research article bioguided fraction and isolation of the...

Research ArticleBioguided Fraction and Isolation of the Antitumor Componentsfrom Phyllanthus niruri L

Zhi-Zhong Zheng123 Liang-Hua Chen12 Shao-Song Liu12 Yuan Deng3

Guo-Hua Zheng13 Yue Gu2 and Yan-Lin Ming123

1Xiamen Key Laboratory for Plant Introduction Quarantine and Natural ProductsXiamen Overseas Chinese Subtropical Plant Introduction Garden Xiamen 361002 China2Fujian Key Laboratory of Physiology and Biochemistry for Subtropical Plant Fujian Province Institute of Subtropical BotanyXiamen China3College of Chemical Engineering Huaqiao University Xiamen Fujian 361021 China

Correspondence should be addressed to Yan-Lin Ming xmyanlingmailcom

Received 8 June 2016 Revised 18 August 2016 Accepted 29 August 2016

Academic Editor Adair Santos

Copyright copy 2016 Zhi-Zhong Zheng et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Phyllanthus niruri L a well-knownmedicinal plant has been used as a folk antitumor remedy in the worldwide scale However theantitumor components in P niruri have not been reported In order to verify the antitumor components of P niruri and the plantswhich have the high content of these components we isolated the antitumor components with bioguided fraction and isolation bydifferent chromatographic methods from the ethyl acetate fraction of P niruri and identified them as ethyl brevifolincarboxylateand corilagin by 1H-NMR 13C-NMR 2D-NMR and mass spectrometric analyses Cell cytotoxicity assays showed that corilaginhas broad-spectrum antitumor activity a better antitumor potential and lower toxicity in normal cells Besides the coefficient ofdrug interaction (CDI) of 10 120583M corilagin and 20 120583M cDDP reached up to 077 which means corilagin can promote the antitumoractivity of cDDP Furthermore by the extensive screening among 10 species of plants reported to contain corilagin we found thatDimocarpus longan Lour has themaximum content of corilagin In conclusion corilagin is themajor active antitumor compositionin P niruri L on HCC cells and has high content in D longan

1 Introduction

Phyllanthus niruri L (P niruri) is a member of the Euphor-biaceae family that consists of over 600 species of shrubstrees and annual or biennial herbs [1] It usually occursas a winter weed throughout the tropical and subtropicalareas including China India and America P niruri is usedas medicated food to nurse peoplersquos health especial hair[2] intestine [3] liver [4] and kidney [5] problems andhas provoked much interest In the folk P niruri has beenclaimed to present beneficial therapeutic effects in treatingkidney and gallbladder stones liver related diseases such asjaundice and liver cancer viral infections such as hepatitisand tuberculosis malaria diabetes and fever [6 7]

In recent years systematic studies on the constituents andmedicinal effects of P niruri have been reported gradually

P niruri has been found to exhibit antitumor [8] antiviral[4] antioxidant [9] anti-inflammatory [10] and antidiabetic[11] activities and radiation protection [12] Particularlyin antitumor activity P niruri extract shows potential inreducing chemically induced skin papillomas by enhancingantioxidant defense systems [13] and shows potential inthe management of a two-stage skin carcinogenesis modelin mice The methanol extracts of P niruri exhibited a595 inhibition of rat aortic vascular growth and showeda significant decrease of 379 in the tube formation assayinvolving human umbilical vein endothelial cells (HUVECs)on Matrigel [14]

Hepatocellular carcinoma (HCC) is one of the deadliestcancers in the world and was ranked the second cause ofcancer death [15] The main treatment of HCC chemothera-peutic treatment suffers from increasing resistance responses

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 9729275 7 pageshttpdxdoiorg10115520169729275

2 BioMed Research International

[16 17] among patients to existing drugs lack of wideractivities and selectivity To overcome these the developmentof novel anticancer agents is an immense demand Previousreports had implied that P niruri is a candidate folk medicine[13 18 19]Howevermost of previous studies in vitrovivo justfocused on the crude extracts from P niruri [3 20]Themajoractive compounds in the crude extracts remain unknownLittle work has been performed to determine which activeconstituents from P niruri have antitumor activity Fujikiindicated that to investigate P niruri a useful and practicalmethod for screening preventive cancer agents is needed[21]

Thus due to the presence of variousmedicinal propertiesthe present study was designed to isolate screen and identifythe structure of really active components from P niruri anddetermine their biological activity on HCC

2 Materials and Methods

21 Plant Material Whole P niruri plants were collectedfrom Gulangyu Islet Fujian province China in October2009 and identified by Professor Yong-Tian Zhang FujianProvince Institute of Subtropical Botany China A voucherspecimen (YZY20091026) was deposited at XiamenOverseasChinese Subtropical Plant Introduction Garden China

