45 leukemia cellline [8 38]

Varamini et al [39] examined five quinoline alkaloidsisolated from Haplophyllum canaliculatum Boiss (7-isopen-tenyloxy-120574-fagarine atanine skimmianine flindersine andperfamine) against resistant (HL-60MX1) and sensitive (HL-60) acute leukemia cell lines 7-Isopen-tenyloxy-120574-fagarineappeared to have the highest IC

50value equal to 22

and 316 120583gmL for HL-60MX1 and HL-60 respectivelyTayarani-Najaran et al [40] have investigated the cytotoxiceffect of CH

2Cl2extract on HL-60 with the IC

50value of

6883 120583gmLwhile the intractum tested in our work exhibited7-fold lower IC

50value (1041 120583gmL) Omosa et al [41] tested

145 extracts from 91 Kenyan flora towards CCRFCEM celllines The highest cytotoxic activity exhibited the ethanolextract of the berries of Solanumaculeastrum and 50MeOHin CH

2Cl2extract of the stem bark of Albizia schimperiana

with IC50

values of 136 and 297 120583gmL The other extractsthat showed good activity included the extracts of the stembarks of Zanthoxylum gilletii (5 MeOH-H

2O) and Bridelia

micrantha 50CH2Cl2-MeOHand leaves of Strychnos usam-

barensis (50 MeOH in CH2Cl2) with IC

50values of 904

943 and 1109 120583gmL Similar results were obtained for theextract from the leaves of Vepris soyauxii (IC

50928120583gmL)

the whole plant of Gladiolus quartinianus (IC501057 120583gmL)

[42] and the bark of Nauclea pobeguinii (IC501462 120583gmL)

[43]The results obtained in this work against CCRFCEM cell

lines (IC501811 120583gmL) are consistent with those of the leaves

of Anonidium mannii (IC501732 120583gmL) [43]

According to the National Cancer Institute (UnitedStates) plant screening program a crude extract is generallyconsidered to have in vitro cytotoxic activity if the IC

50is

lt20120583gmL In turn the criteria of the ATCChave establisheda concentration of 30 120583gmL as the upper IC

50limit [41] On

the basis of this threshold the intractum tested in our studycan be considered as a promising cytotoxic agent towardsHL-60 HL-60MX1 and CCRFCEM cell lines (IC

50lt

20120583gmL)

36 Antioxidant Activity and the Structure-Activity Rela-tionship The human diet provides antioxidants from plantsources in order to protect the body from the radical damageat the biochemical and molecular levels Antioxidant capaci-ties of the extracts were expressed in terms of EC

50value It

was found that the intractum was able to effectively reducefree radicals with the EC

50value of 52 120583gmL and ascorbic

acid and 120572-tocopherol 40 and 50 120583gmL respectively Free

Figure 4 A ldquodot-blotrdquo TLC-DPPHlowast test performed on silica gelplate 1min after immersion in DPPHlowast methanolic solution

OH

O

HO

HO

Figure 5 Structure of trans-caffeic acid (groups marked in redparticipate in an interaction of free radical with caffeic acid)

radical scavenging activity has been confirmed by means ofTLC-DPPHlowast dot-blot test using silica gel as the stationaryphase (Figure 4) Regions of the TLC plate which containDPPHlowast inhibitors show up as yellow spots against a purplebackground We observed the plate after 1 5 10 and 30minfrom the time of immersion of the plate in 02mmol DPPHlowastsolution After 1min the intractum showed area of activityat concentration 10 120583gspot It is noteworthy that in thespectrophotometric assay the intractum showed strong anti-radical properties and this was confirmed in the autographytest It suggests the presence of the compounds which are ableto donate hydrogen or electron and may be considered asantioxidants The antioxidative molecular mechanism can bebased on the chemical structure of phenolic acids especiallytrans-caffeic that possess in their structure the two OHgroups one in the -para position and known as a strongantioxidant (Figure 5) [44] The propenyl chain can alsoimprove the efficiency due to the presence of the doubleC=C bond In our previous study we reported that the leavesand fruits of Eleutherococcus species have a high antioxidantcapability [8 9 19]


