Life Sciences 71 (2002) 1779 1791

www.elsevier.com/locate/lifescie

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,*
a

Institute of Pharmaceutical Sciences of the First Medical University of PLA, PLA Key Lab for Drug Screening,
Guangzhou, Guangdong 510515, China
b
Lindsley 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

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).
0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 9 3 9 - 2

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major inhibitors of the HIV-1 gp41 six-helix bundle formation in the herb extracts and that tannin may inhibit HIV1 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.
HIV entry 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 Cpeptides), 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].

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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. 3hydroxyphthalic 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

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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 20 ml) for testing. The columns were washed with 20 ml 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 sixhelix 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.

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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-incubated
with 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, rabbit
IgG 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,5,5Vtetramethylbenzidine (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 NC1 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)

Glycyrrhiza uralensis (GU)
Viola yedoensis (VY)
Prunella vulgaris (PV)
Andrographis paniculata (AP)
Morus alba (MA)
Lonicera japonica (LJ)
Bupleurum Chinese (BC)
Rhizoma cibotte (RC)

4.6
28.7
14.7
86.2
21.0
4.5
6.8
7.9
98.3

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

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

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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.

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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.

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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 PV and 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).

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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.

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 minicolumns and these recovered polyphenols, like tannin, had potent inhibitory activity on the gp41 sixhelix 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 sixhelix bundle formation. As shown in Fig. 7, both tannins inhibited the interaction between N- and C-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

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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):
1046 50.

1790

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

[2] Richman DD. Antiretroviral drug resistance: mechanisms, pathogenesis, clinical significance. Advances In Experimental
Medicine and Biology 1996;394:383 95.
[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):78 86.
[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):2093 9.
[5] Williamson K, Reboli AC, Manders SM. Protease inhibitor-induced lipodystrophy. Journal of the American Academy of
Dermatology 1999;40(4):635 6.
[6] Blower SM, Aschenbach AN, Gershengorn HB, Kahn JO. Predicting the unpredictable: transmission of drug-resistant
HIV. Nature Medicine 2001;7(9):1016 20.
[7] Moore JP, Jameson BA, Weiss RA, Sattentau QJ. The HIV-cell fusion reaction. In: Bentz J, editor. Viral Fusion Mechanisms. Boca Raton: CRC Press; 1993. p. 233 89.
[8] Berger EA. HIV entry and tropism: the chemokine receptor connection. AIDS 1997;11(suppl A):S3 S16.
[9] Chan DC, Kim PS. HIV entry and its inhibition. Cell 1998;93:681 4.
[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):407 15.
[11] Wyatt R, Sodroski JG. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 1998;280(5371):
1884 8.
[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):276 9.
[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):404 9.
[14] Chan DC, Fass D, Berger JM, Kim PS. Core structure of gp41 from the HIV envelope glycoprotein. Cell 1997;89:263 73.
[15] Weissenhorn W, Dessen A, Harrison SC, Skehel JJ, Wiley DC. Atomic Structure of the Ectodomain from HIV-1 gp41.
Nature 1997;387(6631):426 8.
[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):9770 4.
[18] Lu M, Blacklow SC, Kim PS. A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nature Structural
Biology 1995;2(12):1075 82.
[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):1302 7.
[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):641 6.
[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):10213 7.
[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):85 96.
[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):3203 9.
[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):153 7.
[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):L345 51.
[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):163 75.

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

1791

[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):791 6.
[29] Neurath AR, Jiang S, Strick N, Lin K, Li Y-Y, Debnath AK. Bovine h-lactoglobulin modified by 3-hydroxyphthalic
anhydride blocks the CD4 cell receptors for HIV-1. Nature Medicine 1996;2(2):230 4.
[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):1687 94.
[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;22:27 55.
[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):1389 93.
[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(5 6):263 73.
[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(1 3):107 16.
[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):143 54.
[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):762 8.
[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):1072 6.
[39] Yoshida T, Hatano T, Ito H. Chemistry and function of vegetable polyphenols with high molecular weights. Biofactors
2000;13(1 4):121 5.
[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):289 94.