can ethnophamacology contribute to antiviral drugs

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Journal of Ethnapharmacofogy, 32 (1991) 141-153Elsevier Scientific Publishers Ireland Ltd.

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Can eth~opha~acology contribute toantiviral drugs? the development of

Arnold J. Vlietinck and Dirk A. Vanden BergheFaculty of Medicine, University ojAntwerp (UIA), B-2610 Antwerp (Belgium)

In recent years, many compounds having potent antiviral activity in cell cultures and in experimental animals have been detected,but only a few have been approved by Western health authorities for clinical use. Nevertheless, some of these compounds are currentlyundergoing either preclinicat or clinical evaluation, and perspectives for finding new interesting antiviral drugs are promising. Amongthese antiviral substances are several natural compounds isolated from plants used in traditional medicine including polysaccharides,Bavonoids, terpenes, alkaloids, phenolics and amino acids. Some of these plant compounds exhibit a unique antiviral mechanism ofaction and are good candidates for further clinical research. What follows is a brief summary of the selection methods of plants forantiviral screening and in vitro and in vivo assays, which are currently used for detecting this activity in plant extracts. The importanceof the plant kingdom as a source of new antiviral substances will be illustrated by presenting a survey on plant-derived antirhinovirusand anti-HIV agents.

introductionThe identification of a retrovirus, human im-

munode~cien~y virus (HIV) as the causative agentof AIDS (acquired immunedeficiency syndrome),the steadily increasing incidence of various viralinfections caused by viruses such as herpes simplexvirus (HSV), varicella-zoster virus (VZV),cytomegalovirus (CMV) and Epstein-Barr virus(EBV), in immunode~cient patients, the increasingevidence for the pathogenic role of a number ofviruses viz. human papilloma virus (HPV) in thepathogenesis of primary hepatocellular carcinomaand the socioeconomic impact of virus infectionsof the respiratory tract (influenza, adeno, coronaand rhinoviruses) and gastrointestinal tract(rotaviruses) have all been important factors inboosting the search for new antiviral agents andnew modalities of antiviral chemotherapy.

Although many compounds having potent an-tiviral activity in cell cultures and in experimentalPresented at the First International Congress on Ethnophar-macology, Strasbourg, 5-9 June, 1990.Correspondence to: Arnold J. Vlietinck, Faculty of Medicine,University of Antwerp (UIA), B-2610 Antwerp, Belgium.

animals have been detected, at present, only sevensynthetic compounds and alpha interferon havebeen approved by the FDA for therapy of viral infections in humans. Four of these compounds arefor the therapy of infections caused by members othe herpes family including 5-iodo-2 deoxyuridine(idoxuridine), 5-trifluoromethyl-2-deoxyuridine(trifluridine), 9-fi-D-arabinofuranosyladenine (vidarabine) and 9-(2-hydroxyethoxymethyl)guanine(acyclovir).

A fifth compound, I-aminoadamantane (aman-tadine), is approved for the therapy and pro-phylaxis of respiratory infections caused by the influenza virus A. A sixth antiviral agentl-~-~r~bofuranosyl-l,2,4-triazole-3-carboxamide(ribavirin) is approved for therapy of severe respi-ratory infections in children caused by the respira-tory syncytial virus. The seventh antiviral agent,3 -azido-3 -deoxythymidine (zidovudine, AZT),has been approved for therapy of AIDS. Alpha interferon has been approved for treatment ogenital warts caused by papilloma virus and for thetherapy of hairy cell leukemia and Kaposi sarcoma(Prusoff et al., 1989). The chemical structures othe FDA-approved drugs are given in Fig. 1.

0378-8741~03.50 0 1991 Elsevier Scientific Publishers Ireland Ltd.Published and Printed in Ireland


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0

,l 0H-Na - CHs

OH3

I HOC&I n I

4OH OH NJ6 7

Fig. 1. Chemical structures of antiviral drugs approved by the FDA for use in humans. I, Amantadine; 2. idoxuridine; 3, trifluridine;4, acyclovir; 5, vidarabine; 6, ribavirin; 7, zidovudine (AZT).

None of these drugs, however, is without tox-icities and hence there is a strong need not only toimprove the actual antiviral armamentarium, butalso to find an effective therapy of viral infectionsfor which at present no clinically useful drugs orvaccines are available.