4 Conclusion

We assume that this study provides the better understandingof pharmacological activity of E senticosus and confirmsethnopharmacological reports on rightness of using thatspecies in traditional medicine The administration of theintractum could help to improve the bodyrsquos shape A specialattention should be paid to an incorporation of the speciesin commercial or domestic cultivations which would allowfor their valuable exploitation Further studies are needed tofocus on exploring the mechanism of action of constituentsin in vivomodel

Abbreviation

TPC Total phenolics contentTFC Total flavonoids contentDPPHlowast 22-Diphenyl-1-picrylhydrazylDNPH 24-DinitrophenylhydrazineFeCl3 Aluminium chloride

GA Gallic acidHE Hesperetin equivalentQE Quercetin equivalentTLC-DB-DPPHdot-blot test

TLC-Direct Bioautography dot-blottest

AAS Atomic absorption spectroscopyHyal HyaluronidaseHA Hyaluronic acidHL-60 Human Caucasian promyelocytic

leukemia from American Type CultureCollection (ATCC CCL-240)

HL-60MX1 Human Caucasian acutepromyelocytic leukemia fromAmerican Type Culture Collection(ATCC CRL-2258)

HL60MX2 Human Caucasian acutepromyelocytic leukemia fromAmerican Type Culture Collection(ATCC CRL-2257)

CEMC1 Human Caucasian acute lymphoblasticleukemia from American Type CultureCollection (ATCC CRL-2265)

CCRFCEM Human Caucasian acute lymphoblasticleukemia from American Type CultureCollection (ATCC CCL-119)

Competing Interests

The authors declare no competing financial interests

Acknowledgments

The paper was developed using the equipment purchasedwithin the Projects The Equipment of Innovative Labo-ratories Doing Research on New Medicines Used in theTherapy of Civilization and Neoplastic Diseases within theOperational Program Development of Eastern Poland 2007ndash2013 Priority Axis I Modern Economy Operations I3 Inno-vation Promotion and ldquoNutri-Pharmacological Activity of

Eleutherococcus spprdquo funded by Ministry of Science andHigher Education Poland

References

[1] R Verpoorte ldquoPharmacognosy in the new millennium lead-finding and biotechnologyrdquo Journal of Pharmacy and Pharma-cology vol 52 no 3 pp 253ndash262 2000

[2] J Yang S Zhao C Yu and C Li ldquoLarge-scale plantlet conver-sion and ex vitro transplantation efficiency of siberian ginsengby bioreactor culturerdquo The Scientific World Journal vol 2013Article ID 829067 8 pages 2013

[3] A Panossian G Wikman and H Wagner ldquoPlant adaptogensIII Earlier andmore recent aspects and concepts on their modeof actionrdquo Phytomedicine vol 6 no 4 pp 287ndash300 1999

[4] L Jin F Wu X Li et al ldquoAnti-depressant effects of aqueousextract from Acanthopanax senticosus in micerdquo PhytotherapyResearch vol 27 no 12 pp 1829ndash1833 2013

[5] D H Watson Performance Functional Foods Woodhead Pub-lishing in Food Science and Technology Woodhead Cam-bridge UK 2003

[6] A Arouca and D M Grassi-Kassisse ldquoEleutherococcus sentico-sus studies and effectsrdquoHealth vol 5 no 9 pp 1509ndash1515 2013

[7] M Schmidt M Thomsen O Kelber and K Kraft ldquoMyths andfacts in herbal medicines Eleutherococcus senticosus (Siberianginseng) and its contraindication in hypertensive patientsrdquoBotanics Targets and Therapy vol 4 pp 27ndash32 2014

[8] D Załuski H D Smolarz and U Gawlik-Dziki ldquoBioactivecompounds and antioxidative antileukemic and anti-MMPsactivity of Eleutherococcus species cultivated in PolandrdquoNaturalProduct Communications vol 7 no 11 pp 1483ndash1486 2012

[9] D Załuski E Mendyk and H D Smolarz ldquoIdentification ofMMP-1 and MMP-9 inhibitors from the roots of Eleutherococ-cus divaricatus and the PAMPA testrdquoNatural Product Researchvol 30 no 5 pp 595ndash599 2016

