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

141

Can eth~opha~acology

contribute to the development of antiviral drugs?

Arnold J. Vlietinck and Dirk A. Vanden Berghe

Faculty 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 currently undergoing either preclinicat or clinical evaluation, and perspectives for finding new interesting antiviral drugs are promising. Among these 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 of action and are good candidates for further clinical research. What follows is a brief summary of the selection methods of plants for antiviral screening and in vitro and in vivo assays, which are currently used for detecting this activity in plant extracts. The importance of the plant kingdom as a source of new antiviral substances will be illustrated by presenting a survey on plant-derived antirhinovirus and anti-HIV agents.

introduction The identification of a retrovirus, human immunode~cien~y virus (HIV) as the causative agent of AIDS (acquired immunedeficiency syndrome), the steadily increasing incidence of various viral infections caused by viruses such as herpes simplex virus (HSV), varicella-zoster virus (VZV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV), in immunode~cient patients, the increasing evidence for the pathogenic role of a number of viruses viz. human papilloma virus (HPV) in the pathogenesis of primary hepatocellular carcinoma and the socioeconomic impact of virus infections of the respiratory tract (influenza, adeno, corona and rhinoviruses) and gastrointestinal tract (rotaviruses) have all been important factors in boosting the search for new antiviral agents and new modalities of antiviral chemotherapy. Although many compounds having potent antiviral activity in cell cultures and in experimental
Presented at the First International Congress on Ethnopharmacology, Strasbourg, 5-9 June, 1990. Correspondence to: Arnold J. Vlietinck, Faculty of Medicine, University of Antwerp (UIA), B-2610 Antwerp, Belgium. 0378-8741~03.50 0 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

animals have been detected, at present, only seven synthetic compounds and alpha interferon have been approved by the FDA for therapy of viral infections in humans. Four of these compounds are for the therapy of infections caused by members of the 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 (amantadine), is approved for the therapy and prophylaxis of respiratory infections caused by the influenza virus A. A sixth antiviral agent, l-~-~r~bofuranosyl-l,2,4-triazole-3-carboxamide (ribavirin) is approved for therapy of severe respiratory infections in children caused by the respiratory 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 of genital warts caused by papilloma virus and for the therapy of hairy cell leukemia and Kaposi sarcoma (Prusoff et al., 1989). The chemical structures of the FDA-approved drugs are given in Fig. 1.

142 0

OH

H-N a I

,l
I

0
CHs

HOC& I n

OH OH

NJ

4
Fig. 1. Chemical structures 4, acyclovir; 5, vidarabine; of antiviral drugs approved 6, ribavirin; 7, zidovudine

6
by the FDA for use in humans. (AZT).

7
I, Amantadine; 2. idoxuridine; 3, trifluridine;

None of these drugs, however, is without toxicities and hence there is a strong need not only to improve the actual antiviral armamentarium, but also to find an effective therapy of viral infections for which at present no clinically useful drugs or vaccines are available. There is also a need for finding new substances with extracellular virucidal activity, since many of the existing antiseptics and disinfectants fail to kill all pathogenic viruses after a 5-min exposure time at room temperature (Springthorpe et al., 1986; Vanden Berghe et al., unpublished results). Such compounds could be very useful to diminish the transfer of viable viruses from infected individuals to uninfected ones, to objects or to the air of enclosed spaces.

Therefore, all possible approaches towards the development of new antiviral and virucidal drugs should be pursued. Besides the rational approach, which in its ultimate form requires a sophisticated knowledge of both the target structure, e.g. the active site of an enzyme, a receptor, a macromolecule such as DNA, RNA or a regulatory protein and the compound to interact with it, the emperical approach still remains valuable (Prusoff et al., 1986; De Clercq, 1988; Montgomery, 1989). Systematic screening of pure substances or extracts of various origins has resulted in the discovery of many active leads. These substances can be improved by repeated structure modification in the hope that the change being made will result in increased selectivity, potency, duration of action,