There is also a need for finding new substanceswith extracellular virucidal activity, since many ofthe existing antiseptics and disinfectants fail to killall pathogenic viruses after a 5-min exposure timeat room temperature (Springthorpe et al., 1986;Vanden Berghe et al., unpublished results). Suchcompounds could be very useful to diminish thetransfer of viable viruses from infected individualsto uninfected ones, to objects or to the air ofenclosed spaces.

Therefore, all possible approaches towards thedevelopment of new antiviral and virucidal drugsshould be pursued. Besides the rational approach,which in its ultimate form requires a sophisticatedknowledge of both the target structure, e.g. the ac-tive site of an enzyme, a receptor, a macromoleculesuch as DNA, RNA or a regulatory protein andthe compound to interact with it, the emperical ap-proach still remains valuable (Prusoff et al., 1986;De Clercq, 1988; Montgomery, 1989). Systematicscreening of pure substances or extracts of variousorigins has resulted in the discovery of many activeleads. These substances can be improved byrepeated structure modification in the hope thatthe change being made will result in increasedpotency, selectivity, duration of action,


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bioavailabiiity and reduced toxicity. By combiningstructure-activity relationships with computer-graphic model building, the sere~ndity approachcan even become a rational serependity approach(Prusoff et al., 1989).

There are many sources of materials for an-tiviral testing, including pure synthetic and naturalsubstances of known structure and naturally oc-curring mixtures of the plant, animal and marineworld.

The purpose of this communication is to discussand review the current activities in the area of me-dicinal plants and plant-derived products relatedto in vitro and in vivo preclinical evaluation forantiviral and virucidal activities.Selection of plants for antiviral screening

Four basic methods are available for selectingplants for a screening programme to seek antiviralleads including: (1) random collection of plantsfollowed by mass screening; (2) selection based onethnomedical uses; (3) follow-up of existingliterature leads; and (4) chemotaxonomic ap-proaches (Suffness and Douros, 1979). The sameadvantages and disadvantages of the various ap-proaches to the selection of plants for screening todiscover anticancer activity can be associated withapproaches to discover antiviral activity in plants(WHO Report, 1989). All factors considered, ap-proaches (2) and (3) would seem to be the mostcost-effective for finding plants with antiviral pro-perties,

Comparison of the different approaches showedthe selection method based on folklore to give afive-times higher percentage (about 25%) of activeleads, although, in some cases, the same activecompounds were isolated from botanically non-related active plants. On the other hand, thescreening of extracts from random collected plantsafforded more novel substances with antiviral pro-perties (Vanden Berghe et al., 1978; Ieven et al.,1982; Van Hoof et al., 1989). Since the screeningcapacity of our research group is rather limited, weprefer a selection of plants based on a combinedanalysis of ethnomedi~al, phytochemical, tax-onomical and toxicological data (Vanden Bergheet al., 1986).

Selection of in vitro antiviral tests for the screeningof plant extracts and natural products

As the methodology used in the determinationof the antiviral activity as well as the interpretationof the results have been virtually specific to eachlaboratory and are consequently not comparableto one another, simple procedures and guidelinesfor evaluating antiviral and/or virucidal activitiesof single componnds are urgently needed. This ieven more true for the antiviral testing of crude ex-tracts, containing a complicated mixture of dif-ferent compounds present in several proportions.

Various cell culture-based assays are currentlyavailable and can be succesfully applied for the an-tiviral (A) or virucidal (V) determination of singlesubstances (S) or mixtures of compounds e.g. plantextracts (M). Antiviral agents interfere with one ormore dynamic processes during virus biosynthesisand are consequently candidates as clinicallyuseful antiviral drugs, whereas virucidal sub-stances inactivate virus infectivity extracellularlyand are rather candidates as antiseptics, exhibitinga broad spectrum of germicidal activities.

Cost, simplicity, accuracy and reproductibilityare the key factors which should determine theselection of the assay system, but also selectivity,sociality and sensitivity should be taken into ac-count, especially for the testing of extracts. Werefer to the appropriate literature for an extensivereview of all factors influencing the design of an-tiviral chemotherapy experiments (Hermann, Jr.,1961; Kaufmann, 1965; Sidwell, 1986; VandenBerghe et al., 1986).

