Anti-HIV Activity of Medicinal Herbs: Usage and Potential Development

Anti-HIV Activity of Medicinal Herbs: Usage and Potential Development

Ji An Wu

(Accepted for publication June 5, 2000)

Abstract: The acquired immunodeficiency syndrome (AIDS) is a result of human immunodeficiency virus (HIV) infection which subsequently leads to significant suppression of immune functions. AIDS is a significant threat to the health of mankind, and the search for effective therapies to treat AIDS is of paramount importance. Several chemical anti-HIV agents have been developed. However, besides the high cost, there are adverse effects and limitations associated with using chemotherapy for the treatment of HIV infection. Thus, herbal medicines have frequently been used as an alternative medical therapy by HIV positive individuals and AIDS patients. The aim of this review is to summarize research findings for herbal medicines, which are endowed with the ability to inhibit HIV. In this article, we will emphasize a Chinese herbal medicine, Scutellaria baicalensis Georgi and its identified components (i.e., baicalein and baicalin), which have been shown to inhibit infectivity and replication of HIV. Potential development of anti-AIDS compounds using molecular modeling methods will also be discussed.

The acquired immunodeficiency syndrome (AIDS), caused by the human immunodeficiency virus (HIV), is an unprecedented threat to nations as well as to global health (Fauci et al., 1985; Fauci and Artlett, 1998). It is estimated that each year HIV infects at least 2 million people in the United States and more than 10 million people worldwide. Thus, the search for effective therapies to treat AIDS is urgently needed. In order to combat HIV, a colossal amount of money and manpower have been dedicated to searching for compounds that can be developed as therapeutic agents. In the last two decades, several chemical anti-HIV agents have been developed. However, herbal medicines have also been used frequently by HIV positive individuals and AIDS patients as an alternative medical therapy (Kassler et al., 1991).

Conventional Chemotherapy for HIV Infection

According to De Clercq the replicative cycle of HIV is comprised of ten steps that may be adequate targets for chemotherapeutical intervention (De Clercq, 1995a; De Clercq, 1995b). Most of the substances that have been identified as anti-HIV agents can be assigned to one of these ten classes of HIV inhibitors based on the stage at which they interfere with the HIV replicative cycle. These ten steps are: (1) Viral adsorption to the cell membrane, (2) Fusion between the viral envelope and the cell membrane, (3) Uncoating of the viral nucleocapsid, (4) Reverse transcription of the viral RNA to proviral DNA, (5) Integration of the proviral DNA to the cellular genome, (6) DNA replication, (7) Transcription of the proviral DNA to RNA, (8) Translation of the viral precursor mRNA to mature mRNA, (9) Maturation of the viral precursor proteins by proteolysis, myristoylation, and glycosylation and (10) Budding, virion assembly and release. Step 4, a key step in the replicative cycle of retroviruses, which makes it distinct from the replicative cycle of other viruses, is the reverse transcription catalyzed by reverse transcriptase. Another target for therapeutic intervention is step 9, particularly the proteolysis of precursor proteins by HIV protease. The majority of chemotherapeutic strategies have, therefore, focused on the development of retroviral enzyme inhibitors.

The U.S. Food and Drug Administration (FDA) has approved a number of anti-HIV drugs for clinical use (De Clercq, 1999). However, these medications have limitations such as high cost, peripheral neuropathy and decreased sensitivity due to the rapid emergence of drug resistant mutant virus strains, and adverse effects like bone marrow suppression, and anemia (Lee and Morris-Natschke, 1999; Vandamme et al., 1998). Thus, more effective and less toxic anti-HIV agents are still needed. In addition, alternative approaches, including herbal therapies, long-term screening of plant extracts, particularly anti-infective or immunomodulating medicinal herbs, and the structural modification of lead compounds, have been attempted.

Studies of Medicinal Herbs on HIV Infection

Use of Herbs in Asia and North America

Herbal medicine has been used in China for centuries. Even after opening its doors to western medicine two centuries ago, China still relies heavily on traditional medicine and herbal therapies because of their efficacy. Indeed, the recent focus of the Chinese government has been to propel research at its institutes and universities towards developing efficacious herbal drugs, particularly as anti-cancer, anti-cardiovascular disease, and immunomodulating agents (Huang, 1999).

In China, medicinal herbs are being used in the treatment of HIV positive subjects and AIDS patients. One example is the traditional Chinese medicinal herb Tian-Hua-Fen (Trichosanthes kirilowii), which appears in the classical Chinese medical reference work Compendium of Materia Medica from the late 14th century. Tian-Hua-Fen has been used in China for hundreds of years to reset menstruation and expel retained placentas. Trichosanthin (TCS), an active protein component isolated from Tian-Hua-Fen, has been shown to inhibit HIV infection and has been used in the clinical treatment of AIDS (Zhao et al., 1999).

In addition to the study of Tian-Hua-Fen, two multiple screening approaches have been applied to aqueous extracts of 19 herbs in Hong Kong (Collins et al., 1997) and 35 herbs in Beijing (Tang et al., 1990) in order to detect anti-viral agents. Also, an oriental remedy called Xiao-Chai-Hu-Tang (Chinese name) or Sho-saiko-to (SST or TJ9, Japanese name), which consists of a mixture of aqueous extracts from seven commonly used herbs, has been used on AIDS patients in China and Japan (Buimovici-Klein et al., 1990; Wu et al., 1995; Piras et al., 1997).

Scientists from both Thailand and Japan, have worked together to screen the anti-HIV activity of 413 plants grown in Thailand. Significant inhibitory activity has been found in 81 of these plants (Yamamoto et al., 1997).

