Identification of inhibitors of the HIV-1 gp41 six-helix bundle

formation from extracts of Chinese medicinal herbs

Prunella vulgaris and Rhizoma cibotte

Shuwen Liu a,b, Shibo Jiang b,*, Zhihua Wu a, Lin Lv a, Jiajie Zhang a,Zhengguang Zhu a, Shuguang Wu a,*

aInstitute of Pharmaceutical Sciences of the First Medical University of PLA, PLA Key Lab for Drug Screening,

Guangzhou, Guangdong 510515, ChinabLindsley F. Kimball Research Institute, The New York Blood Center, 310 E 67th Street, New York, NY 10021, USA

Received 14 March 2002; accepted 29 April 2002

Abstract

An increasing portion of patients with HIV infection and/or AIDS cannot use currently FDA-approved anti-HIV

drugs, including the reverse transcriptase and protease inhibitors, due to the adverse effects and the emergence of

drug resistance. Thus, it is essential to develop new anti-HIV agents with a target different from the HIV reverse

transcriptase and protease. Using a conformation-specific monoclonal antibody NC-1, we previously established a

high throughput screening assay for identification of small molecular organic compounds that disrupt the HIV-1

gp41 six-helix bundle formation, a critical step of membrane fusion between the HIV and the target cell. In the

present study, we used this assay to screen for inhibitors of the gp41 six-helix bundle formation from aqueous

extracts of nine Chinese medicinal herbs with antiviral activity. We found that the extracts of two herbs, Prunella

vulgaris and Rhizoma cibotte, showed potent inhibitory activity. The inhibitory activity of these two herb extracts

significantly decreased after they were passed through polyamide resin mini-columns, which are able to bind

polyphenols including tannin, an HIV-1 inhibitor with multiple mechanisms of action. The bound polyphenols

were eluted from the polyamide columns and also showed potent inhibitory activity on the gp41 six-helix bundle

formation. Tannin purchased from different commercial sources inhibited the gp41 six-helix bundle formation in a

manner similar to the polyphenols isolated from the herb extracts. These results suggest that tannin may be one of

0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.

PII: S0024 -3205 (02 )01939 -2

* Corresponding authors. S. Jiang is to be contacted at Tel.: +1-212-570-3058; fax: +1-212-570-3099. S. Wu, Institute of

Pharmaceutical Sciences of the First Medical University of PLA, PLA Key Lab for Drug Screening,Guangzhou, Guangdong

510515, China. Fax: +86-20-87644781.

E-mail addresses: shibo_jiang@nybc.org (S. Jiang), gzwsgxw@public.guangzhou.gd.cn (S. Wu).

www.elsevier.com/locate/lifescie

Life Sciences 71 (2002) 17791791

major inhibitors of the HIV-1 gp41 six-helix bundle formation in the herb extracts and that tannin may inhibit HIV-

1 entry by disrupting the gp41 six-helix bundle formation.

D 2002 Elsevier Science Inc. All rights reserved.

Keywords: HIV-1 gp41; Anti-HIV-1 agents; Medicinal herbs; Tannin; Polyphenols; High throughput screening

Introduction

The current anti-human immunodeficiency virus (HIV) drugs approved by the Food and Drug

Administration (FDA) of the United States are targeted to the HIV reverse transcriptase and protease [1].

More and more patients with HIV infection and/or AIDS are unable to use these drugs because of the

serious adverse effect and emergence of HIV mutants having single or multiple resistance to the drugs

used [26]. Therefore, it is essential to develop more effective and less toxic anti-HIV drugs targeting

the HIV entry steps.

HIVentry into a target cell is initiated by binding of the envelope glycoprotein (Env) subunit gp120 to

the primary receptor, CD4 [7], and the coreceptor, CXCR4 or CCR5 [8], resulting the conformation

change of the Env transmemberane subunit gp41 from a native state to an intermediate state, and then to

a fusion-active state [9]. In the intermediate state, the N- and C-terminal heptad repeat (designated NHR

and CHR, respectively) regions of gp41 extracellular domain (ectodomain) become exposed and

accessible [8,1013]. In the fusion-active state, three NHR regions of gp41 associate to form the

central trimeric coiled coil and three CHR regions pack obliquely in an antiparallel manner into the

highly conserved hydrophobic grooves on the surface of the coiled coil, resulting in the formation of a

six-helix bundle, which brings both the viral and target cell membranes into proximity for fusion [14,15].