The methods commonly used for evaluation ofin vitro antiviral activities are based on the dif-ferent abilities of viruses to replicate in culturedcells. Some viruses can cause cytopathic effects(CPE) or form plaques. Others are capable of pro-ducing specialized functions or cell transforma-tion. Virus replications in cell cultures may also bemonitored by the detection of viral products, i.eviral DNA, RNA or polypeptides. Thus, the antiviral test selected may be based on inhibition oCPE, reduction or inhibition of plaque formationand reducing virus yield or other viral functions(Hu and Hsiung, 1989; Vanden Berghe andVlietinck, 1991).


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A survey of the various in vitro antiviral testsand their possible suitability for the antiviraland/or virucidal screening of plant extracts andplant products is presented in Table 1.It should be emphasized that the toxic effects ofan antiviral agent on the host cells must be consid-ered since a substance may exhibit an apparent an-tiviral activity by virtue of its toxic effects on thecells. The cytotoxicity assay on cell cultures isusually done by the cell viability assay and the cell

growth rate test, although other parameters suchas destruction of cell morphology undermicroscopic examination or measurement ofcellular DNA synthesis have been used as in-dicators of compound toxicity (Hu and Hsiung,1989).

After the antiviral potency of a test substancetogether with its cytotoxicity is determined, thetherapeutic index of the antiviral compound in agiven virus-cell system can be calculated. Taking

TABLE 1IN VITRO ANTIVIRAL SCREENING ASSAYSDetermination of the viral infectivity in cultured cells during virus multiplication in the presence of a single compound (A-S) or amixture of compounds e.g. plant extracts (A-M) or after extracellular incubation with a single compound (V-S) or a mixture of com-pounds (V-M).(1) Plaque inhibition assayOnly for viruses which form plaques in suitable cell systems.Titration of a limited number of viruses in the presence of a non-toxic dose of the test substance.Applicability: A-S.(2) Plaque reduction assayOnly for viruses which form plaques in suitable cell systems.Titration of residual virus infectivity after extracellular action of test substance(s). Cytotoxicity should be eliminated e.g. by dilution.filtration etc. before the titration.Applicability: V-S; V-M.

(3) Inhibition of virus-induced cytopathic effect (CPE)For viruses that induce CPE but do not readily form plaques in cell cultures.Determination of virus-induced CPE in monolayers, cultured in liquid medium, infected with a limited dose of virus and treated witha non-toxic dose of the test substance(s).Applicability: A-S; A-M.(4) Virus yield reduction assayDetermination of the virus yield in tissues cultures, infected with a given amount of virus and treated with a non-toxic dose of thetest substance(s).Virus titration is carried out after virus multiplication by the plaque test (PT) or the 50% tissue culture dose end point test (TCDse).Applicability: A-S; A-M.(5) End point titration technique (EPTT)Determination of virus titer reduction in the presence of two-fold dilutions of test compound(s).Applicability: AS; A-M. This method has been especially designed for the antiviral screening of crude extracts (Vanden Berghe et al.. 1986).(6) Assays based on measurement of specialized functions and viral productsFor viruses that do not induce CPE or form plaques in cell cultures.Determination of virus specific parameters e.g. hemagglutination and hemadsorption tests (myxoviruses), inhibition of cell transfor-mation (EBV), immunological tests detecting antiviral antigens in cell cultures (EBV, HIV, HSV and CMV).Reduction or inhibition of the synthesis of virus specific polypeptides in infected cell cultures e.g. viral nucleic acids, determinationof the uptake of radioactive isotope labelled precursors or viral genome copy numbers.Applicability: A-S; A-M; V-S; V-M.


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into account that the cell growth rate test has beenclaimed to be the most stringent method formeasuring cytotoxicity (Amtman et al., 1987), thetherapeutic index (TI,) can be defined as the ratioof the maximum drug concentration at which 50%of the growth of normal cells is inhibited (CyD&to the minimum drug concentration at which x%(50%, 90% or 99%) of the virus is inhibited (ED,)(De Meyer et al., 1991).