In the U.S., the use of herbs as an alternative medical treatment for many illnesses has increased steadily over the last decade. Because herbs are categorized as natural food products or dietary supplements, they are not currently subject to strict control by the FDA (Huang, 1999). However, many patients with AIDS are using herbal medical therapies in addition to conventional treatment. One study reported that in 1991, 22% of AIDS patients had used one or more herbs for medicinal purposes in the previous 3 months (Kassler et al., 1991). Recently, this percentage has increased (Phillips et al., 1995). Our group evaluated a new Chinese herbal medicine formulation, Qian-Qun-Ning, which consists of a mixture of aqueous extracts from 14 different herbs, and showed efficacy in a pilot clinical trial of HIV positive subjects (Xue et al., 1999).

Since 1987, the U.S. National Cancer Institute has worked with the Chinese Academy of Sciences to study Chinese medicinal herbs for anti-AIDS application. Over 1000 Chinese traditional medicines were screened using different solvent extraction forms and more than 140 different herbs were found to have HIV inhibition activity. Among them, more than 20 herbs have exhibited significant HIV inhibitory activity (Luo et al., 1999).

In this country experimental studies are in progress to isolate anti-HIV agents from medicinal plants and their natural products. In one such study, conducted by the National Cancer Institute, approximately 4500 plant samples are currently screened per year for in vitro anti-HIV activity, based on a random selection of plants (Vlietinck et al., 1998).

At the University of Illinois at Chicago (Tan et al., 1991), a simple in vitro method has been developed for screening the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitory potential of natural products. More than 100 plant extracts have been evaluated, and 15 of these extracts show significant inhibitory activity. 156 natural products have been examined in this system (Tan et al., 1992).

Bioactive Components from the Herbs

A number of articles that discuss the HIV inhibitory activity of herbs and their natural products (Chu and Cutler, 1992; Ng et al., 1997; Cragg et al., 1997; Vlietinck et al., 1997; Vlietinck et al., 1998; Luo et al., 1999; Lee, 2000) suggest that a variety of chemically disparate molecules, produced by species distributed across the plant kingdom such as algae, pine trees and flowering plants are effective at inhibiting the activity of HIV. These compounds are comprised of (1) Aliphatic Ketones and Aldehydes, (2) Terpenoids, (3) Alkaloids, (4) Coumarin Derivatives, (5) Flavonoids, (6) Xanthone, (7) Flavone-Xanthone C-glucoside, (8) Hyperlein, (9) Tannins, (10) Gossypol Acetic Acid, (11) Polysaccharides and (12) Proteins.

Because of their potential systemic effects and prophylactic action against HIV infection, plant-derived antiviral agents are prime study candidates. They may also be useful as topical agents to inactivate newly formed viruses, or as adjuvants with other antiviral drugs.

In the isolation of natural products, it is essential to adhere to the following steps. First, the plant kingdom as a source of new antiviral lead compounds should continue to be explored. Second, lead compounds which have been shown to inhibit HIV activity should be developed through modern pharmacological methods to increase activity and decrease toxicity. Finally, herbal medicines or natural products as part of drug combination regimens for the treatment of HIV-infections should be encouraged and continued (Vlietinck et al., 1998).

Anti-HIV Activity of Flavonoids and Scutellaria Baicalensis Georgi

Various flavonoids have been shown to inhibit, in vitro, the reverse transcriptase of certain retroviruses, including HIV (Step 4 of replicative cycle), as well as cellular DNA polymerases. These products exhibited selective anti-HIV-I activity (Wang et al., 1998; Mahmood et al., 1993), whereas baicalein (5,6,7 trihydroxyflavone), a constituent isolated from Scutellaria baicalensis Georgi (Huang Qin in Chinese, Worgon in Japanese), specifically inhibited HIV reverse transcriptase (Kitamura et al., 1998; Huang, 1999).

In AIDS treatment, the inhibition of HIV reverse transcriptase is currently considered a useful approach, therefore, natural products that show inhibitory activity have been extensively explored (Ng et al., 1997).

Effects of baicalein and baicalin. Ono et al. showed the effects of baicalein on the activity of various reverse transcriptases. They demonstrated that 1 [micro]g/ml baicalein inhibited 90% of the activity of MLV-reverse transcriptase, and that 2 [micro]g/ml baicalein inhibited 90% of the activity of HIV-reverse transcriptases (Ono et al., 1989).

Tang (Tang et al., 1990) found that baicalin, which is isolated from Scutellaria baicalensis Georgi, inhibited HIV-reverse transcriptase, with an [IC.sub.50] value of 22 [micro]M. Some pharmacological test results have demonstrated noncompetitive, inhibition of retroviral reverse transcriptase activity in HIV-1-infected H9 cells (Zhang et al., 1991), HIV-1 specific core antigen p24 expression, and quantitative focal syncytium formation on CEM-SS monolayer cells (Li et al., 1993). Baicalin, and its derivative 7-glucuronic acid 5, 6-dihyorxyflavone were also efficacious in inhibiting reverse transcriptase of other retroviruses (Baylor et al., 1992). The difference in HIV-1 reverse transcriptase inhibitory activity between baicalein and baicalin has been examined (Zhao et al,, 1998), The results show that the HIV-1 reverse transcriptase inhibitory activity of baicalein was four times higher than baicalin. The inhibition of HIV-1 integration (Step 5 of replicative cycle) by baicalein was investigated biochemically and by means of structure-activity relationships. It was reported that [IC.sub.50] for HIV integrase inhibition by baicalein was 4.3 [micro]M (Raghavan et al., 1995).

An investigation on the metabolism of baicalin has been published (Muto et al., 1998). The results indicated that baicalin was first metabolized into baicalein, and the final metabolite was identified as baicalein 6-O-sulfate by comparing its retention time in high-performance liquid chromatography (HPLC), and electrospray ionization mass spectra (ESI-MS)/MS methods with that of an authentic sample.