22Chemicals andReagents All solvents andchemicals used forextraction in this study were of analytical grade and obtainedfrom Tianjin Reagent Company (Tianjin China) Materi-als for column chromatography included polyamide resin(100ndash200mesh Zhejiang Tetracarboxylic Biochemical Plas-tics Ltd China) and Sephadex LH-20 (Amersham Pharma-cia Biotech Sweden) The SMMC7721 Bel7402 MHCC97-H HepG2 OC316 SGC7901 QBC939 and Chang-livernormal liver cell line were obtained from the Cell Bankof the Chinese Academy of Sciences (Shanghai China)RPMI 1640 medium and Dulbeccorsquos Modified Eagle Media(DMEM)were obtained fromGibco (Grand IslandNY) The 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) reagent was obtained from Sigma (St Louis MO)Trypsin Leibovitzrsquos L-15 medium fetal bovine serum (FBS)and penicillinstreptomycin solution (200x) were obtainedfromMediatech Inc (Herndon VA)

23 Chromatograph Conditions The chromate column wasWaters XBridge Shield RP18 (46mm times 250mm 5 120583m)DAD detector detection wavelength 280 nm column tem-perature 30∘C mobile phase Acetonitrile (A) 01 KH

2PO4

(B) flow speed 1mLmin injection volume 20 120583L Elutionprocedures are as follows 0ndash10min119881(A) 119881(B) = 15 85 10ndash15min119881(A) V(B) = 20 80 15ndash20minV(A) V(B) = 25 75

24 Standard Curve We weighed 25mg corilagin pre-cisely dissolved it in a 10mL measuring flask with 50methanol and prepared a standard solution whose concen-tration is 025mgmL diluted corilagin standard solutioninto 0025 0050 0075 0100 and 0125mgmL injected thediluted solution into HPLC and then determined absorption

spectrums We could get the linear relation from peak area(119884) and concentration (119883)

25 Preparation of Test Solution Five g sample powder wasextracted with 25mL of 95 ethanol at room temperaturefor three times (12 h each time) The solvent was evapo-rated in vacuo and the dried ethanol extract was dissolvedin 50 methanol (100mL) and then we filtered it with045 120583m membrane the subsequent filtrate was the testsolution

26 General Procedure Compounds were purified on poly-amide column D101 MCI gel and Sephadex LH-20 columnanalyzed qualitatively by TLC and quantitatively on ODS-C18 column (150 times 46mm) with a UV detector and MeOH-water (7 3) as mobile phase and determined by HPLC IRspectra were obtained with a Nicolet AVAIAR 360 FT-IRspectrometer with KBr pellets The1H- 13C- and 2D-NMRspectra were recorded on Bruker AV-400 spectrometers atroom temperature (120575 in ppm 119869 in Hz) Mass spectrometrywas carried out on a VG AutoSpec-3000 spectrometer or aFinnigan MAT 90 instrument EI-MS measurements werecarried out on an LCQ-Finnigan instrument at room temper-ature

Column chromatography was performed with polyamideresin (100ndash200mesh) and Sephadex LH-20 Fractions weremonitored using TLC and spots were visualized by heatingsilica gel plates or spraying with 5 H

2SO4in ethanol

27 Extraction Fractionation and Separation of AntitumorCompounds The dried and powdered whole P niruri (3 kg)were extracted with 75 EtOH (3 times 5 L) The 75 EtOHextract was combined and evaporated under reduced pres-sure to yield a residue Then it was suspended in waterand partitioned with petroleum ether CHCl

3 EtOAc and

n-BuOH (3 1 vv for 3 times) resulting in five fractionspetroleum ether fraction (Fr1 95 g) CHCl

3fraction (Fr2

58 g) EtOAc fraction (Fr3 48 g) n-BuOH fraction (Fr41137 g) and water fraction (Fr5 62 g) After that the cyto-toxicity of five fractions on HCC cell lines was determinedby MTT assay Fraction showing the highest cytotoxicity wasfurther fractionated by column chromatography each time

Fr3 was separated by polyamide column chromatographyusing a gradient of H

2O-EtOH (10 0 8 2 6 4 4 6 2 8

and 0 10) and then 35 NH3-H2O to yield seven fractions

(Fr31ndashFr37) For Fr32 the solvent was allowed to slowlyevaporate causing the compound to precipitate which wasthen collected by filtration and recrystallized to yield yellowdiamond flake crystallization (compound 1 126mg) Thefiltrate was then separated by chromatography on a SephadexLH-20 column and eluted with MeOH to obtain compound2 (430mg) (Figure 1)

28 Cell Culture Cells were cultured in RPMI 1640(SMMC7721 Bel7402 SGC7901 OC316 and QBC939) orDMEM (Chang-liver HepG2 and MHCC97-H) supple-mented with 10 FBS penicillin (100UmL) and strepto-mycin (100 120583gmL) in a humidified incubator aerated with5 CO

2and 95 air at 37∘C

BioMed Research International 3

75 EtOH extract

Petroleum ether fraction

Partition with different organic solvent

EtOAc fraction n-BuOH fraction Water fraction

Fr32

Compound 1

Recrystallization

Compound 2

Filtrate

Sephadex LH-20 MeOH

Fr31 Fr33 Fr34 Fr35 Fr36 Fr37

g g g gFr1 95 Fr2 58 Fr3 48 Fr4 1137 Fr5 62 g

16 mg43mg

Polyamide column chromatography EtOHNH3

Five L 75 EtOH extraction three times

Three kg dried and powdered whole P niruri

CHCl3 fraction

Figure 1 Scheme of bioguided fractionation and isolation of compounds from P niruri