It should be noted that the calculation of thetherapeutic index of a mixture of compounds e.g.crude extracts is irrelevant since cytotoxicity andantiviral activity of the mixture are not neccessari-ly due to the same components of the mixture. Onthe contrary, without the cytotoxicity data reportsof antiviral activity of a single compound even atvery low concentrations are of limited value. In ad-dition, the relative potency of a new antiviral pro-duct should also be compared with existing ap-proved drugs. Several years ago, we evaluated andcompared the antiviral data of about 50 plant-derived substances, which were reported in theliterature to exhibit interesting in vitro antiviralproperties, using the EPTT. The results of thestudy showed that, in many cases, the originallyproposed antiviral activity of these substances wastoo low, non-specific or only detectable in toxicconcentrations for the host (Vanden Berghe et al.,1986).Invivo testing ofantiviralagents

Whereas in vitro screening tests are undoubtedlya first choice not only to screen crude extracts, but.also to guide the isolation of the antivirally activecompounds from the crude extracts, in vivo assaysin a suitable animal model of the candidate com-pound remain the stepping-stone between tissueculture antiviral activity and the demonstration ofa corresponding activity in human clinical trials.This model should predict efficacy in man, andmust therefore mimic the natural disease as closelyas possible. On the other hand, antiviral activity tothe exclusion of cytotoxicity under tissue cultureconditons should clearly be demonstrable in theinfected laboratory animal. Too often, thetherapeutic index is inadequate, so that the con-

centration of the antiviral agent in the target tissueof the infected animal will not be sufficient underdosing conditions to inhibit virus, due to toxicityor practical difficulties. Species-specific charact-eristics of absorption, tissue distribution, metabo-lism and excretion are the contributing factors.Animal models for a number of virus infectionsare available (Sidwell, 1986; Vanden Berghe et al.1986). They are helpful in detecting not only if thecandidate compound is an effective viral inhibitorwithout inducing viral resistance, but also if it isable to (1) reach the target organ, (2) be stable inthe form administered, (3) be cleared by the tissuewithin a reasonable time, (4) resist and notadversely affect the immune system and (5) not in-terfere with the normal metabolic processes ofuninfected cells (Galasso, 1984).

Some viral infections, such as herpes simplex inguinea-pigs and mice and cytomegalo- or polio-virus infections in mice, mimic the natural diseasesvery closely (Sidwell, 1986). These animal modelsallow continuous observation of the infectedanimals, and virus spread and pathogenesis alocal sites can be studied thoroughly. It is, how-ever, uncertain whether animal models can bedeveloped for all human infections requiring an-tiviral chemotherapy. The ability of humanrhinovirus to replicate only in the respiratory tractof non-human primates, for instance, has posed aproblem in translating the tissue culture an-tirhinovirus activity of a candidate compound tothe stage of clinical evaluation and consequentlydelayed the finding of an antiviral drug effectivefor the prevention and treatment of the commoncold considerably (Grunert, 1979). Up until nowno animal model is known for the study of HIValthough other retroviruses are known to causeleukemia, lymphoma and other forms of cancer ina wide variety of animals.

We regularly use two herpes animal models, including the rabbit eye model and the mice model,in which lesions are produced on the shaved back,and one coxsackie animal model in which CNS-associated mortality in suckling mice is determinedto evaluate in vivo antiviral activity of plant-derived antiviral agents (Van Hoof et al., 1984DeClerq E., 1984; Sidwell, 1986).

For all viruses used in our tissue culture screen-


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ing system, however, suitable animal models werereported (Vanden Berghe et al., 1986).Plant derived antirhinovirus agents

A few years ago, in their antiviral screening pro-gram of pure microbial and plant products, Ishit-suka et al. (1982a) found 5,4-dihydroxy-3,7,3-trimethoxyflavone (Ro-09-0179) (3,7,3-trimethyl-quercetin or 3,7,3-TMQ), which was originallyisolated from the Chinese medicinal herbAgastache rug;osa Kuntze, to be highly active intissue cultures against all picornaviruses exceptMengovirus. Independently, Van Hoof et al.(1982, 1984) found several 3-methoxyflavones,which were identified as derivatives of the3-methylethers of quercetin (3-MQ) and kaemp-ferol (3-MK), to be responsible for the pro-nounced antiviral properties of the alcoholicextracts prepared from different African Euphor-bia spp. (Fig. 3).