Previous studies demonstrated that peptides derived from the CHR region of gp41 (designated C-

peptides), such as SJ-2176 and DP-178 (also named T-20), inhibit HIV type 1 (HIV-1) mediated

membrane fusion by interacting with the gp41 NHR region at the intermediate state, thus blocking the

six-helix bundle formation [9,12,13,1618]. Clinical studies demonstrated that T-20 could reduce the

viral load at a rate comparable to that resulting from current 3 or 4 drug combination (cocktail) regiment

of reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs) [19], suggesting that T-20 can be

used as a new class of anti-HIV drugs for treatment of patients infected by HIV-1 that are resistant to

RTIs and PIs [20]. However, the future application of T-20 may also be limited because of its lack of oral

availability and high cost of production. Therefore, it is important to develop inexpensive small

molecular HIV entry inhibitors with oral availability as a new class of anti-HIV drugs [21].

We previously developed a monoclonal antibody (mAb), NC-1, which specifically recognizes the

conformational epitopes on the HIV-1 gp41 six-helix bundles [22]. Using this antibody, we established a

sandwich enzyme-linked immunosorbent assay (ELISA) for screening of antiviral compounds that block

the interaction between the gp41 N- and C-peptides to form the six-helix bundle corresponding to the

fusion-active gp41 core, thus inhibiting gp41-mediated fusion between the HIV and target cell

membranes [23]. Using this screening assay in combination with the computer-aided molecular docking

techniques, we have identified one small molecular weight HIV-1 fusion inhibitor, ADS-J1 from a

chemical library [24,25].

S. Liu et al. / Life Sciences 71 (2002) 177917911780

In the present study, we used the sandwich ELISA to screen aqueous extracts of nine Chinese herbs

which have been reported to have anti-HIV-1 activity [26,27] in order to identify the active components

that may disrupt the HIV-1 gp41 six-helix bundle formation. We found that the aqueous extracts of two

herbs, Prunella vulgaris and Rhizoma cibotte, had potent inhibitory activity on the six-helix bundle

formation and their inhibitory activity significantly decreased after the herb extracts were passed through

a polyamide resin mini-column, which is able to absorb polyphenols, such as tannin [28], suggesting that

the polyphenols may be the active components in the herb extracts. Since the tannins obtained

commercially inhibited the gp41 six-helix bundle formation in a manner similar to that of active

components isolated from the herb extracts, tannin may be one of the major inhibitors of the HIV-1 gp41

six-helix bundle formation in the extracts of Chinese medicinal herbs.

Methods

Reagents

The rabbit polyclonal antibody and mouse monoclonal antibody specific for the gp41 six-helix bundle

were prepared and characterized as previously described [22]. Peptides N36, C34 and T22 were

synthesized by a standard solid-phase FMOC method at the MicroChemistry Laboratory, the New York

Blood Center. The N-termini of the peptides were acetylated and their C-termini were amidated. 3-

hydroxyphthalic anhydride modified h-lactoglobulin (3HP-h-LG) was prepared as previously described[29]. ADS-J1 [24] was obtained from ComGenex, Inc. (Budapest, Hungary). Tannin was obtained from

Ouhai Chemical Inc., Wenzhou, China. Tannin purchased from Sigma Chemical Co., St. Louis, MO

(designated Tannin-S) was used as a control in Fig. 7.