143

bioavailabiiity and reduced toxicity. By combining structure-activity relationships with computergraphic model building, the sere~ndity approach can even become a rational serependity approach (Prusoff et al., 1989). There are many sources of materials for antiviral testing, including pure synthetic and natural substances of known structure and naturally occurring mixtures of the plant, animal and marine world. The purpose of this communication is to discuss and review the current activities in the area of medicinal plants and plant-derived products related to in vitro and in vivo preclinical evaluation for antiviral and virucidal activities. Selection of plants for antiviral screening Four basic methods are available for selecting plants for a screening programme to seek antiviral leads including: (1) random collection of plants followed by mass screening; (2) selection based on ethnomedical uses; (3) follow-up of existing literature leads; and (4) chemotaxonomic approaches (Suffness and Douros, 1979). The same advantages and disadvantages of the various approaches to the selection of plants for screening to discover anticancer activity can be associated with approaches to discover antiviral activity in plants (WHO Report, 1989). All factors considered, approaches (2) and (3) would seem to be the most cost-effective for finding plants with antiviral properties, Comparison of the different approaches showed the selection method based on folklore to give a five-times higher percentage (about 25%) of active leads, although, in some cases, the same active compounds were isolated from botanically nonrelated active plants. On the other hand, the screening of extracts from random collected plants afforded more novel substances with antiviral properties (Vanden Berghe et al., 1978; Ieven et al., 1982; Van Hoof et al., 1989). Since the screening capacity of our research group is rather limited, we prefer a selection of plants based on a combined analysis of ethnomedi~al, phytochemical, taxonomical and toxicological data (Vanden Berghe et al., 1986).

Selection of in vitro antiviral tests for the screening of plant extracts and natural products As the methodology used in the determination of the antiviral activity as well as the interpretation of the results have been virtually specific to each laboratory and are consequently not comparable to one another, simple procedures and guidelines for evaluating antiviral and/or virucidal activities of single componnds are urgently needed. This is even more true for the antiviral testing of crude extracts, containing a complicated mixture of different compounds present in several proportions. Various cell culture-based assays are currently available and can be succesfully applied for the antiviral (A) or virucidal (V) determination of single substances (S) or mixtures of compounds e.g. plant extracts (M). Antiviral agents interfere with one or more dynamic processes during virus biosynthesis and are consequently candidates as clinically useful antiviral drugs, whereas virucidal substances inactivate virus infectivity extracellularly and are rather candidates as antiseptics, exhibiting a broad spectrum of germicidal activities. Cost, simplicity, accuracy and reproductibility are the key factors which should determine the selection of the assay system, but also selectivity, sociality and sensitivity should be taken into account, especially for the testing of extracts. We refer to the appropriate literature for an extensive review of all factors influencing the design of antiviral chemotherapy experiments (Hermann, Jr., 1961; Kaufmann, 1965; Sidwell, 1986; Vanden Berghe et al., 1986). The methods commonly used for evaluation of in vitro antiviral activities are based on the different abilities of viruses to replicate in cultured cells. Some viruses can cause cytopathic effects (CPE) or form plaques. Others are capable of producing specialized functions or cell transformation. Virus replications in cell cultures may also be monitored by the detection of viral products, i.e. viral DNA, RNA or polypeptides. Thus, the antiviral test selected may be based on inhibition of CPE, reduction or inhibition of plaque formation and reducing virus yield or other viral functions (Hu and Hsiung, 1989; Vanden Berghe and Vlietinck, 1991).

144

A survey of the various in vitro antiviral tests and their possible suitability for the antiviral and/or virucidal screening of plant extracts and plant products is presented in Table 1. It should be emphasized that the toxic effects of an antiviral agent on the host cells must be considered since a substance may exhibit an apparent antiviral activity by virtue of its toxic effects on the cells. The cytotoxicity assay on cell cultures is usually done by the cell viability assay and the cell
TABLE 1 ANTIVIRAL SCREENING ASSAYS

growth rate test, although other parameters such as destruction of cell morphology under microscopic examination or measurement of cellular DNA synthesis have been used as indicators of compound toxicity (Hu and Hsiung, 1989). After the antiviral potency of a test substance together with its cytotoxicity is determined, the therapeutic index of the antiviral compound in a given virus-cell system can be calculated. Taking