Among the many derivatives of 3,7,3-TMQthat were synthesized and tested one chalcone,2-hydroxy-4-ethoxy-4,6-dimethoxychalcone(Ro-09-0410), emerged as a new type of antiviralagent exclusively active against many humanrhinovirus serotypes and, consequently, a can-didate drug for the treatment of the common cold(Ishitsuka et al., 1982b) (Fig. 2 (2)). The an-tirhinovirus activity of flavan was discoveredserependitously during an in vitro screening pro-gram utilizing the plaque inhibition test. Structure-activity relationship studies led to the4,~dichloro~avon derivative (BW 683C), beingthe most potent agent against several rhinovirusserotypes (Bauer et al., 1981, 1983) (Fig. 2 (1))Subsequently, it was shown that flavans andchalcones inactivate rhinoviruses directly by bin-ding to or interacting with specific sites on theviral capsid proteins. While little or no interferencewith viral attachment or penetration of the hostcell membrane was observed, the uncoating pro-

C14b CH3

3 4 O?>a-(CH,), acH3

~~ N 7;)N CH,COOH5 6

Fig. 2. Chemical structures of antirhinovirus agents recently evaluated in human volunteers. 1. 4&Dichloroflavan; 2,4-ethoxy-2-hydroxy~,6-dimethoxychaicone (Ro-~-~lO); 3,3-methoxy-6-[4-(3-methylphenyl)-l-piperazinyt]pyridazine (R 61837);4, i-(S-tetradecyloxy-2-furanyl)ethanone (RMI 15,731); 5. (S)-(-)-5-~7-[4(4,S-djhydro-4-methy~-2-oxazolyl)phenoxy~hepty~l-3-~thylisox~ole (WIN 52084); 6, 2-I( 1,5,1Oa-tetrahydro-3H-thiazolo[3,Qb)-isoquinolin-3- ylindene)aminol~-thiazoieacetic acid (5) (44-081 R.P.).


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cess in the host cell was inhibited throughstabilization of the protein capsid of the virus andprevention of the conformational changes requiredfor release of viral RNA (Ninomiya et al., 1984;Tisdale et al., 1984).

At the same time, several pharmaceutical com-panies developed a number of synthetic capsid-binding drugs with prominent antirhinovirus pro-perties. Most were rhinovirus specific, but all ofthese agents had substantial serotype relatedvariability in antiviral activity. The concentrationsinhibiting rhinovirus replication in vitro varied upto lOOO-fold or different serotypes, indicating thatthe binding sites on the viral capsid proteins werehighly specific (Sperber and Hayden, 1988). X-raycrystallographic structural analysis has determinedthat the precise binding site of the WIN com-pounds, which have an isoxazole at one end andan oxazolyl-phenoxy group at the other end of analkyl chain (Fig. 2 (5)), to rhinovirus type 14 is theinterior of viral protein 1 (VPl), one of the threeexternal polypeptides of the protomers of the pro-tein shell of the virus (Smith et al., 1986). Changesin the amino acids of this binding pocket may af-fect the ability of a specific agent to bind to thecapsid and thus explain the different suscep-tibilities of different rhinovirus serotypes. The bin-ding sites for some of these agents me be the sameor lie very close to one another (Ninomiya et al.,1985). Another potential limitation with the use ofthese compounds is that drug-resistant mutantscan be selected readily under in vitro conditions(Selway, 1986).

Clinical trials have found discrepancies betweenthe in vivo and in vitro antiviral activities of thesecompounds. Orally administered dichloroflavanand phosphorylated chalcone were ineffective inthe prophylaxis of experimental rhinovirus colds(Phillpotts et al., 1983, 1984). Also, intranasalpreparations of both compounds failed to reduceinfection rates or protect against illness, probablybecause no adequate levels of drugs were achievedin nasal mucosal cells (Al-Nakib et al., 1987a,1987b). Similarly, in vivo test results for the syn-thetic antiviral agents RMI 15,731 (Fig. 2 (4)) and44-081 RP (Fig. 2 (6)) did not show significantprophylactic activity as compared to placebo(Sperber and Hayden, 1988; Zerial et al., 1985).