29 Cell Cytotoxicity Assays The effect of constituents on thegrowth of SMMC7721 Bel7402 and Chang-liver cells wasdetermined using the MTT assay Briefly 5000 cells per wellin 96-well microtiter plates were treated with corilagin at 010 20 40 80 120 and 160 120583gmL or DMSO (01 vv) for48 hThen 20120583L of MTT (5mgmL) was added to each welland the plates were incubated for additional 4 h at 37∘C Themedium was replaced with DMSO to dissolve the formazanproduced from MTT by viable cells Absorbance at 492 nmis proportional to the live cell count and cell survival wasexpressed as the absorbance ofMTT-treated cells relative thatof DMSO-treated controls

210 Drug Combinations As cell cytotoxicity assaysSMMC7721 was treated with corilagin (5 10 and 20120583M)and cis-platinum (cDDP 10 20 and 40 120583M) alone ortogether with SMMC7721 for 48 h The coefficient of druginteraction (CDI) was calculated as follows CDI = AB(Atimes B) According to the absorbance of each group AB isthe absorbance of the combination group A or B is theabsorbance of the single drug group Thus CDI value lt1=1 or gt1 indicates that the drugs are synergistic additive orantagonistic respectively CDI value lt07 indicates that thedrugs are significantly synergistic Thus CDI was used toanalyze effects of drug combinations [22]

211 Statistical Analysis All experiments were repeated atleast three times and each experiment was performed induplicateThe data are expressed as themean plusmn SD Statisticalanalysis was performed using Studentrsquos 119905-test

3 Results

31 Antitumor Activity of Components Extracted from Pniruri Five fractions petroleum ether fraction (Fr1) CHCl

3

fraction (Fr2) EtOAc fraction (Fr3) n-BuOH fraction (Fr4)

and water fraction (Fr5) were extracted from 75 EtOHextract of P niruriWe estimated the antitumor activity of fivefractions by MTT assay and found that the IC

50values of the

Fr3 and Fr4 were lower than others (Table 1) So Fr3 and Fr4will be further fractionated by column chromatography

Fr3 was further separated by polyamide column chro-matography using 1 L H

2O-EtOH (10 0 8 2 6 4 4 6 2 8

and 0 10) and 35 NH3sdotH2O respectively to yield seven

fractions (Fr31ndashFr37) MTT assay showed that the IC50

value of Fr32 was the lowest one (Table 1) So Fr32 will befurther fractionated

Fr32 was dissolved in 1mL EtOH at 50∘C added in 1mLand at rest overnight Compound 1 was recrystallized andsupernatant was eluted by Sephadex LH-20 column with 1 L50 MeOH Compound 2 was separated and purified TheIC50

values for Chang-liver SMMC7721 and Bel7402 cellswere 7349 plusmn 1918 3589 plusmn 399 and 2431 plusmn 066 120583gmLfor compound 1 and 9335 plusmn 1199 1482 plusmn 100 and1552 plusmn 425 120583gmL for compound 2 respectively (Table 1)So compound 2 maybe is the best antitumor compound inthe Fr3

32 Spectrometric Identification of Two Compounds Chro-matographic purification of P niruri led to the isolation ofcompounds 1 and 2 Structural identification was performedby comparing the 1H- and 13C-NMR spectra with thosereported in the literature The structures of the componentsidentified are presented in Figure 2 and their spectral detailsare shown below These compounds were identified as ethylbrevifolincarboxylate (1) and corilagin (2)

Ethyl brevifolincarboxylate (1) 1H-NMR (600MHz DMSO-1198896) 120575 119 (3H t J = 7Hz -OCH

2CH3) 405 (2H q J = 7Hz

-OCH2CH3) 244 (1H dd 119869

119860119861= 18Hz 119869

119860119883= 2Hz H-10)

298 (1H dd 119869119860119861

= 18Hz 119869119861119883

= 7Hz H-10) 440 (1H dd119869119861119883

= 7Hz 119869119860119883

= 2Hz H-9) 729 (1H s Ar-H) 13C-NMR


Table 1 The IC50of Chang-liver and HCC cells treated with organic solvent-soluble fractions of P niruriData represent Means plusmn SE from 3

independent experiments

Components IC50(120583gmL)

Chang-liver SMMC7721 Bel740275 EtOH extract gt100 gt100 7186 plusmn 043Petroleum ether fraction (Fr1) 7463 plusmn 768 6849 plusmn 196 gt100CHCl

3fraction (Fr2) gt100 8442 plusmn 497 gt100

EtOAc fraction (Fr3) 6903 plusmn 533 6492 plusmn 604 5816 plusmn 327Fr31 gt100 4072 plusmn 632 gt100Fr32 8368 plusmn 341 2186 plusmn 060 2780 plusmn 409Fr321 gt100 5963 plusmn 1834 4420 plusmn 1355Fr322 gt100 6339 plusmn 559 6727 plusmn 723Compound 1 7349 plusmn 1918 3589 plusmn 399 2431 plusmn 066Compound 2 9335 plusmn 1199 1482 plusmn 100 1552 plusmn 425