Preparation of aqueous extracts of Chinese medicinal herbs

The following Chinese herbs, Trichosanthes kirilowii (TK), Glycyrrhiza uralensis (GU), Viola

yedoensis (VY), Prunella vulgaris (PV), Andrographis paniculata (AP), Morus alba (MA), Lonicera

japonica (LJ), Bupleurum Chinese (BC), and Rhizoma cibotte (RC), were obtained from the Chinese

Medicinal Herb Inventory of Nangfang Hospital, Guangzhou, China. The aqueous extracts of these

herbs were prepared by boiling the herbs in water, in a way similar to that the pharmaceutists in China

have used to prepare the traditional Chinese herb medicines for thousands years, in order to extract

water-soluble heat-resistant compounds from the herbs. Briefly, each herb (20 g) was cut into fine pieces,

soaked in 200 ml distilled water, and boiled in a flask which was attached to a reflux apparatus to cool

the vapor and return it back to the flask as a liquid. The mixture was then centrifugated at 2000 rpm for

20 minutes. The supernatants were collected and vacuum dried at room temperature. The dried powder

was resuspended in distilled water to 1 mg/ml before use.

Measurement of polyphenol concentrations

The concentrations of polyphenols in herb extracts were measured as previously described [30]. In

brief, 0.5 ml aqueous herb extract was mixed with 1 ml FolinCiocalteus phenol reagent (Sigma, St.

Louis, MO), followed by addition of 3.5 ml 20% NaCO3 and 5 ml distilled water. After incubation at

S. Liu et al. / Life Sciences 71 (2002) 17791791 1781

room temperature for 10 min, the absorbance at 700 nm was recorded using a spectrophotometer

(Model: DU 530, Beckman Inc., Fullerton, CA). The concentrations of polyphenols in the herb extracts

were estimated using tannin as a standard.

Removal of polyphenols from aqueous extracts of Chinese medicinal herbs and recovery of polyphenols

from the polyamide columns

Polyphenols were removed from aqueous extracts of Chinese medicinal herbs as previously described

[28,30]. Briefly, 20 ml of an aqueous extract was loaded onto a mini-column (12 mm diameter and 15 cm

length) packed with polyamide resin (Shanghai Chemical Reagent Inc., Shanghai, China). The flow-out

fractions were collected and loaded again. After three cycles, the final flow-out fractions were collected

(about 20ml) for testing. The columnswerewashedwith 20ml of distill water and then eluted by adding 40

Fig. 1. The specificity of the mAb NC-1. A) As measured by the sandwich ELISA, NC-1 specifically bound to the gp41 six-

helix bundle formed by mixing of N36 and C34, but did not bind to the isolated N36 or C34. B) ADS-J1 specifically inhibited

the six-helix bundle formation, but did not block the binding of NC-1 to the pre-formed gp41 core formed by N36 and C34.

Other HIV-1 entry inhibitors had no effect on the six-helix bundle formation.

S. Liu et al. / Life Sciences 71 (2002) 177917911782

ml 5% ammonia in water (vol/vol) to recover polyphenols as previously described [31]. The eluates

containing polyphenols were adjusted to pH 7.4 before testing.

ELISA

A sandwich ELISA described previously [23] was modified for detecting the inhibitory activity of the

herb extracts on the gp41 six-helix bundle formation. Briefly, peptide N36 (2 AM) was pre-incubatedwith compounds at graded concentrations at 37 jC for 30 min, followed by addition of C34 (2 AM).After incubation at 37 jC for 30 min, the mixture was added to wells of 96-well polystyrene plates(CorningCostar, Acton, MA) which were precoated with mAb NC-1 IgG (0.2 Ag/well). Then, rabbitIgG purified from antisera directed against the gp41 six-helix bundle, horseradish peroxidase (HRP)-

labeled goat-anti-rabbit IgG (Boster Biotechnology, Inc., Wuhan, China), and the substrate, 3,3V,5,5V-tetramethylbenzidine (TMB) (Sigma) were added sequentially. Absorbance at 450 nm was read using an

ELISA reader (BioRad, Hercules, CA). The percentage of inhibition of six-helix bundle formation by

the compounds was calculated as previously described [24] and the concentration for 50% inhibition

(IC50) was calculated [32] using a computer program, designated Calcusyn, kindly provided by Dr. T.

Chou at SloanKettering Cancer Center, New York.