The antirhinovirus compound R61837 (Fig. 2(5)), which is a 6-( 1 piperazinyl)pyridazine deriva-tive, however, caused marked reductions in nasalsymptoms and mucus weights, when it was ad-ministered intranasally in frequent doses begin-ning 1 h before and continuing for 6 days afterexperimental rhinovirus challenge with a verysusceptible serotype (Sperber and Hayden, 1988)

The relative success of the latter experimentmight probably be ascribed to the efficient absorp-tion through the nasal mucosa of the hydrophobicantiviral agent from a pharmaceutical compositioncontaining cyclodextrines (European Patent Ap-plication No. 88201288.3, 1988).The 3-methoxyflavones, 3-MQ and 3-MK, how-ever, have shown not to interact with the capsidproteins of picornaviruses, but rather to interferewith an early stage in the viral RNA synthesis.Although their exact mode of action is not com-pletely understood yet, it has been found that theyprobably inhibit the formation of minus-strandRNA of poliomyelitis virus by interacting with theproteins involved in the binding of the virusreplication complex to vesicular membranes, awhich poliovirus replication takes place (Castrilloet al., 1986; Vrijsen et al., 1987; Lopez Pila et al.1989).

In contrast to the capsid-binding antivirals nodrug-resistant mutants have been detected in thepresence of 3-methoxyflavones (Ninomiya et al.1985).

The attractive mechanism of action, the pro-nounced and broad-spectrum antiviral activity andthe lack of resistance-induction by these flavonesprompted us to explore this class of flavonoids.From a large screening program of naturally-occurring 3-methoxyflavones emerged jaranol andpenduletin (Fig. 3) as the most in vitro activesubstances against polio- and rhinoviruses. Inorder to establish a structure-activity relationshipa series of A-ring substituted analogues o4-hydroxy-3-methoxyflavone (Fig. 3, MF 110were synthetized and tested antivirally. The mostinteresting compound was 4,7-dihydroxy-3-meth-oxy-5,6_dimethylflavone (Fig. 3, MF 142) possess-ing in vitro TIg9 values of > 1000 and 200 againstpoliovirus type 1 and rhinovirus type 15, respec-tively (EPTT). This compound was also active


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R4 A, = R, = R, = R, = H MFllOR2 = R, = H ; R, = OH ; R, = OCH, JoranolR2 = R4 5: H ; R, = R, = OH 3-MKR, = H ; R, = R3 = RI = OH 3-MQR, = H ; Rt = OH ; R, = R, = OCH, PenduletinR, = H ; R, = Rz = CHa ; R, = OH MF142Rz = R, = t-l ; R, = CH, ; R, = OCH, MF140

Fig. 3. Chemical structures of antirhinovirus 3-methoxyflavones. 4-Hydroxy-3-methoxyflavone (MF I IO):4-5-dihydroxy-3,7-dimethoxyflavone tjaranol); 3-methylether of kaempferol (3-MK); 3-methyl~ther of quercetin (3-MQ);4,5-dihydroxy-3,6,7-trimethoxyflavone (penduletin); 4,7-dihydroxy-3-methoxy-5,6-dimethylflavone (MF 142);4-hydroxy-3,7-d~met~oxy-5-methyiflavone (MF 140).

against a representative battery of all otherrhinovirus serotypes having MICse values rangingfrom 0,016 to 0.5 @g/ml. The corresponding valuesof a moderately active analogue such as4-hydroxy-3,7-dimethoxy-5-methylfavone (Fig.3, MF 140) were 10-25 times higher (De Meyer etal., 1990).It was also found that, in contrast to quercetin,MF 142 was not mutagenic in concentrations up to2.5 mg in the Ames test (De Meyer et al., 1991).

