Ali, Abdul Manaf

Lajis, Md Nordin

Mackeen, Muhammad Mukram

Universiti Putra Malaysia Institutional Repository

UPM Research Report 1997-2000, Section 2-Extended AbstractsAntiviral and Cytotoxic Activities of Some Plants Used in Malaysian Indigenous MedicineAl7dul Manaf Ali", Muhammad Mukram Mackeen' and Nordin HJ. Lajis2Department of BiotechnologyFaculty of Food Science and BiotechnologyUniversiti Putra Malaysia43400 UPM. Serdang. SelangorMalaysiaEsmail of CorrespondingAuthor:manaf@putra.upm.edu.myKey words: antiviral activity, cytotoxicity, herpes simplex virus rype-I, plant extracts, vesicular stomatitis virus.IntroductionPlants are an important source oftherapeutics from which 25% of thepharmaceuticals in current use havebeen derived (Farnsworth et al.andBingel. 1977). However. of the esti-mated 250.000 species of higher plantsexisting throughout the world. only afraction has been examined for phar-macological activities (Balick et al.,1990). Phytotherapeutics exhibit awide range of pharmacological activi-ties. which among others include anti-cancer and antiviral activities (Farns-worth et al. and Kaas, 1981; Hudson.1989). Anticancer drugs. such as theindole alkaloids vincristine and vin-blastine. and podophyllotoxin deriva-tives etoposide and teniposide, areprominent chemotherapeutics of plantorigin which were either obtained di-rectly through isolation or derived fromlead structures (Arcamone et al .• 1980).Therefore. the screening of higherplants for antiviral and anticanceragents has been actively pursued on aninternational scale especially by the U.S. National Cancer Institute (Farns-worth et al. and Kaas, 1981; Hudson.1989). Furthermore. mammalian cellculture systems have greatly aided theroutine screening of plant extracts andcompounds for anticancer activity us-ing cytotoxicity and antiviral activity.which previously relied upon time-consuming. expensive and cumber-some in-vivo models. These screeningefforts have resulted in the discovery ofseveral prospective antiviral and anti-cancer compounds currently undergo-ing clinical trials of which taxol is themost notable example (Wiernik et al .•1987).In the present work we screened 61local and introduced plant specieswidely used as anti-infective and anti-cancer agents in Malaysian indigenousmedicine. comprising traditional.232ethno- and folk-medicine. for antiviraland cytotoxic activities. We adoptedan ethnopharmacological approach toscreening because it is more likely toyield a higher number of plants withsignificant biological activity thanscreening by random selection (Balicket al., 1990). The ethanolic extracts ofthese medicinal plants were tested forantiviral activities against both herpessimplex type-I (DNA type) and ve-sicular stomatitis (RNA type) virusesusing Vero cells. and the cytotoxicityassay was done using the HeLa (humancervical carcinoma) cell line.Materials and MethodsPlant ExtractsTwenty mg samples of leaves fromeach plant (and fruits from Cerberamanghas) were sliced into small pieces(ca. I cm x 1 cm) and macerated in 60ml of 80% (v/v) ethanol. After beingleft for one week at room temperature.the extracts were filtered using filterpaper (Whatman No. I) and thenevaporated at 40°C under vacuum.Cultivation of CellsVero and HeLa cell lines were obtainedfrom the RIKEN Cell Bank. Tsukuba,Japan and cultured in RPMI-1640 me-dium supplemented with 5% (v/v) foe-tal calf serum (FCS). 100 IV/ml peni-cillin and lOOug/ml streptomycin as acomplete growth medium (CGM).Virus StocksHerpes simplex virus type-I (HSV-I)and vesicular stomatitis virus (VSV).which are DNA and RNA virus typerespectively were obtained form theDepartment of Medicinal Chemistry.College of Pharmacy. University ofMinnesota. Minnesota. USA. Theantiviral test was performed accordingto the simplified plaque reduction assay(Abou-Karam et al. & Shier et al.,1990).Cytotoxicity AssayThe assay used was the microtitrationcytotoxicity assay (Shier et al., 1983).Using an inverted microscope (lowpower). cytotoxicity was determined asthe concentration of plant extract.which reduced cell number by ca. 50%with reference to the control (COso.mg/ml).Results and DiscussionThe overall results of the 61 plantsfrom 33 families screened for antiviraland cytotoxic activities. 