Results

Specificity of the screening assay

The specificity of the sandwich ELISA for detecting the gp41 six-helix bundle formation between the

N-peptide N36 and the C-peptide C34 using mAb NC-1 was confirmed by showing that: 1) NC-1 only

bound to the complex N36/C34, but did not react with the individual peptides N36 and C34 (Fig. 1A),

and 2) ADS-J1 effectively inhibited the gp41 six-helix bundle formation, but did not block the mAb NC-

1 binding to the pre-formed six-helix bundles. Other HIV-1 entry inhibitors with different targets, such as

3HP-h-LG which inhibits binding of HIV-1 to CD4 [29] and T-22, a coreceptor CXCR4 antagonism[33], did not inhibit the formation of the gp41 six-helix bundles (Fig. 1B).

Table 1

Inhibition of the HIV-1 gp41 six-helix bundle formation by herb extracts

Chinese medicinal herbs (abbreviation) % Inhibition of the gp41 six-helix bundle formation

Trichosanthes kirilowii (TK) 4.6

Glycyrrhiza uralensis (GU) 28.7

Viola yedoensis (VY) 14.7

Prunella vulgaris (PV) 86.2

Andrographis paniculata (AP) 21.0

Morus alba (MA) 4.5

Lonicera japonica (LJ) 6.8

Bupleurum Chinese (BC) 7.9

Rhizoma cibotte (RC) 98.3

S. Liu et al. / Life Sciences 71 (2002) 17791791 1783

Inhibition of the HIV-1 gp41 six-helix bundle formation by aqueous extracts of Chinese medicinal herbs

with antiviral activity

Aqueous extracts of nine Chinese medicinal herbs were tested for inhibition of the gp41 six-helix

bundle formation by the sandwich ELISA using the mAb NC-1. All the extracts have certain inhibitory

activity against the gp41 six-helix bundle formation at the final concentration of 50 Ag/ml. But only twoof them obtained from Prunella vulgaris (PV) and Rhizoma cibotte (RC) had more than 50% inhibition

tested at this concentration (Table 1). Therefore, these two extracts were selected for further studies.

Fig. 2. The inhibitory activity of herb extracts on the gp41six-helix bundle formation is resistant to boiling treatment.

Fig. 3. The concentration of tannin, a polyphenol, is closely correlated with the absorbance at 700 nm.

S. Liu et al. / Life Sciences 71 (2002) 177917911784

The active components in the herb extracts are resistant to boiling treatment

To determine whether the inhibitors are organic compounds or proteins, the extracts of herbs PV and

RC were boiled for 15 min, cooled down to room temperature and then tested for their inhibitory activity

on the gp41 six-helix bundle formation. ADS-J1 was included as a control. As shown in Fig. 2, the

inhibitory activity of the active component in these extracts, like that of ADS-J1, was not suppressed

after the boiling treatment, suggesting that the inhibitors in these extracts may not be proteins or

peptides, but organic compounds that are resistant to boiling treatment.

The inhibitory activity of the herb extracts on the gp41 six-helix bundle formation is correlated with the

concentrations of polyphenols

It was reported that Chinese medicinal herbs contain different amount of polyphenols, such as

tannin [30]. To determine whether the active components in the above aqueous extracts are

Fig. 4. Correlation between the concentrations of polyphenols in herb extracts and their inhibitory activity. A) Measurement of

the concentrations of polyphenols in the aqueous extracts of nine Chinese medicinal herbs. B) The inhibitory activity of herb

extracts on six-helix bundle formation is correlated with the concentrations of polyphenols in these herb extracts.

S. Liu et al. / Life Sciences 71 (2002) 17791791 1785

polyphenols, we adapted a method from Au et al. [30] for measuring the concentrations of

polyphenols in the herb extracts. Using tannin as a standard, the concentrations of tannin is linearly

correlated with the absorbance at 700 nm (r = 0.989), confirming that this method is reliable for

measuring the concentrations of polyphenols including tannin (Fig. 3). Using this assay, we

determined the concentrations of polyphenols in the aqueous extracts of the above nine Chinese

medicinal herbs. Indeed, these herb extracts contained different amount of polyphenols. Two of the

herbs, PV and RC, contained much higher amount of polyphenols than others (Fig. 4A). Strikingly,

the concentrations of polyphenols in these herb extracts are closely correlated with their inhibitory

activity against the gp41 six-helix bundle formation (r = 0.902) (Fig. 4B), suggesting that the

inhibition is mediated by polyphenols in these herb extracts.