[10] J M Lee D G Lee J Kim and S Lee ldquoContent analysis offlavonoids in the stems and roots of Acanthopanax species indifferent countriesrdquo Asian Journal of Chemistry vol 26 no 12pp 3511ndash3514 2014

[11] M Chen F Song M Guo Z Liu and S Liu ldquoAnalysis of flavo-noid constituents from leaves of Acanthopanax senticosusHarms by electrospray tandemmass spectrometryrdquoRapid Com-munications in Mass Spectrometry vol 16 no 4 pp 264ndash2712002

[12] H H S Fong ldquoIntegration of herbal medicine into modernmedical practices issues and prospectsrdquo Integrative CancerTherapies vol 1 no 3 pp 287ndash293 2002

[13] J Tumiłowicz and P Banaszczak ldquoTrees and shrubs of aquifoli-aceae family in rogow glinna arboretardquoRocznik Dendrologicznyvol 55 pp 41ndash56 2007

[14] V L Singleton and J A Rossi ldquoColorimetry of total phenolicswith phosphomolybdic-phosphotungstic acid reagentsrdquo Amer-ican Journal of Enology Viticulture vol 16 pp 144ndash158 1965

[15] C-C Chang M-H Yang H-M Wen and J-C Chern ldquoEsti-mation of total flavonoid content in propolis by two com-plementary colorimetric methodsrdquo Journal of Food and DrugAnalysis vol 10 no 3 pp 178ndash182 2002

[16] N Nowacka R Nowak M Drozd M Olech R Los andA Malm ldquoAnalysis of phenolic constituents antiradical andantimicrobial activity of edible mushrooms growing wild inPolandrdquo LWTmdashFood Science and Technology vol 59 no 2 pp689ndash694 2014


[17] Y A Yahaya and M M Don ldquoEvaluation of Trametes lactineaextracts on the inhibition of hyaluronidase lipoxygenase andxanthine oxidase activities in Vitrordquo Journal of Physical Sciencevol 23 no 2 pp 1ndash15 2012

[18] W Brand-Williams M E Cuvelier and C Berset ldquoUse of a freeradical method to evaluate antioxidant activityrdquo LWTmdashFoodScience and Technology vol 28 no 1 pp 25ndash30 1995

[19] D Załuski and Z Janeczko ldquoVariation in phytochemicals andbioactivity of the fruits of Eleutherococcus species cultivated inPolandrdquoNatural Product Research vol 29 no 23 pp 2207ndash22112015

[20] S Heo H Ahn M Kang J Lee J Cha and Y Cho ldquoAntioxida-tive activity and chemical characteristics of leaves roots stemsand fruits extracts from Acanthopanax senticosusrdquo Journal ofLife Science vol 21 no 7 pp 1052ndash1059 2011

[21] AM Shohael D ChakrabartyM BAli et al ldquoEnhancement ofeleutherosides production in embryogenic cultures of Eleuthe-rococcus sessiliflorus in response to sucrose-induced osmoticstressrdquo Process Biochemistry vol 41 no 3 pp 512ndash518 2006

[22] D Jang J Lee S Eom et al ldquoComposition antioxidantand antimicrobial activities of Eleutherococcus senticosus fruitextractsrdquo Journal of Applied Pharmaceutical Science vol 6 no3 pp 125ndash130 2016

[23] M H Grace D Esposito K L Dunlap and M A Lila ldquoCom-parative analysis of phenolic content and profile antioxidantcapacity and anti-inflammatory bioactivity in wild alaskanand commercial Vaccinium berriesrdquo Journal of Agricultural andFood Chemistry vol 62 no 18 pp 4007ndash4017 2014

[24] R Nowak M Olech Ł Pecio et al ldquoCytotoxic antioxidantantimicrobial properties and chemical composition of rosepetalsrdquo Journal of the Science of Food and Agriculture vol 94no 3 pp 560ndash567 2014

[25] V A Kurkin G G Zapesochnaya and V V Bandyshev ldquoPhe-nolic compounds of Eleutherococcus senticosusrdquo Khimiya Priro-dnykh Soedinenii vol 6 pp 854ndash856 1991