28 species(46%) that gave negative results for allthree tests. 26 species (43%) that ex-hibited antiviral activity and the 18species (30%). which showed cytotox-icity. Eight species (13%). i.e. Calotro-pis gigantea. Costus speciosus,Eugenia michelii, Hedyotis auricularia.Mentha arvensis, Orthosiphon arista-tus, Polygonum minus and Ricinuscommunis showed antiviral activityagainst both HSV-! and VSV. Theextracts of Calotropis gigantea,Eugenia michelii and Ricinus commu-nis showed a similar MIC value ofO.OImg/rnl against both viruses but onlyEugenia michelii demonstrated cyto-toxicity (COso: 0.05 mg/ml). In thecase of Polygonum minus. a similarMIC value against HSV-I was ob-tained but lesser activity was shownagainst VSV (MIC: 0.02 mg/ml) andHeLa cells (COso: 0.1 mg/ml). Verystrong anti HSV-I but weak anti-VSVand cytotoxic activities were observedin the Mentha arvensis extract (MIC:0.002 mg/ml & 0.1 mg/ml: and COso:0.1 mg/ml, respectively). On the otherhand. strong anti-VSV but weak antiHSV-I activities were displayed by theextracts of Costus specious and He-dyotis auricularia. while weak antiviralactivity against both viruses was ob-served in the extract of Orthosiphonaristatus (MIC: 0.1 mg/ml).Selective antiviral activity towardsonly HSV-l was seen in extracts fromBiological Sciencesseven plants (11%). viz. (in order ofdecreasing activity) Altemantherasessilis, Blumea chinensis, Freycinetiamalaccensis. Leea indica. Euphorbiahirta, Eleusine indica. and Solanumamericanum with MIC values withinthe range of 0.001-0.1 mg/m!. Con-versely. 11 plant (18%) extracts pos-sessed selective antiviral activityagainst VSV. viz. (in order of decreas-ing activity) Bertholletia excelsa,Acalypha indica. Piper sarmentosum,Oenanthe javanica, Mirabilis jalapa.Premna odorata. Cerbera manghas,Codiaeum variegatum, Plectranthusamboinicus. Centella asiatica andMorinda elliptica with MIC valuesfrom 0.005 - 0.1 mg/ml, More plantextracts were active against VSV thanHSV-l. The selective antiviral activityof some plant extracts against eitherHSV -Lor VSV implicates the in-volvement of different mechanisms ofaction exploiting the difference in nu-cleic acid composition of the viruses.In the case of the anti HSV-l species.only Freycinetia malaccensis showedcytotoxicity (CDso: 0.1 mg/ml) whereasseven of the anti- VSV extracts showedcytotoxicity. i.e. (in order of decreasingactivity) Cerbera manghas, Morindaelliptica. Acalpyha indica. Centellaasiatica. Codiaeum variegatum, Mi-rabilis jalapa and Piper sarmentosumranging from CDso 0.001-0.1 mg/m!.Since VSV is a RNA-type virus. theconcomitant anti-VSV and cytotoxicactivities may involve a related modeof action. most probably via proteininteraction. Co-existing antiviral andcytotoxic activities were found in theextracts of three species (5%). i.e.Eugenia michelii, Mentha arvensis andpolygonum minus.Of the 18 plants showing cytotoxicity.only three species (Acalypha indica.Cerbera manghas and Morinda ellip-tica) showed significant activity belowthe cut-off value of 0.02 mg/ml sug-gested by Wall et al. (1987) and allthree species exhibited anti- VSV ac-tivity. The strongest cytotoxic activitywas shown by the fruits of Cerberamanghas (CDso: 0.001 mg/ml). Thefruits of Cerbera manghas always ex-hibited stronger cytotoxic (20 times)and anti- VSV activities (twice the ac-tivity) than its leaves. This suggeststhat a higher concentration of the bio-active compound(s) is present in thefruits of Cerbera manghas than theleaves.ConclusionsThe results of this preliminary study toa certain extent scientifically substanti-ate the pharmacological activities of33 plants used in Malaysian indigenousmedicine and point out some plantswith potential for further investigation.In addition. these results may also con-tribute towards the documentation ofpharmacological profiles of Malaysianplants for conservation efforts andprotection of biodiversity rights. In-adequate record of the pharmacologicalactivities of Malaysian plants may leadto the commercial exploitation of tra-ditional knowledge by foreign partieswithout any benefit to the country asexperienced by India in the case of theNeem tree and tumeric (Agarwal et al.and Narain, 1996).Benefits from the studyCytotoxic and antiviral assays wereestablished in the laboratory. In thepresent work 61 species of local andintroduced plant species widely used asanti-infective and anticancer agents inMalaysian indigenous medicine. com-prising traditional. ethno- and folk-medicine. for antiviral and cytotoxicactivities.Literature cited in the textAbou-Karam, M. and. Shier. W.T. 1990. Asimplified plaque reduction assay forantiviral agents from plants.Demonstration of frequent occurrence ofantiviral activity in higher plants. Journalof Natural Products. 