Fig. 5. Removal of polyphenols from herb extracts resulted in the decrease of their inhibitory activity. A) Comparison of

polyphenol concentrations before and after the herb extracts were passed through the plolyamide resin mini-columns. B)

Comparison of the inhibitory activity on the gp41 six-helix bundle formation before and after the herb extracts were passed

through the plolyamide resin mini-columns.

S. Liu et al. / Life Sciences 71 (2002) 177917911786

The active components, like tannin, could be removed from the herb extracts by passing the extracts

through polyamide resin mini-column

It was reported that polyphenols in the herb extracts could be removed by passing the samples through

a polyamide resin mini-column [28,30]. To determine whether the active components can be removed

from the herb extracts, we loaded the aqueous extracts of PVand RC (1 mg/ml) onto the polyamide resin

mini-columns. The flow-out fractions were collected and tested for the concentration of polyphenols and

for their inhibitory activity on the gp41 six-helix bundle formation. The tannin purchased from Ouhai

Chemical Inc., Wenzhou, China was included as a control. As shown in Fig. 5A, after passing through

the polyamide resin columns, 8098% of the polyphenols, as measured by absorbance at 700 nm, in the

herb extracts and the control tannin solution were removed. Accordingly, their inhibitory activity on the

gp41 six-helix bundle formation was also significantly decreased after passing them through the

polyamide resin mini-columns (Fig. 5B), suggesting that the inhibitors in the herb extracts, like tannin,

may bind to the polyamide resins.

Fig. 6. The active components in the herb extracts eluted from the polyamide resin mini-columns contained mainly polyphenols

(A) and showed inhibitory activity on the gp41 six-helix bundle formation (B).

S. Liu et al. / Life Sciences 71 (2002) 17791791 1787

The eluates from the polyamide resin mini-columns contain the inhibitors of gp41 six-helix bundle

formation

Polyphenols bound to the polyamide column could be eluted with ammonia [31]. After the PV and

RC extracts as well as the tannin solution were passed through the polyamide columns, the columns

were washed and treated with 5% ammonia. The eluates were collected and evaluated for the

concentrations of polyphenols and for the inhibitory activity on the gp41 six-helix bundle formation.

As shown in Fig. 6A, 5263% of the polyphenols was recovered from the polyamide resin mini-

columns and these recovered polyphenols, like tannin, had potent inhibitory activity on the gp41 six-

helix bundle formation (Fig. 6B). These results suggest that the inhibitors in the Chinese herb extracts

may be polyphenols.

Tannins purchased commercially inhibited the gp41 six-helix bundle formation in a dose-dependent

manner

Then, we tested whether tannin obtained from different commercial sources (Ouhai Chemical Inc.,

Wenzhou, China and Sigma Chemical Co., St. Louis, MO, USA) have inhibitory activity on the gp41 six-

helix bundle formation.As shown inFig. 7, both tannins inhibited the interactionbetweenN- andC-peptides

to form the gp41 core in a dose-dependent manner with IC50 values at 0.96 and 0.82 Ag/ml, respectively.

Discussion

Chinese medicinal herbs have been widely used in China for thousands years for treatment of human

diseases, including viral infection. Their pharmaceutical and toxic profiles have been scientifically and

empirically accumulated. Therefore, they can be used as important resource for screening of HIV-1 entry

Fig. 7. Tannin inhibited the HIV-1 gp41 six-helix bundle formation in dose-dependent manner. Tannin was purchased from

Ouhai Chemical Inc., Wenzhou, China. Another tannin (Tannin-S) was purchased from Sigma, St. Louis, MO, USA.

S. Liu et al. / Life Sciences 71 (2002) 177917911788

inhibitors. Extracts of several Chinese medicinal herbs have been shown to have anti-HIV-1 activity

[26,30,3436]. It is interesting to know whether the active components in these herb extracts are able

to disrupt the formation of the gp41 six-helix bundle, which represents the fusion-active gp41 core

structure.