Mechanisms of action. As for the mechanism of the anti-HIV-1 effect of baicalin, it was found that baicalin and baicalein have an inhibitory effect on various cellular DNA and RNA polymerases (Ono and Nakane, 1990; Kitamura et al., 1998). In the case of baicalein, the mode of inhibition was of the competitive type (murine leukemia virus reverse transcriptase and HIV-1 reverse transcriptase) with respect to the template primer ((rA)n(dT) 12-18), or mixed type suggesting that baicalein also inhibits HIV-1 reverse transcriptase activity by interfering with the binding of viral RNA to the reverse transcriptase molecule near the active site of the enzyme. Baicalin does not inhibit the activity of HIV-2 reverse transcriptase, or murine leukemia virus reverse transcriptase. Futhermore, baicalin neither inhibited the binding of OKT4A mAb to the gp 120 binding site of CD4, nor interfered with the gp 120-CD4 binding. This definitely rules out the possibility that baicalin interferes with the virus adsorption step (Step 1 of replicative cycle). Flavonoids such as gardennin, myricetin, and baicalein were found to inhibit HIV-1 protease. However, the [IC.sub.50] value of baicalein was 480 [micro]M, almost 44 times that of gardennin ([IC.sub.50] = 11 [micro]M) (Brinkworth et al., 1992).

Efficacy of herbal formulation. As mentioned above, Xiao-Chai-Hu-Tang or Sho-saiko-to consists of a mixture of aqueous extracts from seven different plants. Seven and a half grams of this contains 4.5 g of dried extract, which is prepared from boiled water extracts of seven herbs: 7.0 g of Bupleurum root, 5.0 g of Pinellia tuber, 3.0 g of Scutellaria root, 3.0 g of Jujube fruit, 3.0 g of Ginseng root, 2.0 g of Glycyrrhiza root, and 1.0 g of ginger rhizome (Shimizu et al., 1999; Geerts and Rogiers, 1999). Some research groups demonstrated that among the active components of Sho-saiko-to, baicalein and baicalin were found to be mainly responsible for antioxidative (Shimizu et al., 1999; Yoshino et al., 1997), anti-tumor (Tsutsumi et al., 2000; Wang et al.; 1998, Liu et al., 1998; Kato et al., 1998; Mizushima et al., 1995; Motoo and Sawabu, 1994), anti-proliferative (Inoue and Jackson 1999; Ono et al., 1999; Yagura et al., 2000), and anti-HIV (Buimovici-Klein et al., 1990; Muto et al., 1998) activity.

It is interesting to note that data on antioxidative activity between Sho-saiko-to and Scutellaria root using MeOH extracts were very similar (Shimizu et al., 1999). Our group and other researchers indicated that the water extracts of Scutellaria root also have significant antioxidant activity (Shao et al., 1999). In the four major constituents, the order of antioxidant activity is baicalein > baicalin >> worgonin > wogonoside (Gao et al., 1999). Antioxidant and other mechanisms may also play a role in the anti-HIV effects of baicalin and baicalein (Kitamura et al., 1998).

An oral dose toxicity study of Sho-saiko-to in rats has been reported (Minematsu et al., 1995). Two oral doses (2 and 6.4 g/kg) of Sho-saiko-to were administered to the animal after overnight fasting, and no death was observed.

Combination of herbal medicine and chemotherapy. Combination therapy for AIDS patients has been applied, discussed and standardized (Vandamme et al., 1998). Synergistic anti-HIV-1 effects of baicalin with 3′-Azido-2′,3′-dideoxythymidine (AZT) have been reported (Inada et al., 1994), suggesting that baicalin might be potentially useful as part of a drug combination regimen for the treatment of HIV-1 infections. The use of Sho-saiko-to as an adjuvant with other antiviral drugs such as 3TC has been published (Piras et al., 1997). A patent for anti-AIDS virus, effect-enhancing agents containing Sho-saiko-to or baicalein has been approved in Japan (Maikeru et al., 1996).

Discovery and Development of Plant-derived Natural Products and Their Analogues as Anti-HIV Agents

Although the history of Chinese herbal medicines dates back thousands of years, herb-drug interactions should not be overlooked (Fugh-Berman, 2000). With any anti-AIDS drug, attention must be paid to adverse effects, long-term sustainable effects, and increased toxicity due to drug-drug interaction in a person receiving multiple drug therapies. Thus, the search for effective and less toxic, anti-AIDS agents of single structure still continues. One approach is to modify novel, lead compounds derived from plants. Some promising research developments from different group have been reported (Lee and Morris-Natschke, 1999; Xie et al., 1999; Kashiwada 1999; Luo, et al., 1999).

A successful example is the study by Lee and Morris-Natschke (1999). Through a bioactivity-directed search for plant-derived, naturally occurring compounds, the lead compound sukudorfin was isolated from the fruit of lomatium suksorfii and its structure was identified. Sukudorfin inhibited HIV-1 replication in H9 lymphocytes with an in vitro [IC.sub.50] value of 1.3 [micro]M and a therapeutic index (TI; TI = [LD.sub.50]/[IC.sub.50]) value of over 40. The discovery of sukudorfin led to the syntheses of 42 khellactone derivatives by structure modification. Among these synthetic compounds, the most promising lead compound was 3′, 4′-di-O-(S)-(-)-camphanoyl- (3’R, 4’R)-(+)-cis-khellactone (or DCK), which showed extremely potent activity ([IC.sub.50] = 0.00041 [micro]M) against HIV-1 replication in the H9 cell line, and had a remarkable TI value of 136,719. In comparison, the values of AZT in the same assays were 0.15 [micro]M and 12,500, respectively. As an anti-HIV chemotherapeutic agent, DCK is a candidate for an anti-AIDS clinical trial (Lee and Morris-Natschke, 1999; Kashiwada, 1999; Xie et al., 1999).

