Pertanika J. Trop. Agric. Sci. 19(2/3): 129-136 (1996)ISSN: 0126-6128

© Penerbit Universiti Pertanian Malaysia

Antiviral and Cytotoxic Activities of Som.e Plants Used in MalaysianIndigenous Medicine

ABDUL MANAF ALI,I. MUHAMMAD MUKRAM MACKEEN, SALEH H. EI-SHARKAwy l,

JUNAINAH A. HAMIDI, NOR HADIANI ISMAILI, FAUJAN B. H. AHMADI and NORDIN H. LAJISIDepartment of Biotechnology

1Department of ChemistryUniversiti Pertanian Malaysia

43400 Serdang, Selangor, Malaysia

Keywords: antiviral activity, cytotoxicity, HeLa cell, herpes shnplex virus type-I, plant extracts,vesicular stomatitis virus

ABSTRAK

Ekstrak etanol 61 tumbuhan perubatan yang digunakan di Malaysia telah disaring untuk aktiviti antivirus andsitotoksik. Aktiviti antivirus telah diuji terhadap virus "herpes simplex"-jenis 1 (HSV-1) , dan "vesicularstomatitis" (VSV), dan ujian sitotoksik dijalankan menggunakan sel-sel HeLa. Ekstrak Calotropis gigantea,Costus speciosus, Eugenia rnichelii, Hedyotis auricularia, Mentha arvensis, Orthosiphon aristatus,Polygonum minus dan Ricinus communis menunjukkan aktiviti perencatan terhadap kedua-dua virus (MIG:0.002-0.1 mg/ml). Aktiviti antivirus khusus terhadap virus HSV-1 telah ditunjukkan oleh ekstrak Alternantherasessilis, Blumea chinensis, Eleusine indica, Euphorbia hirta, Freycinetia malaccensis, Leea indica danSolanum americanum (0.001-0.1 mg/ml). Ekstrak Acalypha indica, Bertholletia excelsa, Cerberamanghas, Codiaeum variegatum, Plectranthus amboinicus, Centella asiatica, Mirabilis jalapa,Morinda elliptica, Oenanthe javanica, Piper sarmentosum dan Premna odorata menunjukkan aktivitiantivirus khusus terhadap virus VSV (MIG: 0.005-0.1 mg/ml). Aktiviti sitotoksik pula hadir dalam ekstrakAcalypha indica, Andrographis paniculata, Centella asiatica, Cerbera manghas, Codiaeumvariegatum, Cosmos caudatus, Elephantopus scaber, Etlingera elatior, Eugenia michelii,Freycinetia malaccensis, Hibiscus rosa-sinensis, Lecythis ollaria, Mentha arvensis, Mirabilisjalapa, Morinda elliptica, Ocimum tenuiflorum, Piper sarmentosum dan Polygonum minus(GDso: 0.001-0.1 mg/ml). Kedua-dua aktiviti antivirus and sitotoksik ditunjukkan oleh ekstrak Eugeniamichelii, Mentha arvensis dan Polygonum minus.

ABSTRACT

Ethanolic extracts of 61 medicinal plants used in Malaysia were screened for antiviral and cytotoxic activities.Antiviral activity was tested against the herpes simplex type-1 (HSV-1) and vesicular stomatitis (VSV) viruses,and cytotoxicity was assayed using the HeLa cell line. Antiviral activity against both viruses was present in theextracts from Calotropis gigantea, Costus speciosus, Eugenia michelii, Hedyotis auricularia, Menthaarvensis, Orthosiphon aristatus, Polygonum minus and Ricinus communis (MIG: 0.002-0.1 mg/ml).The extracts from Alternanthera sessilis, Blumea chinensis, Eleusine indica, Euphorbia hirta,Freycinetia malaccensis, Leea indica and Solanum americanum were active in selectively inhibitingHSV-1 (0.001-0.1 mg/ml). Selective activity against VSV was shown by the extracts from Acalypha indica,Bertholletia excelsa, Cerbera manghas, Codiaeum variegatum, Plectranthus amboinicus, Centellaasiatica, Mirabilis jalapa, Morinda elliptica, Oenanthe javanica, Piper sarmentosum and Premnaodorata (MIG: 0.005-0.1 mg/ml). Gytotoxic activity was present in the extracts from Acalypha indica,Andrographis paniculata, Cerbera manghas, Codiaeum variegatum, Cosmos caudatus, Elephanto­pus scaber, Etlingera elatior, Eugenia michelii, Freycinetia malaccensis, Hibiscus rosa-sinensis,

* author to whom all correspondence should be addressed

ABDUL MANAF, MUHAMMAD MUKRAM, SALEH, JUNAINAH, NOR HADIANI, FAUJAN AND NORDIN

Cen~ella asiati~a, Lecythis ollaria, Mentha arvensis, Mirabilis jalapa, Morinda elliptica, OcimumtenUlflorum, PIper sarmentosum and Polygonum minus (CD50: 0.001-0.1 mgjml).Co-existing antiviraland cytotoxic activities were shown by Eugenia michelii, Mentha arvensis and Polygonum minus.