53: 340·344.Agarwal. A. and Narain. S. 1996. Pirates inthe garden of India. New Scientist. 152:14-15.Arcamone, F.• Cassinelli. G. and Casazza.A.M. 1980. New antitumor drugs fromplants. Journal of Ethnopharmacology.2: 149-160.Balick, M.J. 1990. Ethnobotany and theidentification of therapeutic agents fromthe rainforest. In: Bioactive Compoundsfrom Plants. Eds. D. J. Chadwick and J.Marsh. p. 22-29. Chichester: John Wileyand Sons.Farnsworth. N.R. and KAAS. C.J. 1981.An approach utilizing information fromtraditional medicine to identify tumor-inhibiting plants. Journal ofEthnopharmacology. 3: 85-99.Farnsworth. N.R. and. Bingel. A.S. 1977.Problems and prospects of discoveringnew drugs from higher plants bypharmacological screening. In: NewNatural Products and Plant Drugs withPharmacological. Biological orTherapeutical Activity. eds. H. Wagnerand P. Wolff. p. 61-73. Berlin: Springer-Verlag.Shier. W.T. 1983. An undergraduateexperiment to demonstrate the use ofcytotoxic drugs in cancer chemotherapy.American Journal of PharmaceuticalEducation. 47: 216·220.Wiernik, P.H.• Schwartz. EL. Strauman,U.•Dutcher. J.P .• Lipton. R.B. andPaaietta, E. 1987. Phase I clinical andpharrnacokinetic study of taxol-1. CancerResearch. 47: 2486-2493.Project Publications inRefereed JournalsAli. A.M .•Mackeen, M.M .•Aun, Q.B .•Yusuf, Z .•Hawariah, A. & Kawazu, K.1997. Cytotoxicity and cell death inducedby goniothalamin. Planta Medica. 63: 81-83.Ali. A.M .• El-Sharkawy, S.H .. Hamid. l.A .•Mackeen, M.M .•Nur-Hadiani, I.. Ahmad.F.B. & Lajis, N.H. 1995. Antiviral andcytotoxic activities of ethanolic extract ofsome Malaysian medicinal plants.PERTANlKA Journal of TropicalAgriculture Science. 18: 57-61.Ali. A.M .• El-Sharkawy. S.H .• Hamid. J.A ..Nur-Hediani, I. & Lajis, N.H. 1995.Antimicrobial activity of someMalaysian plants. Pertanika Journal ofTropical Agriculture Science. 18: 49-52.Ali. A.M .• EI-Sharkawy. S.H .• Salleh, K.M .•Lajis, N.H. & Kawazu, K. 1997.Anticancer and antimicrobial properties ofsome Malaysian traditional vegetables("Ulam"). International Journal ofPharmacognosy. 35: 1-5.Ali. A.M .•Mackeen, M.M .• Intan-Safinar,I..Hamid. M.• Lajis, N.H .. El-Sharkawy,S.H. & Murakoshi, M. 1996. Anti-tumorpromoting and anti-tumor activities of thecrude extract from leaves of Juniperuschinensis. Journal of Ethnopharmacology53: 165-167.El-Sharkawy, S.•Yusuf, Z .•Pihie, A.H.L .•Ali. A.M. 1996. Metabolism ofgoniothalamin and microbial system. Boll.Chim. Farmaceutico. 135: 35-40. M.M.MackeenEl-Sharkawy, S.H .• Ali. A.M. & Mat, N.B.1995. Supercritical fuid extraction ofcolor from Bixa orellana seeds.Alexandria Journal of PharmaceuticalScience. 9: 155-158.Mackeen, M.M. Ali. A.M .•Abdullah. M.A .•Nasir. R.M .. Mat. N.B .. Razak, A.R. & K.Kawazu .1997. Antinematodal activity ofsome Malaysian plant extracts against the233UPM Researeh Report 1997·2000. 5«tJon 2-ExtendtHI Abstractspine wnod nemalode, B,,,JDpNl,IICI,,..."yluplll/lU. fmk'uU Sc"IIC' ~I: IM·170.Project Publications InConference ProceedingsNone.234Graduate ResearchInllUl SafilllU' "mall. IIJ%. Natural pruductlbtocec:hnol,'IY (MS). Univcraiti PutraMalAy,ill.Muhamrnacl Mukram Mackeen. 1'J9!1.Natural prudu,b biutechnolollY (MSI.Unive"iti Putra Mlllay.ia.Shar Mariam Mohamed. 2(X)O. Naturalproduct~ biolCchnolollY (PhD). UnivcnitiPutra Malaysill.Research funded by the Intenslflceuon of Rehtllrchin Priority llrell~ (lRPA) • Supported by IRPA RM.7(19'J6·20()O), Cycle! 996, Grant O)·()2·04·(J045.

Antiviral and cytotoxic activities of some plants used in Malaysian indigenous medicine

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Antiviral and cytotoxic activities of some plants used in Malaysian indigenous medicine

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