The present studies indicate that the aqueous extracts of two Chinese herbs Prunella vulgaris and

Rhizoma cibotte contain inhibitors of the HIV-1 gp41 six-helix bundle formation. Prunella vulgaris has

been widely used in China for antiviral therapy and was demonstrated to contain active components

against the HIV-1 reverse transcriptase [26]. Prunellin, an anti-HIV compound, was isolated from

aqueous extracts of Prunella vulgaris [34]. Our studies demonstrated that polyphenols, mainly tannin,

presented in the extracts of Prunella vulgaris and Rhizoma cibotte may be the major inhibitors of the

gp41 six-helix bundle formation since: 1) their inhibitory activity is closely correlated with the

concentrations of the polyphenols; 2) removal of the polyphenols from the herb extracts by passing

through polyamide resin columns results in the decrease of the inhibitory activity; and 3) the eluates

from these polyamide resin columns which contain the polyphenols have similar inhibitory activity as

tannins purchased from different commercial sources.

It has been reported that tannin is a potent inhibitor of HIV-1 infection through multiple mechanisms

of action, such as inhibition of the activity of HIV-1 reverse transcriptase [36,37], protease [38], and

intergrase [30] as well as binding of gp120 to CD4 [26,36]. Here it is the first time to demonstrate that

tannin is also a potent inhibitor of the HIV-1 gp41 six-bundle formation, a critical step of HIV-1 fusion

with target cells. Therefore, inhibition of HIV-1 mediated membrane fusion may be another mechanism

of action of tannin for inhibition of HIV-1 infection.

Although tannin is a non-selective anti-HIV agent, it may be developed as a microbicide for

prevention of sexual transmission of HIV since it has following advantages: 1) its presence in a variety

of foods and beverages, such as vegetables [39], tea, beer and wine [40], etc. Thus, it should have no

toxic effect on human; 2) inexpensive and widely available sources; and 3) targeting both the early and

late stages of HIV-1 infection. Thus, it may inhibit HIV-1 entry and replication. Because tannin has

multiple targets in HIV-1, it may not be easy for HIV-1 become resistant to tannin.

In addition of the polyphenols, Chinese medicinal herbs may also contain other small molecular

inhibitors of the HIV-1 gp41 six-helix bundle formation. These inhibitors may be extracted from the

herbs using different solvents or techniques. Tannin and other polyphenols should be removed by

passing the herb extracts through polyamide resin columns before using the sandwich ELISA for

screening in order to identify the non-polyphenolic HIV-1 fusion inhibitors.

Acknowledgements

This work was supported by grants from 863 Project Foundation of China (2001AA214201),

Natural Science Foundation of China (301400220), Chinese Army Medical Science and Technology

Foundation (01Z051), and Guangdong Province Science and Technology Foundation to Shuguang Wu,

and US NIH grants (RO1 AI46221 and PO1 HD41761) to Shibo Jiang.

References

[1] Fauci AS. The AIDS epidemic-considerations for the 21st century. The New England Journal of Medicine 1999;341(14):

104650.

S. Liu et al. / Life Sciences 71 (2002) 17791791 1789

[2] Richman DD. Antiretroviral drug resistance: mechanisms, pathogenesis, clinical significance. Advances In Experimental

Medicine and Biology 1996;394:38395.

[3] Carpenter CC, Fischl MA, Hammer SM, Hirsch MS, Jacobsen DM, Katzenstein DA, Montaner JS, Richman DD, Saag

MS, Schooley RT, Thompson MA, Vella S, Yeni PG, Volberding PA. Antiretroviral therapy for HIV infection in 1998:

updated recommendations of the International AIDS Society-USA Panel. Journal of American Medical Association

1998;280(1):7886.

[4] Carr A, Samaras K, Thorisdottir A, Kaufmann GR, Chisholm DJ, Cooper DA. Diagnosis, prediction, and natural course of

HIV-1 protease-inhibitor-associated lipodystrophy, hyperlipidaemia, and diabetes mellitus: a cohort study. Lancet 1999;

353(9170):20939.

[5] Williamson K, Reboli AC, Manders SM. Protease inhibitor-induced lipodystrophy. Journal of the American Academy of

Dermatology 1999;40(4):6356.

[6] Blower SM, Aschenbach AN, Gershengorn HB, Kahn JO. Predicting the unpredictable: transmission of drug-resistant

HIV. Nature Medicine 2001;7(9):101620.