When baicalein was first found to be a strong inhibitor of reverse transcriptase activity, the question arose as to the necessary structural requirements of the flavonoid for such activity. It is believed that (1) number, (2) position of the putative functional groups (hydroxyl groups), and (3) flavone or flavonoid structure (Oho et al., 1990) are important. As a lead compound, structural modification and structure-activity relationship (SAR) research of baicalin and baicalein has been reported. The results indicated that the flavonoids with hydroxyl groups at C-5 and C-7 in the A-ring, and with a C-2-C-3 double bond were the most potent HIV growth inhibitors. In general, the presence of substituents (hydroxyl and halogen) in the B-ring increased toxicity and/or decreased activity (Wang et al., 1998; Zhao et al., 1997; Hu et al., 1994).

According to the above information on structure activity relationships, structure modification methods can be also used for flavonoid lead compounds, which are derived from plants with possible anti-HIV activity. As a potential target, the heteroatom in position 1 of the C ring of the flavonoid compounds has been considered. Therefore, similar or even new biological activities could be anticipated when the oxygen of bioactive flavonoids is replaced by another atom such as nitrogen or sulfur, which lines up closely with oxygen in the Periodic Table. Thus, a series of 5,6,7,8-substituted-2-phenylthio-chromen-4-ones have been synthesized and evaluated for anti-HIV activity (Wang et al., 1998). Among them, one new compound was the most active ([IC.sub.50] value of 0.65 [micro]M) against HIV in acutely infected H9 lymphocytes, and had a TI of approximately 5.

Using Molecular Modeling Methods to Develop Anti-AIDS Compounds

Computer-Assisted Drug Design and Molecular Modeling

Depending on the lead compound used to develop new, anti-AIDS compounds, an important method that can be applied is computer-assisted drug design (CADD). The earliest method has been called quantitative structure-activity relationships (QSAR) (Garg et al., 1999). The currently used method involves traditional or classic QSAR and 3D QSAR. In the traditional approach to QSAR, the chemical structure can be described with experimental and theoretical steric, electronic, and hydrophobic parameters. 3D QSAR methods were developed as an alternative to traditional QSAR to describe molecules more “realistically”, i.e., with properties of molecules calculated from their three-dimensional structures. These two approaches of QSAR are widely used in the area of drug design and agrochemistry design.

Garg et al. summarized the investigations with QSAR for anti-HIV drug design. The relationship between structure and anti-HIV activities, log P volume (partition coefficient) and stereo effect are very important parameters (Garg et al., 1999). A 3D QSAR research set of 15 flavones, including baicalein, that inhibit HIV-1 integrase-mediated cleavage and integration in vitro were tested using Comparative Molecular Field Analysis (CoMFA) (Raghavan, et al., 1995; Garg et al., 1999; Wang et al., 1998). The results show a strong correlation between the inhibitory activity of these flavones and the steric and electrostatic fields around them. A diversity analysis of 14156 molecules tested by the National Cancer Institute for anti-HIV activity using the quantitative structure-activity relational expert system MCASE has been reported. This study shows that certain structure-activity relationships exist among the anti-HIV-1 agents. They found that log P and the Highest Occupied Molecular Orbital (HOMO) coefficient of hydrogen bond acceptors are important factors for the activity of some biophores. With the help of the resulting model, they have tested 10 highly diverse chemicals that came from different sources, the overall accuracy of their prediction being 80%. This result provides a first glance at the possible predictivity of MCASE (Klopman and Tu, 1999).

Molecular modeling has become a well-established research area during the last decade due to advances in computer hardware and software that have brought high-performance computing and graphics within the reach of most academic and industrial laboratories. It is very important to realize what is really meant by “computer-assisted drug design” (CADD) with the QSAR method. Molecular modeling systems provide powerful tools for building, visualizing, analyzing and storing models of complex molecular systems (i.e. inhibitor binding with receptor) that can help interpret structure-activity relationships (Cohen et al., 1990).

There are the two major molecular modeling strategies currently used in the conception of new drugs for macromolecules (called “direct design”) and small molecules (called “indirect design”). In direct design, the three-dimensional features of a known receptor site are directly considered. Indirect design is based on the comparative analysis of the structural features of known active and inactive molecules that are interpreted in terms of complementarity with a hypothetical receptor site model (Cohen et al., 1990).

Over the past 15 years, molecular modeling combined with database searching has become a part of the drug discovery and design process. An increasing number of applications for molecular modeling combined with database searching has led to the discovery of new lead compounds used in drug design (Martin, 1992; Bures, 1998). Database searching programs have been developed and are being widely used in an integrated fashion with molecular modeling systems such as DOCK, which depicts a small molecule docking to the macromolecule (Kuntz, 1992; Briem and Kuntz, 1996; Wang et al., 1999). DOCK is typically used to generate proposed binding orientations of small molecules, (ligands, such as an inhibitor) and macromolecules, (receptor, such as an enzyme) with an X-ray crystallographic structure of the macromolecule as the starting point. DOCK can find many orientations for a single molecule, or it can be used to search a database to identify compounds that may bind with a macromolecular site. The output compounds from DOCK are uniformly oriented in the target site and can be viewed by most molecular modeling programs (Bures, 1998; Shoichet and Kuntz, 1996).

Molecular Modeling in Anti-HIV Studies

The application of molecular modeling to develop new anti-HIV drugs is just unfolding (Bures, 1998; DesJarlais et al., 1990; Schafer et al., 1993; Rutenber et al., 1993; Gussio et al., 1996; Smith et al., 1995; Wang et al., 1996; Kireev et al., 1997; Huang et al., 1999). The discovery of a novel, non-peptide, HIV-1 protease inhibitor has been reported (Wang et al., 1996). Fifteen novel, non-peptide HIV-1 protease inhibitors were identified by flexible 3D database pharmacophore searching of the National Cancer Institute Drug Information System (DIS) database. The pharmacophore query used in the search was derived directly from the X-ray-determined structures of protease/inhibitor complexes. These 15 inhibitors, belonging to nine different chemical classes, are promising leads for further development. The two best inhibitors found, NSC 32180 and NSC 117027, had [IC.sub.50] values of 0.32 and 0.75 [micro]M, respectively, for HIV-1 protease inhibition (Wang et al., 1996).