IN VITRO

Determination of the viral infectivity in cultured cells during virus multiplication in the presence of a single compound (A-S) or a mixture of compounds e.g. plant extracts (A-M) or after extracellular incubation with a single compound (V-S) or a mixture of compounds (V-M). (1) Plaque inhibition assay Only for viruses which form plaques in suitable cell systems. Titration of a limited number of viruses in the presence of a non-toxic Applicability: A-S. (2) Plaque reduction assay Only for viruses which form plaques in suitable cell systems. Titration of residual virus infectivity after extracellular action of test substance(s). 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, a non-toxic dose of the test substance(s). Applicability: A-S; A-M. (4) Virus yield reduction assay Determination of the virus yield in tissues cultures, test substance(s). Virus titration is carried out after virus multiplication Applicability: A-S; A-M. Cytotoxicity should be eliminated e.g. by dilution.

dose of the test substance.

infected with a limited dose of virus and treated with

infected

with a given amount

of virus and treated

with a non-toxic

dose of the

by the plaque

test (PT) or the 50% tissue culture

dose end point test (TCDse).

(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 products For 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 transformation (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, determination of the uptake of radioactive isotope labelled precursors or viral genome copy numbers. Applicability: A-S; A-M; V-S; V-M.

14.5

into account that the cell growth rate test has been claimed to be the most stringent method for measuring cytotoxicity (Amtman et al., 1987), the therapeutic index (TI,) can be defined as the ratio of 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 the therapeutic index of a mixture of compounds e.g. crude extracts is irrelevant since cytotoxicity and antiviral activity of the mixture are not neccessarily due to the same components of the mixture. On the contrary, without the cytotoxicity data reports of antiviral activity of a single compound even at very low concentrations are of limited value. In addition, the relative potency of a new antiviral product should also be compared with existing approved drugs. Several years ago, we evaluated and compared the antiviral data of about 50 plantderived substances, which were reported in the literature to exhibit interesting in vitro antiviral properties, using the EPTT. The results of the study showed that, in many cases, the originally proposed antiviral activity of these substances was too low, non-specific or only detectable in toxic concentrations for the host (Vanden Berghe et al., 1986). In vivo testing of antiviral agents Whereas in vitro screening tests are undoubtedly a first choice not only to screen crude extracts, but. also to guide the isolation of the antivirally active compounds from the crude extracts, in vivo assays in a suitable animal model of the candidate compound remain the stepping-stone between tissue culture antiviral activity and the demonstration of a corresponding activity in human clinical trials. This model should predict efficacy in man, and must therefore mimic the natural disease as closely as possible. On the other hand, antiviral activity to the exclusion of cytotoxicity under tissue culture conditons should clearly be demonstrable in the infected laboratory animal. Too often, the therapeutic index is inadequate, so that the con-

centration of the antiviral agent in the target tissue of the infected animal will not be sufficient under dosing conditions to inhibit virus, due to toxicity or practical difficulties. Species-specific characteristics of absorption, tissue distribution, metabolism and excretion are the contributing factors. Animal models for a number of virus infections are available (Sidwell, 1986; Vanden Berghe et al., 1986). They are helpful in detecting not only if the candidate compound is an effective viral inhibitor without inducing viral resistance, but also if it is able to (1) reach the target organ, (2) be stable in the form administered, (3) be cleared by the tissue within a reasonable time, (4) resist and not adversely affect the immune system and (5) not interfere with the normal metabolic processes of uninfected cells (Galasso, 1984). Some viral infections, such as herpes simplex in guinea-pigs and mice and cytomegalo- or poliovirus infections in mice, mimic the natural diseases very closely (Sidwell, 1986). These animal models allow continuous observation of the infected animals, and virus spread and pathogenesis at local sites can be studied thoroughly. It is, however, uncertain whether animal models can be developed for all human infections requiring antiviral chemotherapy. The ability of human rhinovirus to replicate only in the respiratory tract of non-human primates, for instance, has posed a problem in translating the tissue culture antirhinovirus activity of a candidate compound to the stage of clinical evaluation and consequently delayed the finding of an antiviral drug effective for the prevention and treatment of the common cold considerably (Grunert, 1979). Up until now, no animal model is known for the study of HIV, although other retroviruses are known to cause leukemia, lymphoma and other forms of cancer in a 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 CNSassociated mortality in suckling mice is determined to evaluate in vivo antiviral activity of plantderived antiviral agents (Van Hoof et al., 1984; DeClerq E., 1984; Sidwell, 1986). For all viruses used in our tissue culture screen-