Since some 3-methoxyflavones, when ad-ministered intraperitoneally, have been shown toprotect mice from lethal infections from coxsackieB_, (Van Hoof et al., 1984), the most antivirallyactive substance of this study viz. MF 142 shouldbe considered as a promising candidate for clinicalstudies in human volunteers.Plantderived anti-HIV agents

Acquired i~unede~ciency syndrome is apandemic immunosuppressive disease whichresults in life-threatening opportunistic infectionsand malignancies. Since a retrovirus, designatedhuman i~unode~ciency virus (HIV), has beenisolated and identified as the etiologic agent of thisdisease, numerous compounds have beenevaluated for their inhibitory effects on HIVreplication in vitro (DeClercq, 1987). Moreover,the AIDS saga has resuscitated several substances,

especially reverse transcriptase inhibitors and in-terferon inducers, which were mentioned manyyears ago as antiviral agents, then forgotten to bediscovered again as potential candidates for thetreatment of AIDS (DeClercq, 1989).Besides the inhibition of reverse transcriptase, amultifunctional enzyme that transcribes the viralRNA genome to DNA, the replication cycle ofHIV offers a wealth of possible targets for antiviralagents, and so might several viral regulatory genessuch as tat and rev, which regulate respectively thetransactivation and the expression of viral proteinsand vis, which determines virus infectivity (Oxfordet al., 1989).

A wide range of biochemical and cell culture-based assays are currently available for the detec-tion of in vitro anti-HIV activity. However, aminimum of a single T-cell culture assay system(eg. H9, ATH8, MT-2, MT-4 etc.) should be usedas a first-line screen of crude extracts or to guidethe antiviral activity during the isolation. Once theactive component(s) are identified, more detailedconfirmatory assays should be carried out (WHOReport, 1989).

Recently, several single, sensitive and rapid testsfor the screening of a large number of sampleshave been described. Among these tests, a col-orimetric assay based upon the transformation ofa tetrazolium salt to a coloured formazan by livingcells but not by dead cells or culture medium has


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shown to be very useful in the large scale evalua-tion of anti-HIV agents (Pauwels et al., 1988;Schwartz et al., 1988).Up until now, only one study on the screeningof plant extracts for anti-HIV activity has ap-peared (Chang and Yeung, 1988). Out of 27 medic-inal plants used in traditional Chinese medicine asanti-infectives, 11 showed in vitro inhibitory activ-ity against HIV. Of two of these active plants, i.e.Viola yeodensis and Prune&r ~~gur~~, the activeprinciples were isolated and identified as sulphatedpolysaccharides (Ngan et al., 1988; Tabba et al.,1989).

CH20S0,0H

I ASO~OH hS020HL1

OH

HO RHO R = CH3

R = CH20H

1

Other sulphated polysaccharides of differentorigins including heparin, A-carrageenan, dextransulphate, pentosan polysulphate and an aqueousextract of the red alga, ~c~~~~~e~j~ paciJca haveproven to be very potent and selective inhibitors ofHIV replication in TJymphocyte cultures (Ito etal., 1987a; Nahashima et al., 1987; Ueno andKuno, 1987; Baba et al., 1988). These substanceswere inhibitory to HIV at a concentration whichwas lo-fold (heparin) or more than lOO-fold (dex-tran sulphate, pentosan polysuiphate) below theiranticoagulant threshold (De Clercq, 1989). It wasalso shown that their anti-HIV activity should be

RO()cz/ 2COOH o-

OH OH CHOHO

CHJO

6Fig. 4. Chemical structures of natural products as potential drugs for the treatment of AIDS. 1, heparin; 2, glycyrrhiin: 3, castanospermk4, (-)-gossypol; 5, hype&in; and pseudohypericin. 6, papaverine.


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readily attributed to an inhibitory effect on virusabsorption rather than to their inhibitory actionon HIV-associated reverse transcriptase (Baba etal., 1988).

Other studies have provided additional informa-tion on natural products with experimental anti-HIV activity. Their chemical structures aredepicted in Fig. 3.

Glycyrrhizin, one of the main saponins ofGlycyrrhiza glabra, inhibits the growth of anumber of DNA and RNA viruses includingHIV-l in vitro (Ito et al., 1987b). In Japan, glycyr-rhizin has been studied in AIDS patients. It wasclaimed that, when given orally to asymptomaticHIV carriers, the substance delayed the progres-sion of symptoms related to HIV. It was alsoclaimed that, when administered intravenously fora period of more than a month to hemophilics withAIDS, viral antigen considerably decreased, sug-gesting that glycyrrhizin might inhibit HIVreplication in vivo. In another claim, simultaneousadministration of the compound appeared todecrease the adverse reactions of zidovudine (Hat-tori et al., 1989). Studies on the mechanism of ac-tion of glycyrrhizin suggested that the compound,being a polyanionic substance, interferes withvirus adsorption, which is further complementedby an inhibitory effect on protein kinase C. Thisenzyme seems to be required for the binding ofHIV-l particles to the cellular CD4 receptors (Itoet al., 1988). Also, other saponins includingsaponin B, from soybean exhibited potent anti-HIV effects in vitro (Nakashima et al., 1989).