Baicalin and Baicalein Anti-HIV Studies Using Molecular Modeling Methods

In order to develop new structures based on baicalin and baicalein lead compounds, our group has conducted several preliminary studies using molecular modeling methods. The Insight II program (version 98, Molecular Simulation Inc., CA) was used at SGI (Silicon Graphics Inc.) superworkstation (model 02) under the UNIX system. We carried out a search for the possible active site of the receptor (reverse transcriptase), and for the binding site of the inhibitor (baicalin and baicalein). The modeling calculations were based on the X-ray structure coordinates of HIV-1 reverse transcriptase (HIV-1 with nevirapine) and obtained from the Protein Data Bank. A modified site surrounding the inhibitor binding pocket was constructed for the complex from the respective X-ray structure coordinates of the enzyme (Briem and Kuntz, 1996).

DOCK calculation results show a possible active fit site of baicalin and baicalein for binding to HIV-1 reverse transcriptase. The graphics indicate that Insight II is a very useful tool for molecular modeling of natural products and the fit site is one orientation for binding between baicalin/baicalein and HIV-1 reverse transcriptase. An X-ray crystallography study will be performed on the HIV-1 reverse transcriptase-baicalein complex. Based on our data we will conduct further molecular modeling studies on a series of flavone compounds using Insight II software and DOCK calculations to understand why anti-HIV activity differs between baicalein and baicalin or other flavonoid compounds. We will also modify the structure of baicalein, which is a lead compound for anti-HIV agents in herbal medicines, in order to design new anti-AIDS drugs.

Summary

Medicinal herbs may have practical value as an alternative medical therapy in the inhibition of HIV activity. There is considerable evidence that sukudorfin, baicalin and baicalein are important lead compounds for the development of antiviral and/or virucidal drugs against HIV. Presently, baicalin and baicalein might be useful as topical agents to deactivate a newly formed virus, or act as an adjuvant with other antiviral drugs. However, it is essential that the herbal medicine kingdom, as a source of new anti-HIV leads, should be explored further and that these investigations should be encouraged and continued. The molecular modeling method may be a potential tool in the development of new anti-HIV agents from medicinal herbs.

Acknowledgments

This work is supported in part by Enwei Institute of Traditional Chinese Medicine, Chengdu, China. The authors wish to thank Tasha K. Lowell for her technical assistance.

References

[1.] Baylor, N.W., T. Fu, Y.D. Yan and F.W. Ruscetti. Inhibition of human T cell leukemia virus by the plant flavonoid baicalin (7-Glucuronic acid, 5,6-dihydroxyflavone). J. Infect. Dis. 165: 433-437, 1992.

[2.] Briem, H. and I.D. Kuntz. Molecular similarity based on DOCK-generated fingerprints. J. Med Chem. 39: 3401-3408, 1996.

[3.] Brinkworth, R.I., M.J. Stoermer and D.P. Fairlie. Flavones are inhibitors of HIV-1 proteinase. Biochem. Biophys. Res. Commun. 188: 631-637, 1992.

[4.] Buimovici-Klein, E., V. Mohan, M. Lange, E. Fenamore, Y. Inada and L.Z. Copper. Inhibition of HIV replication in lymphocyte cultures of virus-positive subjects in the presence of Sho-saiko-to, an oriental plant extract. Antivir. Res. 14: 279-286, 1990.

[5.] Bures, G.M. Integration of molecular modeling and database searching. In: Designing Bioactive Molecules: Three-Dimensional Techniques and Applications. Martin, Y.C. and P. Willett. (Ed) American Chemical Society. 1998, pp. 97-117.

[6.] Chu, C.K. and H.G. Cutler. Natural products as antiviral agents. Plenum Press. 1992.

[7.] Cohen, N.C., J.M, Blaney, C. Humblet, P. Gund and D.C. Barry. Molecular modeling software and methods for medicinal chemistry. J. Med. Chem.. 33: 883-894, 1990.

[8.] Collins, R.A., T.B. Ng, W.P. Fong, C.C. Wan and H.W. Yeung. A comparison of human immunodeficiency virus type 1 inhibition by partially purified aqueous extracts of Chinese medicinal herbs. Life Sci. 60: 345-351, 1997.

[9.] Cragg, G.M., M.R. Boyd, M.A. Christini, R. Kneller, T.D. Mays, K.D. Mazza, D.J. Newman and E.A. Sausville. Screening of natural products of plant, microbial and marine origin: the NCI experience. Spec. Publ. R. Soc. Chem. 200: 1-29, 1997.

[10.] De Clercq, E. Antiviral therapy for human immunodeficiency virus infections. Clin. Microbiol. Rev. 8: 200-239, 1995a.

[11.] De Clercq, E. Toward improved anti-HIV chemotherapy: Therapeutic strategies for intervention with HIV infections. J. Med. Chem. 38: 2491-2517, 1995b.

[12.] De Clercq, E. Perspectives of non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the therapy of HIV-1 infection, II Farmaco. 54: 26-45, 1999.

[13.] DesJarlais, R.L., G.L. Seibel, I.D. Kuntz, P.S. Furth, J.C. Alvarez, P.R. Montellan, D.L. DeCamp, L.M. Babe and C.S. Craik. Structure-based design of nonpeptide inhibitors specific for the human immunodeficiency virus 1 protease. Proc. Natl. Acad Sci. USA. 87: 6644-6648, 1990.

[14.] Fauci, A.S. and J.G. Artlett. Guideline for the use of antiretroviral agents in HIV-infected adults and adolescents. Annal. Internal. Med. 128:1079-1100, 1998.