[26] Q Li Y Jia L Xu et al ldquoSimultaneous determination of proto-catechuic acid syringin chlorogenic acid caffeic acid lirioden-drin and isofraxidin in Acanthopanax senticosus HARMS byHPLC-DADrdquo Biological and Pharmaceutical Bulletin vol 29no 3 pp 532ndash534 2006

[27] K Bączek ldquoAccumulation of biologically active compoundsin Eleuthero (Eleutherococcus senticosus Rupr et MaximMaxim) grown in Polandrdquo Herba Polonica vol 55 pp 7ndash132009

[28] K Bączek ldquoDiversity of Eleutherococcus genus in respect of bio-logically active compounds accumulationrdquoHerba Polonica vol60 no 3 pp 1ndash10 2014

[29] M Drozd M Thomas and R Nowak ldquoDetermination ofphenolic acids in raw garlic (Allium sativum L) and onion(Allium cepa L) bulbsrdquo Annales Universitatis Mariae Curie-Sklodowska Sectio DDD Pharmacia vol 24 no 1 pp 121ndash1272011

[30] P Leterme A Buldgen F Estrada and A M Londono ldquoMin-eral content of tropical fruits and unconventional foods of theAndes and the rain forest of Colombiardquo Food Chemistry vol 95no 4 pp 644ndash652 2006

[31] M M Ozcan A Unver T Ucar and D Arslan ldquoMineral con-tent of some herbs and herbal teas by infusion and decoctionrdquoFood Chemistry vol 106 no 3 pp 1120ndash1127 2008

[32] C A Gonzalez G Pera A Agudo et al ldquoFruit and vegetableintake and the risk of stomach and oesophagus adenocarcinoma

in the European Prospective Investigation into Cancer andNutrition (EPICndashEURGAST)rdquo International Journal of Cancervol 118 no 10 pp 2559ndash2566 2006

[33] WHOUNICEFUnited Nations University Iron DeficiencyAnaemia Assessment Prevention and Control A Guide forProgramme Managers (document WHONHD013) WorldHealth Organization Geneva Switzerland 2001

[34] S Kazaz H Baydar and S Erbas ldquoVariations in chemical com-positions of Rosa damascena Mill and Rosa canina L FruitsrdquoCzech Journal of Food Sciences vol 27 no 3 pp 178ndash184 2009

[35] E Bralley P Greenspan J L Hargrove and D K HartleldquoInhibition of hyaluronidase activity byVitis rotundifolia (Mus-cadine) berry seeds and skinsrdquo Pharmaceutical Biology vol 45no 9 pp 667ndash673 2007

[36] S-H Moon K-T Kim N-K Lee et al ldquoInhibitory effects ofnaringenin and its novel derivatives on hyaluronidaserdquo FoodScience and Biotechnology vol 18 no 1 pp 267ndash270 2009

[37] M Och A Och Ł Ciesla et al ldquoStudy of cytotoxic activ-ity podophyllotoxin and deoxypodophyllotoxin content inselected Juniperus species cultivated in Polandrdquo PharmaceuticalBiology vol 53 no 6 pp 831ndash837 2015

[38] D Zaluski H D Smolarz and A Bogucka-Kocka ldquoCytotoxicactivity of ethanolic extracts of Eleutherococcus species culti-vated in Poland on HL60 leukemia cell linerdquo Current Issues inPharmacy and Medical Sciences vol 27 no 1 pp 41ndash45 2014

[39] P Varamini K Javidnia M Soltani A R Mehdipour and AGhaderi ldquoCytotoxic activity and cell cycle analysis of quinolinealkaloids isolated from Haplophyllum canaliculatum BoissrdquoPlanta Medica vol 75 no 14 pp 1509ndash1516 2009

[40] Z Tayarani-Najaran M Sareban A Gholami S A Emamiand M Mojarrab ldquoCytotoxic and apoptotic effects of differentextracts of Artemisia turanica Krasch on K562 and HL-60 celllinesrdquoThe Scientific World Journal vol 2013 Article ID 6280736 pages 2013

[41] L K Omosa J O Midiwo V M Masila et al ldquoCytotoxicity of91 Kenyan indigenous medicinal plants towards human CCRF-CEM leukemia cellsrdquo Journal of Ethnopharmacology vol 179pp 177ndash196 2016