Castanospermine, an indolizidine alkaloid fromthe seeds of Castanospermum australe, blocksglycoprotein processing via inhibition of o-glucosidase I located in the endoplasmaticreticulum. This alkaloid has been reported to havein vitro anti-HIV activy and has been shown to beactive in vivo when administered orally to mice.There are also indications that a combination ofcastanospermine and zidovudine inhibits HIVsynergistically in vitro (Ruprecht et al., 1989).

The naphtobianthrones hypericin andpseudohypericin, which occur in plants of theHypericum family, are highly effective in preven-ting viral-induced manifestations that follow infec-tions with a variety of retroviruses in vivo and in

vitro. These compounds interfere with viral releasefrom infected cells and possibly inactivate virions.Preliminary in vitro studies have shown thatpseudohypericin reduce the spread of HIV(Meruelo et al., 1988). Other plant-derived com-pounds with inhibitory effect on HIV replicationin vitro are the (-) enantiomer of gossypol, a poly-phenolic aldehyde extracted from cottonseed(Polsky et al., 1989; Lin et al., 1989) and the well-known alkaloid papaverine (Turano et al., 1989).

Many patent applications for treatment andpropylaxis of retroviral infections including HIV-infections have been deposited for plant productse.g. hydrolysable tannins from Coriaria, Oenotheraand Agrimonia spp., lectins from Ulex europeus,Maackia amurensis, Glycine max and Lensculinaris, Japanese pine cone extract, protein-polysaccharide complexes (Kurestin) and sugar-protein-inorganic elements mixture of differentBasidiomycetes including Coriolus spp. and Len-tinus edodes. It is questionable, however, whethersuch preparations will have therapeutic efficacyagainst retrovirus infections in animal models.

Only a few experimental studies to discover anti-HIV agents from medicinal plants and other natu-ral products are in progress. The major pro-gram of this type is being carried out at the NationalCancer Institute in the U.S.A. The program willscreen about 4500 plant samples per year for thenext 5 years for in vitro anti-HIV activity, based ona random selection of plants (WHO Report, 1989).

Although the number of anti-HIV screeningstudies of plant extracts or plant-derivedsubstances has been rather limited in comparisonto the mass screening programs of syntheticcompounds, nevertheless several naturalsubstances have emerged as promising leads forthe development of anti-HIV agents. Whetherthese compounds will have any clinical potential inthe therapy of AIDS remain to be determined. Itis, however, essential that these studies should beencouraged and continued.

Conclusions

The question if ethnopharmacology can con-tribute to the development of antiviral drugs can


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be answered in a positive sense without too muchpremature optimism.Indeed, the screening of a relatively low numberof randomly collected plant substances has afford-ed a remarkably high ratio of active leads in com-parison with the screening programs ofsynthetic compounds. On the other hand, thetesting of plants, selected on the basis ofethnopharmacological data, has been among themost succesful programs of screening plants forantiviral activity. Moreover, contrary to an-tibacterial and antifungal plant substances, severalantiviral plant compounds have exhibited compet-itive in vitro and in vivo antiviral activities withthose found for synthetic antiviral drugs, whichare currently in various stages of development.Finally, at present natural products have beenshown to interfere with many viral targets rangingfrom adsorption of the virus to the host cell torelease from it, which can result in mechanisms ofaction complemental to those of existing antiviraldrugs.Of course, mass screening of plant extractsshould be started and/or continued and naturally-occurring leads should by improved by structure-activity studies until optimal antiviral activity isobtained, but with an acceptable therapeuticindex.

Development of effective clinically useful an-tiviral agents will, however, only be made possibleby the willing collaboration of governments,academiae and pharmaceutical industries and thisgoal will only be obtainable, when a much closerworking relationship and willingness than is cur-rently in practice, could be reaiized.

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can ethnophamacology contribute to antiviral drugs

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