[15.] Fauci, A.S., H. Masur, E.P. Gelmann, P.D. Markham, B.H. Hahn and H.C. Lane. NIH Conference. The acquired immunodeficiency syndrome: an update. Annal. Internal. Med. 102: 800-813, 1985.

[16.] Fugh-Berman, A. Herb-drug interactions. Lancet. 355: 134-138, 2000.

[17.] Gao, Z., K. Huang, X. Yang and H. Xu. Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi. BBA-Biomembranes. 1472: 643-650, 1999.

[18.] Garg, R., S.P. Gupta, H. Gao, M.S. Babu, A.K. Debnath and C. Hansch. Comparative quantitative structure-activity relationship studies on anti-HIV drugs. Chem. Rev. 99: 3525-3601, 1999.

[19.] Geerts, A. and V. Rogiers. Sho-saiko-to: The right blend of traditional oriental medicine and liver cell biology. Hepatology (Philadelphia). 29: 282-284,1999.

[20.] Gussio, R., N. Pattabiraman, D.W. Zaharevitz, G.E. Kellogg, I.A. Topol, W.G. Rice, C.A. Schaeffer, J.W. Erickson and S.K. Burt. All-atom models for non-nucleoside binding site of HIV-1 reverse transcriptase complexed with inhibitors: a 3D QSAR approach. J. Med. Chem. 39: 1645-1650, 1996.

[21.] Hu, C.Q., K. Chen, Q. Shi, R.E. Kilkuskie, Y.C. Cheng and K.H. Lee. Anti-AIDS agents 10. Acacetin-7-O-D-galactopyranoside, an anti-HIV principle from chrysanthemum morifolium and a structure-activity correlation with some related flavonoids, J. Natur. Prod. 57: 42-51, 1994.

[22.] Huang, K.C. The pharmacology of Chinese herbs. CRC Press Boca Raton, 1999.

[23.] Huang, L., G.X. Tao, L.Y. Li, and H.L. Chi. Computer-aided molecular design of HIV-1 protease dissociative inhibitors. Acta Pharmaceut. Sin. 34: 353-357, 1999.

[24.] Inada, Y., K. Watanabe, K. Miyamoto, U. Maitra, E.B. Klein and M. Lange. Regulatory activities of Sho-saiko-to in immune responses, eicosanoid pathway and HIV production. In: Proceedings of the Tenth International Conference on AIDS Satellite Symposium. Yokohama Japan. 1994.

[25.] Inoue, T. and E.K. Jackson. Strong antiproliferative effects of baicalein in cultured rat hepatic stellate cells. Eur. J. Pharm. 378: 129-135, 1999.

[26.] Kashiwada, Y. Studies on bioactive natural products: plant-derived natural products and analogues as anti-HIV agents. Natur. Med. 53:153-158, 1999.

[27.] Kassler, W.J., P. Blanc and R. Greenlatt. The use of medicinal herbs by human immunodeficiency virus-infected patients. Arch. Intern. Med. 151: 2281-2288, 1991.

[28.] Kato, M., W. Liu, H. Yi, N. Asai, A. Hayakawa, K.-I. Kozaki, M. Takahashi and I. Nakashima. The herbal medicine Sho-saiko-to inhibits growth and metastasis of malignant melanoma primarily developed in ret-transgenic mice. J. Invest. Deramatol. 111: 640-644, 1998.

[29.] Kireev D.B., J.R. Chretien, D.S. Grieson and C. Monneret. A 3D QSAR study of a series of HEPT analogues: the influence of conformational mobility on HIV-1 reverse transcriptase inhibition. J. Med. Chem. 40: 4257-4264, 1997.

[30.] Kitamura, K., M. Honda, H. Yoshizaki, S. Yamamoto, H. Nakane, M. Fukushima, K. Ono and T. Tokunaga. Baicalin, an inhibitor of HIV-1 production in vitro. Antivir. Res. 37:131-140, 1998.

[31.] Klopman, G. and M. Tu. Diversity analysis of 14,156 molecules tested by the National Cancer Institute for anti-HIV activity using the quantitative structure-activity relational expert system MCASE. J. Med. Chem. 42: 992-998, 1999.

[32.] Kuntz, I.D. Structure-based strategies for drug design and discovery. Sciences 257: 1078-1082, 1992.

[33.] Lee, K.H. Antitumor agents. 188. Highlights of research on plant-derived natural products and their analogs with antitumor, anti-HIV, and antifungal activity. In: Biol. Act. Nat. Prod. Cutler, S. and H.G. Cutler (Ed), 2000, pp. 73-94.

[34.] Lee, K.H. and S.L. Morris-Natschke. Recent advances in the discovery and development of plant-derived natural products and their analogs as anti-HIV agents. Pure Appl. Chem. 71: 1045-1051, 1999.

[35.] Li, B.Q., T. Fu, Y.D. Yan, N.W. Baylor, F.W. Ruscetti and H.F. Kung. Inhibition of HIV infection by baicalin-a flavonoid compound purified from Chinese herbal medicine. Cell. Mol. Biol. Res. 39:119-124, 1993.

[36.] Liu, W., M. Kato, A.A. Akhand, A. Hayakawa, M. Takemura, S. Yoshida, H. Suzuki and I. Nakashima. The herbal medicine Sho-saiko-to inhibits the growth of maligment melanoma cells by upregulating Fas-mediated apoptosis and arresting cell cycle through downregulation of cyclein-depedent kinases. Int. J. Oncol. 12: 1321-1326, 1998.

[37.] Luo, S.D., J.J. Chen and H.Y. Wang. Natural compounds with anti-HIV activity. Chin. Tradit. Herb. Drug. (Supplement) 30: 40-43, 1999.