146

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

A few years ago, in their antiviral screening program of pure microbial and plant products, Ishitsuka et al. (1982a) found 5,4-dihydroxy-3,7,3trimethoxyflavone (Ro-09-0179) (3,7,3-trimethylquercetin or 3,7,3-TMQ), which was originally isolated from the Chinese medicinal herb Agastache rug;osa Kuntze, to be highly active in tissue cultures against all picornaviruses except Mengovirus. Independently, Van Hoof et al. (1982, 1984) found several 3-methoxyflavones, which were identified as derivatives of the 3-methylethers of quercetin (3-MQ) and kaempferol (3-MK), to be responsible for the pronounced antiviral properties of the alcoholic extracts prepared from different African Euphorbia spp. (Fig. 3).

Among the many derivatives of 3,7,3-TMQ that were synthesized and tested one chalcone, 2-hydroxy-4-ethoxy-4,6-dimethoxychalcone (Ro-09-0410), emerged as a new type of antiviral agent exclusively active against many human rhinovirus serotypes and, consequently, a candidate drug for the treatment of the common cold (Ishitsuka et al., 1982b) (Fig. 2 (2)). The antirhinovirus activity of flavan was discovered serependitously during an in vitro screening program utilizing the plaque inhibition test. Structureactivity relationship studies led to the 4,~dichloro~avon derivative (BW 683C), being the most potent agent against several rhinovirus serotypes (Bauer et al., 1981, 1983) (Fig. 2 (1)). Subsequently, it was shown that flavans and chalcones inactivate rhinoviruses directly by binding to or interacting with specific sites on the viral capsid proteins. While little or no interference with viral attachment or penetration of the host cell membrane was observed, the uncoating pro-

C14b

CH3

?>a
5

0-(CH,),

acH ~~
3

7;)
N CH,COOH

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); (RMI i-(S-tetradecyloxy-2-furanyl)ethanone 15,731); 5. (S)-(-)-5-~7-[4(4,S-djhydro-4-methy~-2-oxazolyl) 4, phenoxy~hepty~l-3-~thylisox~ole (WIN 52084); 6, 2-I( 1,5,1Oa-tetrahydro-3H-thiazolo[3,Qb)-isoquinolin-3ylindene)aminol~-thiazoie acetic acid (5) (44-081 R.P.).