Fr33 gt100 3028 plusmn 033 3488 plusmn 200Fr34 gt100 4152 plusmn 046 5741 plusmn 034Fr35 7698 plusmn 632 5073 plusmn 986 5676 plusmn 870Fr36 7070 plusmn 1267 gt100 gt100Fr37 7320 plusmn 651 gt100 gt100

n-BuOH fraction (Fr4) 9607 plusmn 1094 6855 plusmn 607 5425 plusmn 373Water fraction (Fr5) gt100 gt100 gt100

OO

OOO

O

O

OH

OHOH OH OH

OH

OH

HO

HO

HO

HO

1 2

OO O

O

O

OH

OH

HO

H3C

Figure 2 Chemical structures of isolated constituents from P niruri

(126MHz DMSO-1198896) 120575C 14577 (C=2) 13849 (C=3) 11493

(C=3a) 14363 (C=14020) 14960 (C=6) 10802 (C=7) 11298(C=7a) 16011 (C=8) 4069 (C=9) 3701 (C=10) 19302 (C=11)17199 (C=O) 6056 (C=12) and 1386 (C=13)

Corilagin (2) 1H-NMR (500MHz DMSO-1198896) 120575H 701 (2H

s H-21015840 and H-61015840) 655 (1H s H-610158401015840) 649 (1H s H-6101584010158401015840) 620(1H d J = 75Hz H1) 458 (1H br s H-3) 435 (1H t J =85Hz H-5) 422 (1H br s H-4) 421 (1H dd J = 110 85HzH-6) 394 (1H dd J = 110 85Hz H-6) and 387 (1H d J =71Hz H-2) 13C-NMR (126MHz DMSO-119889

6) 120575C 1672 and

1675 (C=O) 1456 (C-3101584010158401015840) 1453 (C-310158401015840) 1444 (C-5101584010158401015840) 1448(C-510158401015840) 1359 (C-4101584010158401015840) 1360 (C-410158401015840) 1160 (C-1101584010158401015840) 1163 (C-110158401015840)1235 (C-2101584010158401015840) 1244 (C-210158401015840) 1066 (C-6101584010158401015840) and 1074 (C-610158401015840)galloyl 120575C 1653 (C=O) 1460 (C-31015840 and C-51015840) 1395 (C-41015840)1192 (C-11015840) and 1095 (C-21015840 and C-61015840) glucose 120575C 927 (C-1)780 (C-3) 768 (C-5) 721 (C-2) 644 (C-6) and 626 (C-4)

33 Two Compounds Inhibited the Proliferation of CellLines To evaluate the cytotoxicity of corilagin Chang-liver

SMMC7721 Bel7402HepG2MHCC97-HOC316 SGC7901and QBC-939 cells were treated with increasing concentra-tions of corilagin (0 10 20 40 80 160 and 320120583M) for 48 hand the cell viability was detected by MTT assay Figure 3shows that corilagin induced a dose-dependent decrease inHCC cell viability and had a lower cytotoxicity to Chang-liver normal cell line (IC

5014716 plusmn 189 120583M) Figure 4 illus-

trates that corilagin has a strong broad-spectrum antitumoreffect

34 Corilagin Enhancing Drug Interaction with cDDP onSMMC7721 In Vitro Subsequently we analyzed the natureof the interaction between corilagin and the positive controlcDDP using CDI which quantitatively measures the inter-action of two drugs As shown in Figure 5 corilagin andcDDP yielded synergistic interactions on SMMC7721 acrossa wide concentration range The synergistic effect was mostprominent when 10 120583M corilagin was combined with 20120583McDDP (CDI = 077) This result suggests that corilagin canpromote the antitumor activity of cDDP


Chang-liver

Corilagin cDDP Ethyl brevifolincarboxylate

SMMC7721 Cell lines

Bel7402 0

100

200

300

400

IC50

(120583M

)

Figure 3 IC50value of two compounds and positive control cDDP

on HCC cell lines

SGC7901OC316 QBC939MHCC97-HHepG2Cell lines

0

10

20

30

40

IC50

(120583M

)

Figure 4 IC50value of corilagin on different cancer cell lines

35 The Content of Corilagin in Different Plants Accordingto the linear relation from peak area (119884) and concentration(119883) we could get the equation of the standard curve 119884 =103333 + 247423119883 (1198772 = 09998) The linear range was 0ndash125 120583gmL So we get the data of different corilagin contentof plants (Table 2) Results indicated that Dimocarpus longanLour seed contents the maximum corilagin up to 643 120583gg(Table 3)

4 Discussion

Over the last few years the P niruri has gained a reputationin folk and traditional medicine for its multiple healingproperties Previous study tried to induce liver cancer inmicethat had been pretreated with a water extract of P niruriThe results indicated the P niruri extract dose dependentlylowered tumor incidence levels of carcinogen-metabolizingenzymes levels of liver cancer markers and liver injury

cDDP

CDI

coeffi

cien

t of d

rug

inte

ract

ion

Corilagin 5120583M

Corilagin 10 120583M


402010

00

02

04

06

08

10

12

Figure 5 The synergistic antitumor activity of corilagin combinedwith cDDP on SMMC7721 cells CDI for the combination treatmentof corilaginwith cDDP on SMMC7721 cells Data representMeans plusmnSE from 3 independent experiments

markers [23] So the plant had a prospective antiproliferativeand antimetastasis effect against cancer compared to a directantitumor effect or selective ability to kill a cancer cell

