Journal List > J Bacteriol Virol > v.46(4) > 1034228

J Bacteriol Virol. 2016 Dec;46(4):239-247. English.
Published online December 31, 2016.  https://doi.org/10.4167/jbv.2016.46.4.239
Copyright © 2016 The Korean Society for Microbiology and The Korean Society of Virology
Cytotoxic Effects of Gallic Acid and its Derivatives Against HIV-I-infected Microglia
Jin-Ju Jeong, Yong-Sup Lee and Dong-Hyun Kim
Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, Seoul, Korea.

Corresponding author: Dong-Hyun Kim. Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung-Hee University, 26, Kyungheedaero, Dongdaemun-gu, Seoul 02447, Korea. Phone: +82-2-961-0374, Fax: +82-2-957-5030, Email: dhkim@khu.ac.kr
Received November 11, 2016; Revised November 27, 2016; Accepted December 05, 2016.

This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/).


Abstract

In the previous study, we found that flavonoids and ginsenosides exhibited high eliminate rates of human immunodeficiency virus type 1 (HIV-1) D3-transfected macrophages. Based on these findings, here we synthesized the derivatives of gallic acid, including methyl gallate, methyl 4-O-methyl gallate, methyl 3,4-O-dimethyl gallate, and methyl 3,4,5-O-trimethyl gallate and measured their cellular toxic effects against HIV-1-infected macrophages. Of these, treatment with methyl 4-O-methyl gallate in the presence of lipopolysaccharide (LPS) and cycloheximide (CHX) most effectively eliminated HIV-1-transfected cytoprotective human microglial CHME5 cells and HIV-1-D3-infected human primary macrophages. Furthermore, these strongly inhibited LPS/CHX-induced phosphorylation of phosphoinositide 3-kinase (PI3K), pyruvate dehydrogenase lipoamide kinase isozyme 1 (PDK1), Akt, and glycogen synthase kinase-3β (GSK-3β) in the Tat-transfected cells and HIV-1-D3-infected human primary macrophages. These findings suggest that methyl 4-O-methyl gallate may be a promising candidate for eliminating HIV-1 infected macrophages by blocking PI3K/Akt signaling pathway.

Keywords: HIV-1; Macrophage; Gallic acid; Methyl 4-O-methyl gallate

INTRODUCTION

The infection of human macrophages by human immunodeficiency virus type 1 (HIV-1) activates the phosphoinositide 3-kinase (PI3K)/Akt cell survival pathway, resulting in macrophages resisting to cytotoxic attacks, e.g., some HIV-1 antigens such as Tat protein and gp120 activate PI3K/ Akt signaling pathway in macrophages (1, 2). PI3K/Akt signaling pathway regulates the expression of mammalian target of rapamycin (mTOR) and glycogen synthase kinase- 3β (GSK-3β), resulting in the potentiation of cell survival and growth (3). In addition, the transfection of HIV-1 Tat into microglial CHME5 cells activates the PI3K/Akt signaling pathway by suppressing the expression of phosphatase and tensin homolog (PTEN), which is acting as a negative regulator. The expression of Tat protein from HIV-1 in the human microglial cell line, CHME5 as well as primary human macrophages, activates the PI3K/Akt pathway under the cellular stresses by reducing the level of PTEN, leading to a strong resistance to extracellular stresses such as LPS or nitric oxide (1, 4, 5). The Tat-transfected cells become resistant to extracellular cytotoxic insults such as LPS (6). HIV-1 Tat-expressed microglia in humans becomes long-lived HIV-1 reservoirs in the central nervous system (4, 6, 7), and causes neuronal death and neurodegenerative diseases (8, 9). Therefore, to control these diseases, many studies have been conducted to eliminate the HIV-1-infected macrophages by searching for synthetic chemicals such as miltefosine (3), and phytochemicals such as arctigenin (5), oroxylin A and tectorigenin (10). These eliminated the HIV-1-infected macrophages by inhibiting PI3K/Akt cell survival signaling pathway.

Based on these findings, in the present study, we synthetized the derivatives of gallic acid and confirmed their anti-HIV-1 mechanism in CHME5 cells and primary macrophages.

MATERIALS AND METHODS

Reagents and antibodies

Cycloheximide (CHX), propidium iodide (PI), polybrene, LPS purified from Escherichia coli O26:B6 and miltefosine were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies for p-PI3K p85α (Tyr 508), PI3K C2γ (M-228), p-Akt1/2/3 (Ser 473), Akt 1 (G-5), p-GSK-3β (Ser 9), GSK-3β (L-17), and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for p-PDK1 (Ser 241), PDK1, mTOR, and p-mTOR (Ser 2448) were purchased from Cell Signaling Technology (Beverly, MA, USA). Human recombinant GM-CSF was purchased from R&D Systems (Minneapolis, MN, USA). Live/Dead Viability/Cytotoxicity Kit and Calcein AM and lipofectamine 2000 transfection reagent were purchased from Invitrogen (Carlsbad, CA, USA). HIV-1 vectors (D3) pseudotyped with the p-CMV-VSV-g envelope proteins (11) were kindly donated by Dr. Baek Kim (Emory University, Atlanta, GA, USA).