147

cess in the host cell was inhibited through stabilization of the protein capsid of the virus and prevention of the conformational changes required for release of viral RNA (Ninomiya et al., 1984; Tisdale et al., 1984). At the same time, several pharmaceutical companies developed a number of synthetic capsidbinding drugs with prominent antirhinovirus properties. Most were rhinovirus specific, but all of these agents had substantial serotype related variability in antiviral activity. The concentrations inhibiting rhinovirus replication in vitro varied up to lOOO-foldfor different serotypes, indicating that the binding sites on the viral capsid proteins were highly specific (Sperber and Hayden, 1988). X-ray crystallographic structural analysis has determined that the precise binding site of the WIN compounds, which have an isoxazole at one end and an oxazolyl-phenoxy group at the other end of an alkyl chain (Fig. 2 (5)), to rhinovirus type 14 is the interior of viral protein 1 (VPl), one of the three external polypeptides of the protomers of the protein shell of the virus (Smith et al., 1986). Changes in the amino acids of this binding pocket may affect the ability of a specific agent to bind to the capsid and thus explain the different susceptibilities of different rhinovirus serotypes. The binding sites for some of these agents me be the same or lie very close to one another (Ninomiya et al., 1985). Another potential limitation with the use of these compounds is that drug-resistant mutants can be selected readily under in vitro conditions (Selway, 1986). Clinical trials have found discrepancies between the in vivo and in vitro antiviral activities of these compounds. Orally administered dichloroflavan and phosphorylated chalcone were ineffective in the prophylaxis of experimental rhinovirus colds (Phillpotts et al., 1983, 1984). Also, intranasal preparations of both compounds failed to reduce infection rates or protect against illness, probably because no adequate levels of drugs were achieved in nasal mucosal cells (Al-Nakib et al., 1987a, 1987b). Similarly, in vivo test results for the synthetic antiviral agents RMI 15,731 (Fig. 2 (4)) and 44-081 RP (Fig. 2 (6)) did not show significant prophylactic 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 derivative, however, caused marked reductions in nasal symptoms and mucus weights, when it was administered intranasally in frequent doses beginning 1 h before and continuing for 6 days after experimental rhinovirus challenge with a very susceptible serotype (Sperber and Hayden, 1988). The relative success of the latter experiment might probably be ascribed to the efficient absorption through the nasal mucosa of the hydrophobic antiviral agent from a pharmaceutical composition containing cyclodextrines (European Patent Application No. 88201288.3, 1988). The 3-methoxyflavones, 3-MQ and 3-MK, however, have shown not to interact with the capsid proteins of picornaviruses, but rather to interfere with an early stage in the viral RNA synthesis. Although their exact mode of action is not completely understood yet, it has been found that they probably inhibit the formation of minus-strand RNA of poliomyelitis virus by interacting with the proteins involved in the binding of the virus replication complex to vesicular membranes, at which poliovirus replication takes place (Castrillo et al., 1986; Vrijsen et al., 1987; Lopez Pila et al., 1989). In contrast to the capsid-binding antivirals no drug-resistant mutants have been detected in the presence of 3-methoxyflavones (Ninomiya et al., 1985). The attractive mechanism of action, the pronounced and broad-spectrum antiviral activity and the lack of resistance-induction by these flavones prompted us to explore this class of flavonoids. From a large screening program of naturallyoccurring 3-methoxyflavones emerged jaranol and penduletin (Fig. 3) as the most in vitro active substances against polio- and rhinoviruses. In order to establish a structure-activity relationship a series of A-ring substituted analogues of 4-hydroxy-3-methoxyflavone (Fig. 3, MF 110) were synthetized and tested antivirally. The most interesting compound was 4,7-dihydroxy-3-methoxy-5,6_dimethylflavone (Fig. 3, MF 142) possessing in vitro TIg9 values of > 1000 and 200 against poliovirus type 1 and rhinovirus type 15, respectively (EPTT). This compound was also active

148 A, = R, R, = = R, H ; = R, = H MFllO

R4

R2 =

R, = OH ; R, = OCH,

Joranol 3-MK 3-MQ

Penduletin MF142 MF140

R2 = R4 5: H ; R, = R, = OH R, = H ; R, = R3 = RI = OH R, = H ; Rt = OH ; R, = R, = OCH,
R, = H ; R,

R, = Rz = CHa ; R, = OH
= t-l ; R, = CH,

Rz =

; R, = OCH,

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 other rhinovirus serotypes having MICse values ranging from 0,016 to 0.5 @g/ml. The corresponding values of a moderately active analogue such as 4-hydroxy-3,7-dimethoxy-5-methylfavone (Fig. 3, MF 140) were 10-25 times higher (De Meyer et al., 1990). It was also found that, in contrast to quercetin, MF 142 was not mutagenic in concentrations up to 2.5 mg in the Ames test (De Meyer et al., 1991). Since some 3-methoxyflavones, when administered intraperitoneally, have been shown to protect mice from lethal infections from coxsackie B_, (Van Hoof et al., 1984), the most antivirally active substance of this study viz. MF 142 should be considered as a promising candidate for clinical studies in human volunteers.
Plantderived anti-HIV agents