In this study with bioguided fraction and isolationEtOAc fraction significantly inhibits the growth of HCC celllines Two compounds were obtained and identified as ethylbrevifolincarboxylate and corilagin Between them corilaginhad a lower IC

50value on HCC cell lines which indicates

that corilagin is the antitumor compound from P niruri [24]So bioguided fraction and isolation will make sure that theantitumor components from P niruri can be easier to beseparated

Corilagin a tannin was mostly focused on the antioxi-dant activity [25 26] whereas the antitumor activity was notmuch accounted for In this study our research suggestedthat corilagin has better antitumor activity broad-spectrumantitumor activity and less toxicity to normal cells and canpromote the antitumor activity of cDDP These data indicatethat corilagin can significantly inhibit the growth of HCCcells in vitro which is consistent with the most recent reportthat corilagin could retard the in vivo growth of xenograftedHep3B HCC cells [27] and induce G2M phase arrest [24]But the mechanisms of these synergistic interactions need tobe further studied

Besides the favorable antitumor activity corilagin also hasthe maximum content in Dimocarpus longan Lour among10 species of plants reported to contain corilagin up to643 120583gg which means that corilagin has rich sources andis easy to be acquired

5 Conclusions

With bioguided fraction and isolation we found thatP niruriL has great inhibiting effect on HCC cells Corilagin is the


Table 2 Experimental samples

Family Plant Part Acquisition place

Euphorbiaceae

Phyllanthus urinaria L Whole plant Gulangyu Islet ChinaP tenellus Roxb Whole plant Gulangyu Islet China

P debilis Klein ex Willd Whole plant Gulangyu Islet ChinaAcalypha australis L Whole plant Gulangyu Islet China

Polygonaceae Polygonum chinense L Leafy shoot Gulangyu Islet ChinaSaururaceae Saururus chinensis (Lour) Bail Leaf Gulangyu Islet ChinaCombretaceae Terminalia catappa L Leaf Danzhou China

Sapindaceae Dimocarpus longan Lour Fruit rind and seed Xiamen ChinaLitchi chinensis Sonn Fruit rind and seed Xiamen China

Punicaceae Punica granatum L Seed Xiamen China

Table 3 Content of corilagin from different samples (ND notdetected)

Plant Content (120583gg)Acalypha australis L 216Polygonum chinense L 376Saururus chinensis (Lour) Bail 573Terminalia catappa L 271Dimocarpus longan Lour (fruit rind) 569Dimocarpus longan Lour (seed) 643Phyllanthus urinaria L 619P tenellus Roxb 159P debilis Klein ex Willd 679Punica granatum L (Seed) NDLitchi chinensis Sonn (Fruit rind) NDLitchi chinensis Sonn (Seed) ND

major antitumor component that can significantly inhibit thegrowth of HCC cells and that shows low toxicity to normalcells So corilagin is the major active antitumor compositionin P niruri L on HCC cells with growth inhibition

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

Acknowledgments

This work was supported by grants from the National NaturalScience Foundation of China (no 81274149) and the XiamenMunicipal Science and Technology Innovation Fund Project(no 3502Z20130038) Lastly The authors would like torecognize the support of Professor Yong-Tian Zhang FujianInstitute of Subtropical Botany Xiamen 361006 China

References

[1] P Sharma J Parmar P Verma P Sharma and P Goyal ldquoAnti-tumor activity of Phyllanthus niruri (a medicinal plant) onchemical-induced skin carcinogenesis in micerdquo Asian PacificJournal of Cancer Prevention vol 10 no 6 pp 1089ndash1094 2009

[2] S Patel V Sharma N S Chauhan M Thakur and V K DixitldquoEvaluation of hair growth promoting activity of PhyllanthusnirurirdquoAvicenna Journal of Phytomedicine vol 5 no 6 pp 512ndash519 2015

[3] M N de Melo L A Lira Soares C R da Costa Porto et alldquoSpray-dried extract of Phyllanthus niruri L reduces mucosaldamage in rats with intestinal inflammationrdquo The Journal ofPharmacy and Pharmacology vol 67 no 8 pp 1107ndash1118 2015

[4] M Mohan P James R Valsalan and P Nazeem ldquoMoleculardocking studies of phytochemicals from Phyllanthus niruriagainst Hepatitis B DNA Polymeraserdquo Bioinformation vol 11no 9 pp 426ndash431 2015

[5] N Giribabu P V Rao K P Kumar S Muniandy S SRekha and N Salleh ldquoAqueous extract of Phyllanthus nirurileaves displays in vitro antioxidant activity and prevents theelevation of oxidative stress in the kidney of streptozotocin-induced diabetic male ratsrdquo Evidence-Based Complementaryand Alternative Medicine vol 2014 Article ID 834815 10 pages2014

[6] J B Calixto A R S Santos V Cechinel Filho and R A YunesldquoA review of the plants of the genus Phyllanthus their chemistrypharmacology and therapeutic potentialrdquo Medicinal ResearchReviews vol 18 no 4 pp 225ndash258 1998