INTRODUCTION

Plants are an important source of therapeu­tics from which 25% of the pharmaceuticalsin current use have been derived (Farns­worth and Bingel 1977). However, of theestimated 250,000 species of higher plantsexisting throughout the world, only afraction have been examined for pharmaco­logical activities (Balick 1990). Phytothera­peutics exhibit a wide range of pharmaco­logical activities, including anticancer andantiviral activities (Farnsworth and Kaas1981; Hudson 1989). Anticancer drugs, suchas the indole alkaloids vincristine andvinblastine, and podophyllotoxin deriva­tives etoposide and teniposide, are promi­nent chemotherapeutics of plant originwhich were obtained either directlythrough isolation or derived from lead

,structures (Arcamone et al. 1980). There­fore, the screening of higher plants forantiviral and antic'ancer agents has beenactively pursued on an international scale,especially by the US National CancerInstitute (Farnsworth and Kaas 1981;Hudson 1989). Furthermore, mammaliancell culture systems have greatly aided theroutine screening of plant extracts andcompounds for anticancer activity usingcytotoxicity and antiviral activity, whichpreviously relied upon time-consuming,expensive and cumbersome in-vivo models.These screening efforts have resulted in thediscovery ofseveral prospective antiviral andanticancer compounds currently undergoingclinical trials. Taxol is the most notableexample of these compounds (Wiernik et al.1987). "

Although extensive phytochemical sur­veys have been carried out on the flora ofMalaysia (Goh et ai. 1993 and referencescited therein), only a few reports deal with

screening for pharmacological activitiessuch as antimicrobial, antitumour, anti­tumour-promoting and cardiovascular­related activities (Nakanishi et ai. 1965;Yadav et ai. 1989; Ali et ai. 1995; Goh et ai.1995). Reports by Teo et ai. (1990) andreferences cited therein, Ahmad et ai. (1992,1993), Chan et ai. (1992), Kashman et ai.(1992), Mahmud et ai. (1993), Patil et ai.(1993), Alias et ai. (1995), Ali et ai. (1996),and Wong and Tan (1996) are examples ofstudies confined to the antiviral andcytotoxic activities of extracts and com­pounds from one or two plant species;consequently, these studies cannot beconsidered as screening reports.

In the present work we screened 61local and introduced plant species widelyused as anti-infective and anticancer agentsin Malaysian indigenous medicine (tradi­tion~.L ethno- and folk-medicine) for anti­viFal and cytotoxic activities. We adoptedan ethnopharmacological approach toscreening because it is more likely to yielda higher number of plants with significantbiological activity than screening by ran­dom selection (Balick 1990). The ethanolicextracts of these medicinal plants weretested for antiviral activities against bothherpes simplex type-1 (DNA type) andvesicular stomatitis (RNA type) virusesusing Vero cells, and the cytotoxicity assaywas done using the HeLa (human cervicalcarcinoma) cell line.

MATERIALS AND METHODS

Piants

Plant parts were collected from the Medi­cinal Plant Garden, Universiti PertanianMalaysia, and identified by A. GhaniYunus.

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ANTIVIRAL AND CYTOTOXIC ACTIVITIES OF SOME PLANTS USED IN MALAYSIAN INDIGENOUS MEDICINE

Plant Extracts

Samples (20 mg) of leaves from each plant(and fruits from Cerbera manghas) were slicedinto small pieces (ca. 1 cm x 1 cm) andmacerated in 60 ml of 80% (v/v) ethanol.After being left for one week at room tem­perature, the extracts were filtered usingWhatman No. 1 filter paper and thenevaporated at 40°C under vacuum. Theresidues were then stored as stock solutionsof 10 mg/ml in 90% (v/v) ethanol at 4°C.