Acquired i~unede~ciency syndrome is a pandemic immunosuppressive disease which results in life-threatening opportunistic infections and malignancies. Since a retrovirus, designated human i~unode~ciency virus (HIV), has been isolated and identified as the etiologic agent of this compounds have been disease, numerous evaluated for their inhibitory effects on HIV replication in vitro (DeClercq, 1987). Moreover, the AIDS saga has resuscitated several substances,

especially reverse transcriptase inhibitors and interferon inducers, which were mentioned many years ago as antiviral agents, then forgotten to be discovered again as potential candidates for the treatment of AIDS (DeClercq, 1989). Besides the inhibition of reverse transcriptase, a multifunctional enzyme that transcribes the viral RNA genome to DNA, the replication cycle of HIV offers a wealth of possible targets for antiviral agents, and so might several viral regulatory genes such as tat and rev, which regulate respectively the transactivation and the expression of viral proteins and vis, which determines virus infectivity (Oxford et al., 1989). A wide range of biochemical and cell culturebased assays are currently available for the detection of in vitro anti-HIV activity. However, a minimum of a single T-cell culture assay system (eg. H9, ATH8, MT-2, MT-4 etc.) should be used as a first-line screen of crude extracts or to guide the antiviral activity during the isolation. Once the active component(s) are identified, more detailed confirmatory assays should be carried out (WHO Report, 1989). Recently, several single, sensitive and rapid tests for the screening of a large number of samples have been described. Among these tests, a colorimetric assay based upon the transformation of a tetrazolium salt to a coloured formazan by living cells but not by dead cells or culture medium has

149

shown to be very useful in the large scale evaluation of anti-HIV agents (Pauwels et al., 1988; Schwartz et al., 1988). Up until now, only one study on the screening of plant extracts for anti-HIV activity has appeared (Chang and Yeung, 1988). Out of 27 medicinal plants used in traditional Chinese medicine as anti-infectives, 11 showed in vitro inhibitory activity against HIV. Of two of these active plants, i.e. Viola yeodensis and Prune&r ~~gur~~, the active principles were isolated and identified as sulphated polysaccharides (Ngan et al., 1988; Tabba et al., 1989).

Other sulphated polysaccharides of different origins including heparin, A-carrageenan, dextran sulphate, pentosan polysulphate and an aqueous extract of the red alga, ~c~~~~~e~j~ paciJca have proven to be very potent and selective inhibitors of HIV replication in TJymphocyte cultures (Ito et al., 1987a; Nahashima et al., 1987; Ueno and Kuno, 1987; Baba et al., 1988). These substances were inhibitory to HIV at a concentration which was lo-fold (heparin) or more than lOO-fold (dextran sulphate, pentosan polysuiphate) below their anticoagulant threshold (De Clercq, 1989). It was also shown that their anti-HIV activity should be

CH20S0,0H

I
L

ASO~OH
1
OH

hS020H

RO()cz/
1
COOH

2
o-

CHO

OH

OH

CHO

HO HO

R CHJO R = CH3 R = CH20H

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

150

readily attributed to an inhibitory effect on virus absorption rather than to their inhibitory action on HIV-associated reverse transcriptase (Baba et al., 1988). Other studies have provided additional information on natural products with experimental antiHIV activity. Their chemical structures are depicted in Fig. 3. Glycyrrhizin, one of the main saponins of Glycyrrhiza glabra, inhibits the growth of a number of DNA and RNA viruses including HIV-l in vitro (Ito et al., 1987b). In Japan, glycyrrhizin has been studied in AIDS patients. It was claimed that, when given orally to asymptomatic HIV carriers, the substance delayed the progression of symptoms related to HIV. It was also claimed that, when administered intravenously for a period of more than a month to hemophilics with AIDS, viral antigen considerably decreased, suggesting that glycyrrhizin might inhibit HIV replication in vivo. In another claim, simultaneous administration of the compound appeared to decrease the adverse reactions of zidovudine (Hattori et al., 1989). Studies on the mechanism of action of glycyrrhizin suggested that the compound, being a polyanionic substance, interferes with virus adsorption, which is further complemented by an inhibitory effect on protein kinase C. This enzyme seems to be required for the binding of HIV-l particles to the cellular CD4 receptors (Ito et al., 1988). Also, other saponins including saponin B, from soybean exhibited potent antiHIV effects in vitro (Nakashima et al., 1989). Castanospermine, an indolizidine alkaloid from the seeds of Castanospermum australe, blocks glycoprotein processing via inhibition of oglucosidase I located in the endoplasmatic reticulum. This alkaloid has been reported to have in vitro anti-HIV activy and has been shown to be active in vivo when administered orally to mice. There are also indications that a combination of castanospermine and zidovudine inhibits HIV synergistically in vitro (Ruprecht et al., 1989). and hypericin The naphtobianthrones pseudohypericin, which occur in plants of the Hypericum family, are highly effective in preventing viral-induced manifestations that follow infections with a variety of retroviruses in vivo and in