[7] M Markom M Hasan W R W Daud H Singh and J MJahim ldquoExtraction of hydrolysable tannins from Phyllanthusniruri Linn effects of solvents and extraction methodsrdquo Sep-aration and Purification Technology vol 52 no 3 pp 487ndash4962007

[8] R F de Araujo Junior T P de Souza J G L Pires et al ldquoA dryextract of Phyllanthus niruri protects normal cells and inducesapoptosis in human liver carcinoma cellsrdquo Experimental Biologyand Medicine vol 237 no 11 pp 1281ndash1288 2012

[9] R Sailaja and O H Setty ldquoProtective effect of Phyllanthusfraternus against allyl alcohol-induced oxidative stress in livermitochondriardquo Journal of Ethnopharmacology vol 105 no 1-2pp 201ndash209 2006

[10] C-H Lai S-H Fang Y K Rao et al ldquoInhibition ofHelicobacterpylori-induced inflammation in human gastric epithelial AGScells by Phyllanthus urinaria extractsrdquo Journal of Ethnopharma-cology vol 118 no 3 pp 522ndash526 2008

[11] C O Okoli I C Obidike A C Ezike P A Akah and O ASalawu ldquoStudies on the possible mechanisms of antidiabeticactivity of extract of aerial parts of Phyllanthus nirurirdquo Pharma-ceutical Biology vol 49 no 3 pp 248ndash255 2011

[12] J S Londhe T P A Devasagayam L Y Foo and S SGhaskadbi ldquoRadioprotective properties of polyphenols from


Phyllanthus amarusLinnrdquo Journal of RadiationResearch vol 50no 4 pp 303ndash309 2009

[13] P Sharma J Parmar P Verma and P K Goyal ldquoModulatoryinfluence of Phyllanthus niruri on oxidative stress antioxidantdefense and chemically induced skin tumorsrdquo Journal of Envi-ronmental Pathology Toxicology and Oncology vol 30 no 1 pp43ndash53 2011

[14] K-W Ng S M Salhimi A M S A Majid and K-L ChanldquoAnti-angiogenic and cytotoxicity studies of some medicinalplantsrdquo Planta Medica vol 76 no 9 pp 935ndash940 2010

[15] M Sherman ldquoHepatocellular carcinoma epidemiology riskfactors and screeningrdquo Seminars in Liver Disease vol 25 no2 pp 143ndash154 2005

[16] A Mullard ldquo2010 FDA drug approvalsrdquo Nature Reviews DrugDiscovery vol 10 no 2 pp 82ndash85 2011

[17] H Daub K Specht and A Ullrich ldquoStrategies to overcomeresistance to targeted protein kinase inhibitorsrdquoNature ReviewsDrug Discovery vol 3 no 12 pp 1001ndash1010 2004

[18] V T Boeira C E Leite A A Santos Jr et al ldquoEffects of thehydroalcoholic extract of Phyllanthus niruri and its isolatedcompounds on cyclophosphamide-induced hemorrhagic cysti-tis inmouserdquoNaunyn-Schmiedebergrsquos Archives of Pharmacologyvol 384 no 3 pp 265ndash275 2011

[19] GAAsare K Bugyei A Sittie et al ldquoGenotoxicity cytotoxicityand toxicological evaluation of whole plant extracts of themedicinal plant Phyllanthus niruri (Phyllanthaceae)rdquo Geneticsand Molecular Research vol 11 no 1 pp 100ndash111 2012

[20] A Mediani F Abas A Khatib et al ldquoRelationship betweenmetabolites composition and biological activities of phyllanthusniruri extracts prepared by different drying methods andsolvents extractionrdquo Plant Foods for Human Nutrition vol 70no 2 pp 184ndash192 2015

[21] H Fujiki ldquoTwo stages of cancer prevention with green teardquoJournal of Cancer Research and Clinical Oncology vol 125 no11 pp 589ndash597 1999

[22] S-S Cao and Y-S Zhen ldquoPotentiation of antimetabolite anti-tumor activity in vivo by dipyridamole and amphotericin BrdquoCancer Chemotherapy and Pharmacology vol 24 no 3 pp 181ndash186 1989

[23] K J Jeena K L Joy and R Kuttan ldquoEffect of Emblicaofficinalis Phyllanthus amarus and Picrorrhiza kurroa on N-nitrosodiethylamine induced hepatocarcinogenesisrdquo CancerLetters vol 136 no 1 pp 11ndash16 1999

[24] Y Ming Z Zheng L Chen et al ldquoCorilagin inhibits hepato-cellular carcinoma cell proliferation by inducing G2M phasearrestrdquo Cell Biology International vol 37 no 10 pp 1046ndash10542013

[25] S Kinoshita Y Inoue S Nakama T Ichiba and Y Aniya ldquoAnti-oxidant and hepatoprotective actions of medicinal herb Termi-nalia catappa L from Okinawa Island and its tannin corilaginrdquoPhytomedicine vol 14 no 11 pp 755ndash762 2007

[26] Y Chen and C Chen ldquoCorilagin prevents tert-butylhydroperoxide-induced oxidative stress injury in culturedN9 murine microglia cellsrdquo Neurochemistry International vol59 no 2 pp 290ndash296 2011