[42] V Kuete L P Sandjo A TMbaveng J A Seukep B T Ngadjuiand T Efferth ldquoCytotoxicity of selected Cameroonian medici-nal plants andNauclea pobeguinii towardsmulti-factorial drug-resistant cancer cellsrdquo BMC Complementary and AlternativeMedicine vol 15 no 1 article 309 2015

[43] V Kuete A G Fankam BWiench and T Efferth ldquoCytotoxicityand modes of action of the methanol extracts of six Cameroo-nian medicinal plants against multidrug-resistant tumor cellsrdquoEvidence-Based Complementary and Alternative Medicine vol2013 Article ID 285903 10 pages 2013

[44] X Li J Lin Y Gao W Han and D Chen ldquoAntioxidant activityand mechanism of Rhizoma Cimicifugaerdquo Chemistry CentralJournal vol 6 article 140 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the players of the Old Soviet Union have increased recordsafter administering the E senticosus roots products [6 7]According to Załuskirsquos previous studies the fruits of thatspecies cultivated in Poland act as antioxidants inductorsof the apoptosis in Jurkat 45 and HL60 leukemic cell linesand inhibitors of MMP-1 MMP-2 MMP-3 and MMP-9[8 9]

The Eleutherococcus senticosus products attract globalattention as a novel medicinal plant and since a few yearshave become popular as dietary supplement in the UnitedStates and European countries Imported products of thisplant have become available in North America with amarketshare of 31 of the $12 billion medicinal herbal industryThe 1994DSHEA (Dietary SupplementHealth and EducationAct) regulation allows a direct commercialization of Esenticosus as a supplement for consumption in the UnitedStates without the regulation of the FDA (Food and DrugAdministration)With the increased awareness of developingcountries that the study of traditional medicines and findingnew leads is important there is a need to avoid importedexpensive eastern medicines and to estimate a new sourceof some eastern herbs in Europe Preparations of the rootsof E senticosus are given in cases of asthenia with weaknessand fatigue for example in convalescence This indicationhas been officially accepted by the Community HerbalMonograph on Eleutherococcus senticosus (Rupr et Maxim)Maxim Radix (EMEAHMPC2445692006) published bythe European Medicines Agency The clinical applicationof E senticosus is generally considered safe however theEuropean Community Herbal Monograph states ldquoarterialhypertensionrdquo as a contraindication [5ndash7]

The roots of E senticosus are source of phenols calledeleutherosides (derivatives of lignans coumarins and phe-nylpropanoids) flavonoids (hyperin rutin afzelin quercetinand kaempferol) phenolic acids triterpenic acids and antho-cyanins (Figure 1) Compounds isolated from the fruitsbelong to eleutherosides (eleutherosides B and E) flavonoidsphenolic acids and essential oil (03 vdw) The driedfruits consumed as food are rich in Ca Mg Mn Zn andCu In the leaves flavonoids (quercetin quercitrin and rutin)have been identified [8ndash11]

Eleutherococcus senticosus is an example of one suchspecies whose activity and chemistry are yet to be studiedin more detail especially the species harvested in differentplaces The Eleutherococcus senticosus products which areavailable in the herbal drugs market are imported fromChina Because of a lack of the assessment of plant materialthere have been many cases of the poor quality of plantssupplied by Chinese traders leading to financial lossesfor some pharmaceutical companies For example 26 ofproducts prepared among others from E senticosus did notmeet label claims with respect to the claimed eleuthero-sides content [12] To avoid that the establishment of thenew source of important medicinal plants in Europe isrequired As a major part of western medicine has beendeveloped from traditional knowledge it makes sense tofirst of all look to our ancestorsrsquo knowledge and study thatwith all the novel concepts and source of the plant mate-rial

The chemical compounds and biological activity of plantsdepend on the geographical zone of the growth This speciesis successfully cultivated at the botanical garden in Rogowwhich lies in the Central Polish Lowlands region with geo-graphic data such as 51∘ 491015840N and 19∘ 531015840EThe average long-term temperature is minus201∘C what classified the garden to the6bth subclimate (according to USDA Frost Hardiness Zones)and to the second zone according to the Kornikrsquos categoryThese plants are grown on the acidic luvic and sandy soils[13]