[38.] Mahmood, N., C. Pizza, R. Aquino, N.D. Tommasi, S. Piacente, S. Colman, A. Burke and A.J. Hay. Inhibition of HIV by flavonoids. Antivir. Res. 22: 189-199, 1993.

[39.] Maikeru, R., B.K. Erena, M. Utopare, Maitora and R. Inada. Anti-AIDS virus effect-enhancing agents containing Shosaikoto. Jpn. Kokai Tokkyo Koho pp 5, 1996.

[40.] Martin, Y.C. 3D database searching in drug design. J. Med. Chem. 35: 2145-2154, 1992.

[41.] Minematsu, S., H. Takei, K. Sudo, K. Honda, Y. Fujii and T. Oyama. A single oral dose toxicity study of TSUMURA Sho-saiko-to (TJ-9) in rats. Jpn. Pharmacol. Ther. 23: 29-32, 1995.

[42.] Mizushima, Y., T. Kashii, Y. Tokimitsu and M. Kobayashi. Cytotoxic effect of herbal medicine Sho-saiko-to on human lung cancer cell lines in vitro. Oncol. Rep. 2:91-94, 1995.

[43.] Motoo, Y. and N. Sawabu. Antitumor effects of saikosaponins, baicalin and baicalein on human hepatoma cell lines. Cancer Letters. 86: 91-95, 1994.

[44.] Muto, R., T. Motozuka, M. Nakano, Y. Tatsumi, F. Sakamoto and N. Kosaka. The chemical structure of new substance as the metabolite of baicalin and time profiles for the plasma concentration after oral administration of Sho-saiko-to in human. Yakugaku Zasshi. 118: 79-87. 1998.

[45.] Ng, T.B., B. Huang, W.P. Fong and H.W. Yeung. Anti-human immunodeficiency virus (anti-HIV) natural products with special emphasis on HIV reverse transcriptase inhibitors. Life Sci. 61: 933-949, 1997.

[46.] Ono, M., M. Miyamura, S. Kyotani, T. Saibara, S. Ohnishi and Y. Nishioka. Effects of Sho-saiko-to extract on liver fibrosis in relation to the changes in hydroxyproline and retinoid levels of the liver in rats. J. Pharm. Pharmacol. 51: 1079-1084, 1999.

[47.] Oho, K., H. Nakane, M. Fukushima, J.C. Chermann and F. Baffe-Sinoussi. Differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular DNA and RNA polymerases. Eur. J. Biochem. 190: 469-476, 1990.

[48.] Ono, K. and H. Nakane. Mechanisms of inhibition of various cellular DNA and RNA polymerases by several flavonoids. J. Biochem. 108:609-613, 1990.

[49.] Ono, K., H. Nakane, M. Fukushima, J.C. Chermann and F. Baffe-Sinoussi. Inhibition of reverse transcriptase activity by a flavonoid compound, 5, 6, 7, Trihydroxyflavone. Biochem. Biophy. Res. Commu. 160: 982-987, 1989.

[50.] Phillips, L.G., M.H. Nichols and W.D. King. Herbs and HIV: the health food industry’s answer. South. Med. J. 88:911-913, 1995.

[51.] Piras, G., M. Makino and M. Baba. Sho-saiko-to, a traditional kampo medicine, enhances the anti-HIV-1 activity of Lamivudine (3TC) in vitro. Microbiol. Immunol. 41: 835-839, 1997.

[52.] Raghavan, K., J.K. Buolamwini, M.R. Fesen, Y. Pommier, K.W. Kohn and J.N. Weinstein. Three-dimensional quantitative structure-activity relationship (QSAR) of HIV integrase inhibitors: A comparative molecular field analysis (CoMFA). J. Med. Chem. 38: 890-897, 1995.

[53.] Rutenber, E., E.B. Faumant, R.J. Keenan, S. Fong, P.S. Furth, P.R. Ortiz de Montellano, E. Meng, I.D. Kuntz, D.L. DeCamp, R. Salto, J.R. Rose, C.S. Craik and R.M. Stroud. Structure of a non-peptide inhibitor complexed with HIV-1 protease. J. Biol. Chem. 268:15343-15346, 1993.

[54.] Schafer, W., W.G. Friebe, H. Leinert, A. Mertens, T. Poll, W. van der Saal, H. Zilch, B. Nuber and M.L. Ziegker. Non-nucleoside inhibitors of HIV-1 reverse transcriptase: molecular modeling and x-ray structure investigations. J. Med. Chem. 36: 726-732, 1993.

[55.] Shao, Z.H., C.Q. Li, T.L. Vanden Hock, B. Becker, P.T. Schumacker, J.A. Wu, A.S. Attele and C.S. Yuan. Extract from Scutellaria baicalensis Georgi attenuates oxidant stress in cadiomyocytes. J. Mol. Cadiol. 31: 1885-1895, 1999.

[56.] Shimizu, I., Y.R. Ma, Y. Mizobuchi, F. Liu, T. Miura, Y. Nakai, M. Yasuda, M. Shiba, T. Horie, S. Amagaya, N. Kawada, H. Hori and S. Ito. Effects of Sho-saiko-to, a Japanese herbal medicine, on hepatic fibrosis in rats. Hepatology (Philadelphia) 29:149-160, 1999.

[57.] Shoichet, B.K. and I.D. Kuntz. Predicting the structure of protein complexes: a step in the right direction. Chem. Biol. 3: 151-156, 1996.

[58.] Smith, M.K., C.A. Rouzer, L.A. Taneyhill, N.A. Smith, S.H. Hughes, P.L. Boyer, P.A.J. Janssen, H. Moereels, L. Koymans, E. Arnold, J. Ding, K. Das, W. Zhang, C.J. Michejda and R.H. Smith Jr. Molecular modeling studies of HIV-1 reverse transcriptase nonnucleoside inhibitors: total energy of complexation as a predictor of drug placement and activity. Protein Sci. 4: 2203-2222, 1995.