[27] D K-P Hau G-Y Zhu A K-M Leung et al ldquoIn vivoanti-tumour activity of corilagin on Hep3B hepatocellularcarcinomardquo Phytomedicine vol 18 no 1 pp 11ndash15 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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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


[16 17] among patients to existing drugs lack of wideractivities and selectivity To overcome these the developmentof novel anticancer agents is an immense demand Previousreports had implied that P niruri is a candidate folk medicine[13 18 19]Howevermost of previous studies in vitrovivo justfocused on the crude extracts from P niruri [3 20]Themajoractive compounds in the crude extracts remain unknownLittle work has been performed to determine which activeconstituents from P niruri have antitumor activity Fujikiindicated that to investigate P niruri a useful and practicalmethod for screening preventive cancer agents is needed[21]

Thus due to the presence of variousmedicinal propertiesthe present study was designed to isolate screen and identifythe structure of really active components from P niruri anddetermine their biological activity on HCC

2 Materials and Methods

21 Plant Material Whole P niruri plants were collectedfrom Gulangyu Islet Fujian province China in October2009 and identified by Professor Yong-Tian Zhang FujianProvince Institute of Subtropical Botany China A voucherspecimen (YZY20091026) was deposited at XiamenOverseasChinese Subtropical Plant Introduction Garden China

22Chemicals andReagents All solvents andchemicals used forextraction in this study were of analytical grade and obtainedfrom Tianjin Reagent Company (Tianjin China) Materi-als for column chromatography included polyamide resin(100ndash200mesh Zhejiang Tetracarboxylic Biochemical Plas-tics Ltd China) and Sephadex LH-20 (Amersham Pharma-cia Biotech Sweden) The SMMC7721 Bel7402 MHCC97-H HepG2 OC316 SGC7901 QBC939 and Chang-livernormal liver cell line were obtained from the Cell Bankof the Chinese Academy of Sciences (Shanghai China)RPMI 1640 medium and Dulbeccorsquos Modified Eagle Media(DMEM)were obtained fromGibco (Grand IslandNY) The 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT) reagent was obtained from Sigma (St Louis MO)Trypsin Leibovitzrsquos L-15 medium fetal bovine serum (FBS)and penicillinstreptomycin solution (200x) were obtainedfromMediatech Inc (Herndon VA)

23 Chromatograph Conditions The chromate column wasWaters XBridge Shield RP18 (46mm times 250mm 5 120583m)DAD detector detection wavelength 280 nm column tem-perature 30∘C mobile phase Acetonitrile (A) 01 KH

2PO4

(B) flow speed 1mLmin injection volume 20 120583L Elutionprocedures are as follows 0ndash10min119881(A) 119881(B) = 15 85 10ndash15min119881(A) V(B) = 20 80 15ndash20minV(A) V(B) = 25 75

24 Standard Curve We weighed 25mg corilagin pre-cisely dissolved it in a 10mL measuring flask with 50methanol and prepared a standard solution whose concen-tration is 025mgmL diluted corilagin standard solutioninto 0025 0050 0075 0100 and 0125mgmL injected thediluted solution into HPLC and then determined absorption

spectrums We could get the linear relation from peak area(119884) and concentration (119883)

25 Preparation of Test Solution Five g sample powder wasextracted with 25mL of 95 ethanol at room temperaturefor three times (12 h each time) The solvent was evapo-rated in vacuo and the dried ethanol extract was dissolvedin 50 methanol (100mL) and then we filtered it with045 120583m membrane the subsequent filtrate was the testsolution

26 General Procedure Compounds were purified on poly-amide column D101 MCI gel and Sephadex LH-20 columnanalyzed qualitatively by TLC and quantitatively on ODS-C18 column (150 times 46mm) with a UV detector and MeOH-water (7 3) as mobile phase and determined by HPLC IRspectra were obtained with a Nicolet AVAIAR 360 FT-IRspectrometer with KBr pellets The1H- 13C- and 2D-NMRspectra were recorded on Bruker AV-400 spectrometers atroom temperature (120575 in ppm 119869 in Hz) Mass spectrometrywas carried out on a VG AutoSpec-3000 spectrometer or aFinnigan MAT 90 instrument EI-MS measurements werecarried out on an LCQ-Finnigan instrument at room temper-ature

Column chromatography was performed with polyamideresin (100ndash200mesh) and Sephadex LH-20 Fractions weremonitored using TLC and spots were visualized by heatingsilica gel plates or spraying with 5 H

2SO4in ethanol

27 Extraction Fractionation and Separation of AntitumorCompounds The dried and powdered whole P niruri (3 kg)were extracted with 75 EtOH (3 times 5 L) The 75 EtOHextract was combined and evaporated under reduced pres-sure to yield a residue Then it was suspended in waterand partitioned with petroleum ether CHCl

3 EtOAc and

n-BuOH (3 1 vv for 3 times) resulting in five fractionspetroleum ether fraction (Fr1 95 g) CHCl

3fraction (Fr2

58 g) EtOAc fraction (Fr3 48 g) n-BuOH fraction (Fr41137 g) and water fraction (Fr5 62 g) After that the cyto-toxicity of five fractions on HCC cell lines was determinedby MTT assay Fraction showing the highest cytotoxicity wasfurther fractionated by column chromatography each time