vitro. These compounds interfere with viral release from infected cells and possibly inactivate virions. Preliminary in vitro studies have shown that pseudohypericin reduce the spread of HIV (Meruelo et al., 1988). Other plant-derived compounds with inhibitory effect on HIV replication in vitro are the (-) enantiomer of gossypol, a polyphenolic aldehyde extracted from cottonseed (Polsky et al., 1989; Lin et al., 1989) and the wellknown alkaloid papaverine (Turano et al., 1989). Many patent applications for treatment and propylaxis of retroviral infections including HIVinfections have been deposited for plant products e.g. hydrolysable tannins from Coriaria, Oenothera and Agrimonia spp., lectins from Ulex europeus, Maackia amurensis, Glycine max and Lens culinaris, Japanese pine cone extract, proteinpolysaccharide complexes (Kurestin) and sugarprotein-inorganic elements mixture of different Basidiomycetes including Coriolus spp. and Lentinus edodes. It is questionable, however, whether such preparations will have therapeutic efficacy against retrovirus infections in animal models. Only a few experimental studies to discover antiHIV agents from medicinal plants and other natural products are in progress. The major program of this type is being carried out at the National Cancer Institute in the U.S.A. The program will screen about 4500 plant samples per year for the next 5 years for in vitro anti-HIV activity, based on a random selection of plants (WHO Report, 1989). Although the number of anti-HIV screening studies of plant extracts or plant-derived substances has been rather limited in comparison to the mass screening programs of synthetic compounds, nevertheless several natural substances have emerged as promising leads for the development of anti-HIV agents. Whether these compounds will have any clinical potential in the therapy of AIDS remain to be determined. It is, however, essential that these studies should be encouraged and continued. Conclusions

The question if ethnopharmacology tribute to the development of antiviral

can condrugs can

151

be answered in a positive sense without too much premature optimism. Indeed, the screening of a relatively low number of randomly collected plant substances has afforded a remarkably high ratio of active leads in comparison with the screening programs of synthetic compounds. On the other hand, the testing of plants, selected on the basis of ethnopharmacological data, has been among the most succesful programs of screening plants for antiviral activity. Moreover, contrary to antibacterial and antifungal plant substances, several antiviral plant compounds have exhibited competitive in vitro and in vivo antiviral activities with those found for synthetic antiviral drugs, which are currently in various stages of development. Finally, at present natural products have been shown to interfere with many viral targets ranging from adsorption of the virus to the host cell to release from it, which can result in mechanisms of action complemental to those of existing antiviral drugs. Of course, mass screening of plant extracts should be started and/or continued and naturallyoccurring leads should by improved by structureactivity studies until optimal antiviral activity is obtained, but with an acceptable therapeutic index. Development of effective clinically useful antiviral agents will, however, only be made possible by the willing collaboration of governments, academiae and pharmaceutical industries and this goal will only be obtainable, when a much closer working relationship and willingness than is currently in practice, could be reaiized.

The authors are pleased to acknowledge the Belgian National Fund for Scientific Research (NFWO) for financial support of the work under grant no. 3.000486. References
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