[59.] Tan, G.T., J.M. Pezzuto and A.D. Kinghorn. Evaluation of natural products as inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. J. Natur. Prod. 54: 143-154, 1991.

[60.] Tan, G.T., J.F. Miller, A.D. Kinghom, S.H. Hughes and J.M. Pezzuto. HIV-1 and HIV-2 reverse transcriptases: a comparative study of sensitivity to inhibition by selected natural products. Biochem. Biophy. Res. Commu. 185: 370-378, 1992.

[61.] Tang, X.S., H.S. Chen and X.Q. Zhang. Inhibition of human immunodeficiency virus reverse transcriptase by Chinese medicines in vitro. Proc. CAMS PUMC 5:140-144, 1990.

[62.] Tsutsumi, M., H. Kitada, K. Shiraiwa, M. Takahama, T. Tsujiuchi, H. Sakitani, Y. Sasaki, K. Murakawa, M. Yoshimoto and Y. Konishi. Inhibitory effects of combined administration of antibiotics and anti-inflammatory drugs on lung tumor development initiated by N-nitrosobis(2-hydroxypropyl)amine in rats. Carcinogenesis. 21:251-256, 2000.

[63.] Vandamme, A.M., K. Van Vaerenbergh and E. D’ Clercq. Anti-human immunodeficiency virus drug combination strategies. Antivir. Chem. Chemother. 9:187-203, 1998.

[64.] Vlietinck, A.J., T. De Bruyne and D.A. Vanden Berghe. Plant substances as antiviral agents. Curr. Org. Chem. 1: 307-344, 1997.

[65.] Vlietinck, A.J., T.D. Bruyne, S. Apers and L.A. Pieters. Plant-derived leading compounds for chemotherapy of human immunodeficiency virus (HIV) infection. Planta Med. 64: 97-109, 1998.

[66.] Wang, H.K., Y. Xie, Z.Y. Yang, S.L. Morris-Natschke and K.H. Lee. Recent advances in the development of flavonoids and their analogues as antitomor and anti-HIV agents. In: Flavonoids in the living system Manthey, J.A. and B.S. Buslig (Ed). Plenum Press 1998, pp. 191-225.

[67.] Wang, J., P.A. Kollman and I.D. Kuntz. Flexible ligand docking: a multistep strategy approach. Protein-Struct. Funct. Genet. 36: 1-19, 1999.

[68.] Wang S., G.W.A. Milne, X. Yan, I.J. Posey, M.C. Nicklaus, L. Graham and W.G. Rice. Discovery of novel, non-peptide HIV-1 protease inhibitors by pharmacophore searching. J. Med. Chem. 39: 2047-2054, 1996.

[69.] Wu, X.S., H. Akatsu and H. Okada. Apoptosis of HIV-infected cells following treatment with Sho-saiko-to and its components. Jpn. J. Med. Sci. Biol., 48: 79-87, 1995.

[70.] Xie, L., Y. Takeuchi, L.M. Cosentino and K.-H. Lee. Anti-AIDS agents. 37. Synthesis and structure-activity relationships of (3’R,4’R)-(+)-cis-Khellactone derivatives as novel potent anti-HIV agents. J. Med. Chem. 42:2662-2672,1999.

[71.] Xue, Y.X., C.H. Liu, L. Zhang and C.S. Yuan. Traditional Chinese medicine and AIDS. Am. J. Compreh. Med. 1: 542-544, 1999.

[72.] Yagura, M., S. Murai, H. Kojima, H. Tokita, H. Kamitsukasa and H. Harada. Changes of liver fibrosis in chronic hepatitis C patients with no response to interferon-[Alpha] therapy: including quantitative assessment by a morphometric method. J. Gastroenterol. 35:105-111, 2000.

[73.] Yamamoto, T., H. Takahashi, K. Sakai, T. Kowithayakorn and T. Koyano. Screening of Thai plants for anti-HIV-1 activity. Nat. Med. 51: 541-546, 1997.

[74.] Yoshino, M., M. Ito, H. Okajima, M. Haneda and K. Murakam. Role of baicalein compounds as antioxidant in the traditional herbal medicine. Biomed. Res. 18: 349-352, 1997.

[75.] Zhang, X.Q., X.S. Tang and H.S. Chen. Inhibition of HIV replication by baicalin and S. Baicalensis extract in H9 cell culture. Chin. Med. Sci. J. 6: 230-232, 1991.

[76.] Zhao, J., L.H. Ben, Y.L. Wu, W. Hu, K. Ling, S.M. Xin, H.L. Nie, L. Ma and G. Pei. Anti-HIV agent trichosanthin enhances the capabilities of chemokines to stimulate chemotaxis and G protein activation, and this is mediated through interaction of trichosanthin and chemokine receptors. J. Exp. Med. 190:101-111, 1999.

[77.] Zhao, J., Z.P. Zhang, H.S. Chen, X.H. Chen and X.Q. Zhang. Preparation and anti-HIV activity study of baicalein and its benzylated derivates. Acta Pharmaceu. Sin. 32:140-143, 1997.

[78.] Zhao, J., Z.P. Zhang, H.S. Chen, X.Q. Zhang and X.H. Chen. Synthesis ofbaicalin derivatives and evaluation of their anti-human immunodeficiency virus (HIV-1) activity. Acta Pharmaceu. Sin. 33: 22-27, 1998.

Ji An Wu, Anoja S. Attele, Liu Zhang and Chun-Su Yuan(*) Tang Center for Herbal Medicine Research, Committee on Clinical Pharmacology, and Department of Anesthesia & Critical Care, The Pritzker School of Medicine, The University of Chicago, Chicago, IL 60637 (*) Corresponding author

COPYRIGHT 2001 Institute for Advanced Research in Asian Science and Medicine

COPYRIGHT 2001 Gale Group