Fr3 was separated by polyamide column chromatographyusing a gradient of H

2O-EtOH (10 0 8 2 6 4 4 6 2 8

and 0 10) and then 35 NH3-H2O to yield seven fractions

(Fr31ndashFr37) For Fr32 the solvent was allowed to slowlyevaporate causing the compound to precipitate which wasthen collected by filtration and recrystallized to yield yellowdiamond flake crystallization (compound 1 126mg) Thefiltrate was then separated by chromatography on a SephadexLH-20 column and eluted with MeOH to obtain compound2 (430mg) (Figure 1)

28 Cell Culture Cells were cultured in RPMI 1640(SMMC7721 Bel7402 SGC7901 OC316 and QBC939) orDMEM (Chang-liver HepG2 and MHCC97-H) supple-mented with 10 FBS penicillin (100UmL) and strepto-mycin (100 120583gmL) in a humidified incubator aerated with5 CO

2and 95 air at 37∘C


75 EtOH extract




Fr32

Compound 1

Recrystallization

Compound 2

Filtrate

Sephadex LH-20 MeOH



16 mg43mg




CHCl3 fraction





3 Results


3



50values of the



2O-EtOH (10 0 8 2 6 4 4 6 2 8








2CH3) 405 (2H q J = 7Hz

-OCH2CH3) 244 (1H dd 119869

119860119861= 18Hz 119869

119860119883= 2Hz H-10)

298 (1H dd 119869119860119861

= 18Hz 119869119861119883

= 7Hz H-10) 440 (1H dd119869119861119883

= 7Hz 119869119860119883

= 2Hz H-9) 729 (1H s Ar-H) 13C-NMR










OO

OOO

O

O

OH

OHOH OH OH

OH

OH

HO

HO

HO

HO

1 2

OO O

O

O

OH

OH

HO

H3C


(126MHz DMSO-1198896) 120575C 14577 (C=2) 13849 (C=3) 11493




6) 120575C 1672 and








Chang-liver


SMMC7721 Cell lines

Bel7402 0

100

200

300

400

IC50

(120583M

)


on HCC cell lines


0

10

20

30

40

IC50

(120583M

)



4 Discussion


cDDP

CDI

coeffi

cien

t of d

rug

inte

ract

ion

Corilagin 5120583M



402010

00

02

04

06

08

10

12








5 Conclusions





Euphorbiaceae









Competing Interests


Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















75 EtOH extract




Fr32

Compound 1

Recrystallization

Compound 2

Filtrate

Sephadex LH-20 MeOH



16 mg43mg




CHCl3 fraction





3 Results


3



50values of the



2O-EtOH (10 0 8 2 6 4 4 6 2 8








2CH3) 405 (2H q J = 7Hz

-OCH2CH3) 244 (1H dd 119869

119860119861= 18Hz 119869

119860119883= 2Hz H-10)

298 (1H dd 119869119860119861

= 18Hz 119869119861119883

= 7Hz H-10) 440 (1H dd119869119861119883

= 7Hz 119869119860119883

= 2Hz H-9) 729 (1H s Ar-H) 13C-NMR










OO

OOO

O

O

OH

OHOH OH OH

OH

OH

HO

HO

HO

HO

1 2

OO O

O

O

OH

OH

HO

H3C


(126MHz DMSO-1198896) 120575C 14577 (C=2) 13849 (C=3) 11493




6) 120575C 1672 and








Chang-liver


SMMC7721 Cell lines

Bel7402 0

100

200

300

400

IC50

(120583M

)


on HCC cell lines


0

10

20

30

40

IC50

(120583M

)



4 Discussion


cDDP

CDI

coeffi

cien

t of d

rug

inte

ract

ion

Corilagin 5120583M



402010

00

02

04

06

08

10

12








5 Conclusions





Euphorbiaceae









Competing Interests


Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























OO

OOO

O

O

OH

OHOH OH OH

OH

OH

HO

HO

HO

HO

1 2

OO O

O

O

OH

OH

HO

H3C


(126MHz DMSO-1198896) 120575C 14577 (C=2) 13849 (C=3) 11493




6) 120575C 1672 and








Chang-liver


SMMC7721 Cell lines

Bel7402 0

100

200

300

400

IC50

(120583M

)


on HCC cell lines


0

10

20

30

40

IC50

(120583M

)



4 Discussion


cDDP

CDI

coeffi

cien

t of d

rug

inte

ract

ion

Corilagin 5120583M



402010

00

02

04

06

08

10

12








5 Conclusions





Euphorbiaceae









Competing Interests


Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Chang-liver


SMMC7721 Cell lines

Bel7402 0

100

200

300

400

IC50

(120583M

)


on HCC cell lines


0

10

20

30

40

IC50

(120583M

)



4 Discussion


cDDP

CDI

coeffi

cien

t of d

rug

inte

ract

ion

Corilagin 5120583M



402010

00

02

04

06

08

10

12








5 Conclusions





Euphorbiaceae









Competing Interests


Acknowledgments


References



































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Euphorbiaceae









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
















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