In view of several ethnopharmacological medicinal andnutritional value of the fruits described in literature and onthe basis of Załuskirsquos previous studies it was decided to exam-ine the fruits intractum for its phytochemicals and bioactivityMany products which are widely available in the health foodor herbal drugs markets are in the form of capsule powderor tea bag So far the intractum is not available on marketAs part of a program to search for bioactive constituentsfrom Eleutherococcus species this study was focused on theestablishment of phenolic compounds (phenolic acids TPCand TFC) minerals content and anti-Hyal anti-DPPHlowast andcytotoxic properties of the intractumprepared from the fruitsharvested in Poland

2 Materials and Methods

21 Standards and Reagents Folin-Ciocalteu reagent DPPHascorbic acid DMSO bovine albumin hyaluronidase frombovine testes type I-S Streptococcus equi hyaluronic acidand sodium phosphate buffer pH 70 were obtained fromSigma-Aldrich DNPH and FeCl

3and ethanol were obtained

from POCH (Lublin Poland) The acetate buffer pH 45was purchased from J T Baker USA The standards ofphenolic acids flavonoids and aescin were obtained fromChromaDex (SantaAna CA) Liquid chromatography- (LC-)grade methanol (MeOH) and acetonitrile (ACN) were pur-chased from Merck (Darmstadt Germany) LC-grade waterwas prepared using a Milli-Q purification system (MilliporeBedford MA USA) All others reagents were of analyticalgrade

22 Plant Material The fruits of E senticosus (Rupr etMaxim) Maxim were collected at the arboretum in Rogow(Poland) in October 2015 (voucher specimen number ES0914F) The sample was deposited at the Department of Phar-macognosy CollegiumMedicum Bydgoszcz Poland Imme-diately after harvesting the intractum was prepared

23 Preparation of Intractum 20 g of fresh fruits was macer-ated in 100mL 40 ethanol for 30 days at room temperatureAfter that the extract was filtered throughWhatman number4 filter paperThe solvent was dried with an evaporator undervacuum conditions at 45∘C and the residue was subjectedto lyophilisation The extraction yield was calculated basedon the dry weight of the extract and was expressed as apercentage calculated according to the formula

Yield = extract weightsample weight

times 100 (1)


O

OO

O

O

O

(+)-Sesamin

OHO

OH

HO OH

O

OO

OH

HH

Eleutheroside B

OHO

OH

HO OH

OO

O

O O

H

H

OO

O

OHO

HO

HOOH

Eleutheroside E

OHO

OH

HO OH

OO

O

O O

H

H

OO

HO

O

O

HO

O

O

Isofraxidin

HO

H

H

OH

OH

Oleanolic acid

HO

HCOOH

H

Ursolic acid

Eleutheroside E1












3method 20ndash




3was added














50) were


















Time (min)

1

2

3 4

5

6

00

50000

10e4

15e4

20e4

25e4

30e4

35e4

40e4

45e4

50e4

55e4

60e4

65e4

857565554535251505

017027

088118

162 244 282

326

348382

402437 490

500512

575

588

629

652 688714 754783

823843 891

448

40 50

Time (min)


Max 41e4 cps






Ethyl acetatelayer
















0

20

40

60

80

100

120

Inhi

bitio

n (

)







(IC50







50values at





5028120583gmL)



50value equal to 22



50value of





with IC50


2O) and Bridelia



50values of 904


50928120583gmL)







50is


50limit [41] On


50lt

20120583gmL)


50value It





OH

O

HO

HO




4 Conclusion


Abbreviation










Competing Interests


Acknowledgments



References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















O

OO

O

O

O

(+)-Sesamin

OHO

OH

HO OH

O

OO

OH

HH

Eleutheroside B

OHO

OH

HO OH

OO

O

O O

H

H

OO

O

OHO

HO

HOOH

Eleutheroside E

OHO

OH

HO OH

OO

O

O O

H

H

OO

HO

O

O

HO

O

O

Isofraxidin

HO

H

H

OH

OH

Oleanolic acid

HO

HCOOH

H

Ursolic acid

Eleutheroside E1












3method 20ndash




3was added














50) were


















Time (min)