Cultivation of Cells

Vero and HeLa cell lines were obtainedfrom the RIKEN Cell Bank, Tsukuba,Japan and cultured in RPMI-1640 med­ium supplemented with 5% (v/v) foetal calfserum (FCS), 100 IU/ml penicillin and 100Ilg/ml streptomycin as a complete growthmedium (CGM). Cells were maintained in25 cm2 flask with 10 ml of CGM at 37°Cwith 5% (v/v) CO2 until attaining con­fluence. Confluent cells were removed fromthe surface of the flask by tre~tmentwith 1ml of 0.025% (w/v) trypsin prepared inphosphate-buffered saline (PBS) solution.CGM was then-_ added to the trypsin­treated cells to achieve a cell concentrationof 1-2 x 104 cells/ml.

Virus Stocks

Herpes simplex virus type-1 (HSV-1) andvesicular stomatitis virus (VSV), which areDNA and RNA virus type respectively,were obtained from the Department ofMedicinal Chemistry, College of Phar­macy, University of Minnesota, Minneso­ta, USA. Virus stocks were prepared asaliquots of culture medium from Vero cellsinfected at a multiplicity of infection of 0.1and cultured for 3 days at 37°C. Thesealiquots were subsequently stored at -70°C.Working stocks of virus were prepared byserially diluting in culture medium(RPMI-1640) virus stocks to the end­points required for each virus. Serial

dilutions of virus stocks in RPMI-1640medium were assayed to their end-pointsusing Vero monolayers in microtitre plates.These virus working stocks were stored at4°C until further use.

Antiviral Assay

The antiviral test was performed accordingto the simplified plaque reduction assay(Abou-Karam and Shier 1990). Microtitreplates with confluent monolayer cultures ofVero cells were inverted to remove spentmedium. In triplicate, each well was filledwith 100 III of plant extract serially dilutedin RPMI-1640 medium. This was followedby the addition of 100 III of mediumcontaining ca. 30 plaque forming units(pfu) of HSV-1 or 10 pfu of VSV, perwell of confluent Vero cells. In each plate,wells in the last row were used for controls,which consisted of two treatments: (1) cellsnot treated with plant extracts and virus,and (2) cells treated only with virus. Theplates were incubated for 66 h (HSV-1)and 36 h (VSV) at 37°C, with care takennot to disturb the culture during incuba­tion. Antiviral activity was then scoredusing an inverted microscope (low power)as the non-cytotoxic minimum inhibitoryconcentration (MIC, mg/ml) which totallyprevented cytopathic effects (CPE).

Cytotoxicity Assay

The assay used was the microtitrationcytotoxicity assay (Shier 1983). Varyingconcentrations of the plant extracts wereprepared from the stock solutions by serialdilution in RPMI-1640 medium to give atotal volume of 100 III in each well. Eachwell was filled with 100 III of HeLa cellsuspension in CGM at 1-2 x 104 cells/ml.Controls containing only HeLa cells wereincluded for each sample. The assay foreach concentration of plant extract wasperformed in triplicate and the cultureplates were kept at 37°C with 5% (v/v)

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ABDUL MANAF, MUHAMMAD MUKRAM, SALEH, JUNAINAH, NOR HADIANI, FAUJAN AND NORDIN

TABLE 1Species failing to show either antiviral or cytotoxic

*28 activity

Anti-Hela

Fig. 1. The number ofplant species showing antiviraland cytotoxic activities are in the circles. Overlapping

circles indicate co-existing activities. (*Species that didnot show any activity)

Species

MyrtaceaeOxalidaceaePiperaceae

Leguminosae

BixaceaeBombaceaeCommelinaceaeEuphorbiaceaeGramineaeLabiatae

Family

against VSV (MIC: 0.02 mg/ml) andHeLa cells (CDso: 0.1 mg/ml). Very stronganti HSV-l but weak anti-VSV andcytotoxic activities were observed in theMentha arvensis extract (MIC: 0.002 mg/ml& 0.1 mg/ml; and CDso: 0.1 mg/ml,respectively). On the other hand, stronganti-VSV but weak anti HSV-l activitieswere displayed by the extracts of Costusspecious and Hedyotis auricularia, while weakantiviral activity against both viruses wasobserved in the extract of Orthosiphonaristatus (MIC: 0.1 mg/ml).

Selective antiviral activity towards only

Amaranthaceae Aerva lanata (L.) Juss.Anacardiaceae Spondias cytherea SonneratApocynaceae Hunteria zeylanica (Retz.) Gardn.

& Thw.Plumeria rubra L.Bixa orellana L.Ceiba pentandra Gaertn.Tradescantia spathacea Sw.Euphorbia neriifolia L.Cymbopogon citratus (DC.) StapfPlectranthus scutellaroides (L.)R. Br.Caesalpinia pulcherrima (L.) Sw.Cassia alata L.Cassia fistula L.