[7] Moore JP, Jameson BA, Weiss RA, Sattentau QJ. The HIV-cell fusion reaction. In: Bentz J, editor. Viral Fusion Mech-

anisms. Boca Raton: CRC Press; 1993. p. 23389.

[8] Berger EA. HIV entry and tropism: the chemokine receptor connection. AIDS 1997;11(suppl A):S3S16.

[9] Chan DC, Kim PS. HIV entry and its inhibition. Cell 1998;93:6814.

[10] Sattentau QJ, Moore JP. Conformational changes induced in the human immunodeficiency virus envelope glycoprotein by

soluble CD4 binding. Journal of Experimental Medicine 1991;174(2):40715.

[11] Wyatt R, Sodroski JG. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 1998;280(5371):

18848.

[12] Furuta R, Wild CT, Weng Y, Weiss CD. Capture of an early fusion-active comformation of HIV-1 gp41. Nature Structural

Biology 1998;5(4):2769.

[13] Jones P, Korte T, Blumenthal R. Conformational changes in cell surface HIV-1 envelope glycoproteins are triggered by

cooperation between cell surface CD4 and coreceptors. Journal of Biological Chemistry 1998;273(1):4049.

[14] Chan DC, Fass D, Berger JM, Kim PS. Core structure of gp41 from the HIVenvelope glycoprotein. Cell 1997;89:26373.

[15] Weissenhorn W, Dessen A, Harrison SC, Skehel JJ, Wiley DC. Atomic Structure of the Ectodomain from HIV-1 gp41.

Nature 1997;387(6631):4268.

[16] Jiang S, Lin K, Strick N, Neurath AR. HIV-1 inhibition by a peptide. Nature 1993;365(6442):113.

[17] Wild CT, Shugars DC, Greenwell TK, McDanal CB, Matthews TJ. Peptides corresponding to a predictive alpha-helical

domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proceeding of the National

Academy of Science of the United States of America 1994;91(21):97704.

[18] Lu M, Blacklow SC, Kim PS. A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nature Structural

Biology 1995;2(12):107582.

[19] Kilby JM, Hopkins S, Venetta TM, DiMassimo B, Cloud GA, Lee JY, Alldredge L, Hunter E, Lambert D, Bolognesi D,

Matthews T, Johnson MR, Nowak MA, Shaw GM, Saag MS. Potent suppression of HIV-1 replication in humans by T-20,

a peptide inhibitor of gp41-mediated virus entry. Nature Medicine 1998;4(11):13027.

[20] Dove A. New class of HIV drugs shows promise. Nature Medicine 2001;7(12):1265.

[21] Jiang S, Debnath AK. Development of HIV entry inhibitors taregeted to the coiled coil regions of gp41. Biochemical and

Biophysical Research Communications 2000;269(3):6416.

[22] Jiang S, Lin K, Lu M. A conformation-specific monoclonal antibody reacting with fusion-active gp41 from the HIV-1

envelope glycoprotein. Journal of Virology 1998;72(12):102137.

[23] Jiang S, Lin K, Zhang L, Debnath AK. A screening assay for antiviral compounds targeted to the HIV-1 gp41 core

structure using a conformation-specific monoclonal antibody. Journal of Virological Methods 1999;80(1):8596.

[24] Debnath AK, Radigan L, Jiang S. Structure-based identification of small molecule antiviral compounds targeted to the

gp41 core structure of the human immunodecifiency virus type 1. Journal of Medicinal Chemistry 1999;42(17):32039.

[25] Jiang S, Debnath AK. A salt bridge between an N-terminal coiled coil of gp41 and an antiviral agent targeted to the gp41

core is important for anti-HIV-1 activity. Biochemical and Biophysical Research Communications 2000;270(1):1537.

[26] Collins RA, Ng TB, Fong WP, Wan CC, Yeung HW. A comparison of human immunodeficiency virus type 1 inhibition by

partially purified aqueous extracts of Chinese medicinal herbs. Life Sciences 1997;60(23):L34551.

[27] Chang RS, Yeung HW. Inhibition of growth of human immunodeficiency virus in vitro by crude extracts of Chinese

medicinal herbs. Antiviral Research 1988;9(3):16375.