1

2

3 4

5

6

00

50000

10e4

15e4

20e4

25e4

30e4

35e4

40e4

45e4

50e4

55e4

60e4

65e4

857565554535251505

017027

088118

162 244 282

326

348382

402437 490

500512

575

588

629

652 688714 754783

823843 891

448

40 50

Time (min)


Max 41e4 cps






Ethyl acetatelayer
















0

20

40

60

80

100

120

Inhi

bitio

n (

)







(IC50







50values at





5028120583gmL)



50value equal to 22



50value of





with IC50


2O) and Bridelia



50values of 904


50928120583gmL)







50is


50limit [41] On


50lt

20120583gmL)


50value It





OH

O

HO

HO




4 Conclusion


Abbreviation










Competing Interests


Acknowledgments



References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















3method 20ndash




3was added














50) were


















Time (min)

1

2

3 4

5

6

00

50000

10e4

15e4

20e4

25e4

30e4

35e4

40e4

45e4

50e4

55e4

60e4

65e4

857565554535251505

017027

088118

162 244 282

326

348382

402437 490

500512

575

588

629

652 688714 754783

823843 891

448

40 50

Time (min)


Max 41e4 cps






Ethyl acetatelayer
















0

20

40

60

80

100

120

Inhi

bitio

n (

)







(IC50







50values at





5028120583gmL)



50value equal to 22



50value of





with IC50


2O) and Bridelia



50values of 904


50928120583gmL)







50is


50limit [41] On


50lt

20120583gmL)


50value It





OH

O

HO

HO




4 Conclusion


Abbreviation










Competing Interests


Acknowledgments



References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















50) were


















Time (min)

1

2

3 4

5

6

00

50000

10e4

15e4

20e4

25e4

30e4

35e4

40e4

45e4

50e4

55e4

60e4

65e4

857565554535251505

017027

088118

162 244 282

326

348382

402437 490

500512

575

588

629

652 688714 754783

823843 891

448

40 50

Time (min)


Max 41e4 cps






Ethyl acetatelayer
















0

20

40

60

80

100

120

Inhi

bitio

n (

)







(IC50







50values at





5028120583gmL)



50value equal to 22



50value of





with IC50


2O) and Bridelia



50values of 904


50928120583gmL)







50is


50limit [41] On


50lt

20120583gmL)


50value It





OH

O

HO

HO




4 Conclusion


Abbreviation










Competing Interests


Acknowledgments



References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Time (min)

1

2

3 4

5

6

00

50000

10e4

15e4

20e4

25e4

30e4

35e4

40e4

45e4

50e4

55e4

60e4

65e4

857565554535251505

017027

088118

162 244 282

326

348382

402437 490

500512

575

588

629

652 688714 754783

823843 891

448

40 50

Time (min)


Max 41e4 cps






Ethyl acetatelayer
















0

20

40

60

80

100

120

Inhi

bitio

n (

)







(IC50







50values at





5028120583gmL)



50value equal to 22



50value of





with IC50


2O) and Bridelia



50values of 904


50928120583gmL)







50is


50limit [41] On


50lt

20120583gmL)


50value It





OH

O

HO

HO




4 Conclusion


Abbreviation










Competing Interests


Acknowledgments



References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























0

20

40

60

80

100

120

Inhi

bitio

n (

)







(IC50







50values at





5028120583gmL)



50value equal to 22



50value of





with IC50


2O) and Bridelia



50values of 904


50928120583gmL)







50is


50limit [41] On


50lt

20120583gmL)


50value It





OH

O

HO

HO




4 Conclusion


Abbreviation










Competing Interests


Acknowledgments



References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5028120583gmL)



50value equal to 22



50value of





with IC50


2O) and Bridelia



50values of 904


50928120583gmL)







50is


50limit [41] On


50lt

20120583gmL)


50value It





OH

O

HO

HO




4 Conclusion


Abbreviation










Competing Interests


Acknowledgments



References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















4 Conclusion


Abbreviation










Competing Interests


Acknowledgments



References




















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Phytochemical Content and Pharma...

Documents

Transcript of Research Article Phytochemical Content and Pharma...