Liliaceae Allium schoenoprasum L.Lythraceae Lawsonia inermis L.Menispermaceae Tinospora crispa (L.) Hook.

f & Th.Eugenia polyantha WightAverrhoa carambola L.Peperomia pellucida KunthPiper nigrum L.

Plantaginaceae Plantago major L. s.l.Rubiaceae Gardenia augusta (L.) Merr.Sapotaceae Mimusops elengi L.Scrophulariaceae Picria fel-terrae Lour.Solanaceae Datura metel L

Datura inoxia Mill.Zingiberaceae Alpinia galanga (L.) Sw.

Curcuma mangga Val. & Van Zyp,

5

Anti-VSV

6

Anti-HSV-l

RESULTS AND DISCUSSION

The overall results of the 61 plants from 33families screened for antiviral and cytotoxicactivities are summarized in Fig. 1. Table 1lists the 28 species (46%) that gave negativeresults for all three tests. Table 2 lists the 26species (43%) that exhibited antiviralactivity and the 18 species (30%) whichshowed cytotoxicity.

Eight species (13%) (Calotropis gigantea,Costus speciosus, Eugenia michelii, Hedyotisauricularia, Mentha arvensis, Orthosiphon aris­tatus, Polygonum minus and Ricinus communis)showed antiviral activity against bothHSV-l and VSV. The extracts of Calotropisgigantea, Eugenia michelii and Ricinus communisshowed a similar MIC value of 0.01 mg/mlagainst both viruses, but only Eugeniamichelii demonstrated cytotoxicity (CDso:

0.05 mg/ml). In the case of Polygonum minus,a similar MIC value against HSV-l wasobtained but lesser activity was shown

CO2 for 4 days. Using an inverted micro­scope (low power), cytotoxicity was deter­mined as the concentration of plant extractwhich reduced cell number by ca. 50% withreference to the control (CDso, mg/ml).

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TABLE 2The effect of plant extracts on cells as shown by minimum inhibitory concentration values against herpes

simplex virus-type 1 and vesicular stomatitis virus, and CDso values towards HeLa cells

Plant HSV-1 VSV Cytotoxicity

AcanthaceaeAndrographis paniculata Nees

AlDaranthaceaeAlternanthera sessilis (L.) DC.

MIca (mg/ml)

-ve

0.001

-ve

-ve

CDsob (mg/ml)

0.1

-ve

ApocynaceaeCerbera manghas L.

FruitsLeaves

AsclepiadaceaeCalotropis gigantea R. Br.

COlDpositaeBlumea chinensis DC.Cosmos caudatus KunthElephantopus scaber L.

EuphorbiaceaeAcalypha indica L.Codiaeum uariegatum (L.) BI.Euphorbia hirta L.Ricinus communis L.

GralllineaeEleusine indica (L.) Gaertn.

LabiataePlectranthus amboinicus (Lour.) Spreng.Mentha aruensis L.Ocimum tenuijlorum L.Orthosiphon aristatus (BI.) Miq.

LeeaceaeLeea indica (Burm. f.) Merr.

MalvaceaeHibiscus rosa-sinensis L.

MyrtaceaeEugenia michelii Lamk.

LecythidaceaeBertholletia excelsa Hump. & Bonpi.Lecythis ollaria L.

-ve 0.05 0.001-ve 0.1 0.02

0.01 0.01 -ve

0.005 -ve -ve-ve -ve 0.1-ve -ve 0.05

-ve 0.01 0.01-ve 0.1 0.10.1 -ve -ve0.01 0.01 -ve

0.1 -ve -ve

-ve 0.1 -ve0.002 0.1 0.1-ve -ve 0.10.1 0.1 -ve

0.05 -ve -ve

-ve -ve 0.1

0.01 0.01 0.05

-ve 0.005 -ve-ve -ve 0.1

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ABDUL MANAF, MUHAMMAD MUKRAM, SALEH, JUNAINAH, NOR HADIANI, FAUJAN AND NORDIN

NyctaginaceaeMirabilis jalapa L. -ve 0.05 0.1

PandanaceaeFreycinetia malaccensis Ridl. 0.05 -ve 0.1

PiperaceaePiper sarmentosum Roxb. -ve 0.02 0.1

PolygonaceaePolygonum minus Huds. 0.01 0.02 0.1

RubiaceaeHedyotis auricularia L. 0.1 0.05 -veM orinda elliptica Ridl. -ve 0.1 0.003

SolanaceaeSolanum americanum Mill. 0.1 -ve -ve

UDlbelliferaeCentella asiatica (L.) U rb. -ve 0.1 0.1Oenanthe javanica DC. -ve 0.02 -ve

VerbenaceaePremna odorata Blanco -ve 0.05 -ve

ZingiberaceaeCostus speciosus (Koenig) Smith 0.1 0.02 -veEtlingera elatior Uack) R. M. Smith -ve -ve 0.1

aMIC = minimum inhibitory concentration, i.e. the lowest concentration of plant extract which completely inhibitedvirus replication.