S. Liu et al. / Life Sciences 71 (2002) 177917911790

[28] Collins RA, Ng TB, Fong WP, Wan CC, Yeung HW. Removal of polyphenolic compounds from aqueous plant extracts

using polyamide minicolumns. Biochemistry and Molecular Biology International 1998;45(4):7916.

[29] Neurath AR, Jiang S, Strick N, Lin K, Li Y-Y, Debnath AK. Bovine h-lactoglobulin modified by 3-hydroxyphthalicanhydride blocks the CD4 cell receptors for HIV-1. Nature Medicine 1996;2(2):2304.

[30] Au TK, Lam TL, Ng TB, Fong WP, Wan DC. A comparison of HIV-1 integrase inhibition by aqueous and methanol

extracts of Chinese medicinal herbs. Life Sciences 2001;68(14):168794.

[31] Xu RS. Natural product chemistry. Beijing: Science Press; 1993.

[32] Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme

inhibitors. Advances in enzyme regulation 1984;22:2755.

[33] Murakami T, Nakajima T, Koyanagi Y, Tachibana K, Fujii N, Tamamura H, Yoshida N, Waki M, Matsumoto A, Yoshie O,

Kishimoto T, Yamamoto N, Nagasawa T. A small molecule CXCR4 inhibitor that blocks T cell line-tropic HIV-1 infection.

Journal of Experimental Medicine 1997;186(8):138993.

[34] Tabba HD, Chang RS, Smith KM. Isolation, purification, and partial characterization of prunellin, an anti-HIV component

from aqueous extracts of Prunella vulgaris. Antiviral Research 1989;11(56):26373.

[35] Ngan F, Chang RS, Tabba HD, Smith KM. Isolation, purification and partial characterization of an active anti-HIV

compound from the Chinese medicinal herb viola yedoensis. Antiviral Research 1988;10(13):10716.

[36] Tan GT, Pezzuto JM, Kinghorn AD, Hughes SH. Evaluation of natural products as inhibitors of human immunodeficiency

virus type 1 (HIV-1) reverse transcriptase. Journal of Natural Products 1991;54(1):14354.

[37] Nishizawa M, Yamagishi T, Dutschman GE, Parker WB, Bodner AJ, Kilkuskie RE, Cheng YC, Lee KH. Anti-AIDS

agents, 1. Isolation and characterization of four new tetragalloylquinic acids as a new class of HIV reverse transcriptase

inhibitors from tannic acid. Journal of Natural Products 1989;52(4):7628.

[38] Xu HX, Wan M, Dong H, But PP, Foo LY. Inhibitory activity of flavonoids and tannins against HIV-1 protease. Biological

and Pharmaceutical Bulletin 2000;23(9):10726.

[39] Yoshida T, Hatano T, Ito H. Chemistry and function of vegetable polyphenols with high molecular weights. Biofactors

2000;13(14):1215.

[40] Tinkilic N, Uyanik A. Spectrophotometric determination of the tannin contents of various Turkish black tea, beer and wine

samples. International Journal of Food Sciences and Nutrition 2001;52(3):28994.

S. Liu et al. / Life Sciences 71 (2002) 17791791 1791

IntroductionMethodsReagentsPreparation of aqueous extracts of Chinese medicinal herbsMeasurement of polyphenol concentrationsRemoval of polyphenols from aqueous extracts of Chinese medicinal herbs and recovery of polyphenols from the polyamide columnsELISA

ResultsSpecificity of the screening assayInhibition of the HIV-1 gp41 six-helix bundle formation by aqueous extracts of Chinese medicinal herbs with antiviral activityThe active components in the herb extracts are resistant to boiling treatmentThe inhibitory activity of the herb extracts on the gp41 six-helix bundle formation is correlated with the concentrations of polyphenolsThe active components, like tannin, could be removed from the herb extracts by passing the extracts through polyamide resin mini-columnThe eluates from the polyamide resin mini-columns contain the inhibitors of gp41 six-helix bundle formationTannins purchased commercially inhibited the gp41 six-helix bundle formation in a dose-dependent manner

DiscussionAcknowledgementsReferences