.BCDso = cytotoxic dose at 50%, i.e. the concentration of plant extract which reduced the number of HeLa cells by50%.

HSV-1 was seen in extracts from 7 plants(11 %) (in order of decreasing activity,Alternanthera sessilis, Blumea chinensis, Freyci­netia malaccensis, Leea indica, Euphorbia hirta,Eleusine indica, and Solanum americanum) withMIC values within the range of 0.001-0.1mg/ml. Conversely, 11 plant (18%) extractspossessed selective antiviral activity againstVSV (in order of decreasing activity, Ber­tholletia excelsa, Acalypha indica, Piper sarmen­tosum, Oenanthe Javanica, Mirabilis Jalapa,Premna odorata, Cerbera manghas, Codiaeumvariegatum, Plectranthus amboinicus, Centellaasiatica ahd Morinda elliptica) with MICvalues from 0.005 - 0.1 mg/ml. More plantextracts were active against VSV than

HSV-1. The selective antiviral aCtivIty ofsome plant extracts against either HSV-1 orVSV implicates the involvement ofdifferentmechanisms of action exploiting the differ­ence in nucleic acid composition of the viruses.

In the case of the anti HSV-1 species,only Freycinetia malaccensis showed cytotoxi­city (CDso: 0.1 mg/ml) whereas 7 of theanti-VSV extracts (in order of decreasingactivity, Cerbera manghas, Morinda elliptica,Acalpyha indica, Centella asiatica, Codiaeumvariegatum, Mirabilis Jalapa and Pipersarmentosum) showed cytotoxicity rangingfrom CDso 0.001-0.1 mg/ml. Since VSV isa RNA-type virus, the concomitant anti­VSV and cytotoxic activities may involve a

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related mode of action, most probably viaprotein interaction. Co-existing antiviraland cytotoxic activities were found in theextracts of three species (5%), i.e. Eugeniamichelii, Mentha arvensis and Polygonum minus.

Of the 18 plants showing cytotoxicity,only 3 species (Acalypha indica, Cerberamanghas and M orinda elliptica) showedsignificant activity below the cut-off value.of 0.02 mg/ml suggested by Wall et al.(1987) and all three species exhibited anti­VSV activity. The strongest cytotoxicactivity was shown by the fruits of Cerberamanghas (CDso: 0.001 mg/ml). The fruits ofCerbera manghas always exhibited strongercytotoxic (20 times) and anti-VSV activ­ities (twice the activity) than its leaves. Thissuggests that a higher concentration of thebioactive compound(s) is present in thefruits of Cerbera manghas than the leaves.

The in vitro cytotoxicity displayed bythe plant extracts tested is an initialindicator of in vivo antitumour activity.However, since a wide range of phytocom­pounds are capable of exhibiting non­specific cytotoxicity, plant extracts withsignificant cytotoxic activity should befurther assayed using animal models toconfirm antitumour activity, and/or abattery of various cell lines to detectspecific-cytotoxicity. This step is necessaryto eliminate cytotoxic compounds withlittle value for further investigation asanticancer agents.

CONCLUSION

The results of this preliminary studyscientifically substantiate to a certainextent the pharmacological activities of 33plants used in Malaysian indigenous med­icine and point out some plants wi thpotential for further investigation. Inaddition, these results may also contributetowards the documentation of pharmaco­logical profiles of Malaysian plants forconservation efforts and protection of

biodiversity rights. Inadequate recordingof the pharmacological activities of Malay­sian plants may lead to the commercialexploitation of traditional knowledge byforeign parties without any benefit to thecountry as experienced by India in the caseof the Neem tree and turmeric (Agarwaland Narain 1996).

ACKNOWLEDGEMENTS

The authors wish to thank UniversitiPertanian Malaysia (research grant50218-94-01), the National Council forResearch and Development (IRPA 4-07­05-043) and the Japan InternationalCooperation Agency UICA) for financialsupport. The authors also extend theirthanks to Encik Zainudin Samadi andMr. Anthonysamy Sivarimuthu, Facultyof Science and Environmental Studies,U niversiti Pertanian Malaysia for theirassistance in plant collection.

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(Received 9 June 1995)(Accepted 20 January 1997)

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