Gallic acid and its methyl derivatives

Gallic acid derivatives were obtained by following the commonly known procedure available in relevant literature (5). In briefly, gallic acid (compound 1) was transformed to its methyl ester (compound 2) by heating at reflux in methanol in the presence of a catalytic amount of c-H2SO4. The compound 2 was methylated by treating excess methyl iodide in the presence of K2CO3 in dimethylformamide (DMF) to yield its 4-O-methyl, 3,4-O-dimethyl, and 3,4,5- O-trimethyl ethers (compound 3, 4, and 5), respectively (Fig. 1).


Figure 1
Synthesis of gallic acid and its derivatives. (Compound 1), gallic acid; (compound 2), methyl gallate; (compound 3), methyl 4-O-methyl gallate; (compound 4), methyl 3,4-O-dimethyl gallate; (compound 5), methyl 3,4,5-O-trimethyl gallate. cat., catalytic; DMF, dimethylformamide; rt, room temperature.
Click for larger image

Cell and cell culture

A human microglial cell line, CHME5, transfected with plasmid stably expressed full-length Tat, and a control cell line, transfected with plasmid pcDNA3.1-Hygro, were cultured in DMEM medium containing 10% fetal bovine serum (FBS). Human monocytes were isolated from the blood of volunteer donors, which was donated from Korea Red Cross Blood Donation Center (approved by the Committee for the Care and Use of Clinical Study in the center) (2, 10). The peripheral blood mononuclear cells were collected by Ficoll density gradients and peripheral monocytes were isolated by the immunomagnetic selection using with anti- CD14 antibody-conjugated magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany). The monocytes were incubated for four days in a RPMI 1640 medium containing 20% human AB serum (Sigma-Aldrich) in the presence of recombinant GM-CSF (5 ng/ml) and then cultured for an additional three days in the absence of recombinant GM-CSF to differentiate macrophages.

Production of HIV-1-D3 particles

Primary macrophages were infected with a HIV-1-D3. First, D3 and pCMV-VSV-g vectors were transfected in 293- FT cells (2 × 106 cells/well) with the lipofectamine 2000 transfection reagent for three days. Viral production was determined by measuring the level of p24 by enzyme linked-immunosorbent assay (Perkin Elmer Life Sciences, Boston, MA, USA) in the supernatant of the cell culture according to the manufacturer's protocol. The supernatant was treated in primary macrophages in the presence of polybrene (2 µg/ ml) for five days. Virus-expressed primary macrophages were stained with green fluorescence and then detected by a fluorescent microscope because HIV-1-D3 contains green fluorescent protein (GFP) (Axio Vert.A1, Carl Zeiss, Oberkochen, Germany).

Cytotoxicity assay

Tat-transfected CHME5 cells were stressed by treatment with CHX (10 µg/ml) and LPS (50 µg/ml) in the absence or presence of test compounds (5, 10, and 20 µM) for 48 h and then trypsinized and stained with Trypan blue solution (12). The dead and live cells were counted. To analyse the cytotoxicity of test compounds by flow cytometer, cells were trypsinized, stained with calcein AM (0.25 µM) / PI (1 µg/ml), and analyzed by a flow cytometer (C6 Flow Cytometer® System, BD, Ann Arbor, MI, USA). To check cytotoxicity of test compounds using fluorescent microscope, the cells were washed with PBS, stained with calcein AM (0.25 µM) / PI (1 µg/ml), and analyzed by a fluorescent microscope. The primary macrophages were infected with a GFP-conjugated HIV-1-D3 vector and stressed by treatment with CHX (10 µg/ml) and LPS (50 µg/ml) in the absence or presence of test compounds (5, 10 and 20 µM) for 48 h, then stained with PI (1 µg/ml), and analyzed by fluorescent microscopy (10). To detect cytotoxicity of test compounds in HIV-1-D3 infected macrophages, cells were stressed by treatment with CHX (10 µg/ml) and LPS (50 µg/ml) in the absence or presence of test compounds (5, 10 and 20 µM) for 48 h and trypsinized cells were stained with PI (1 µg/ml), and analyzed by flow cytometer. When treatment with LPS/CHX alone induced a significant amount of the untransduced/control cell death within 24 h, as previously reported (4), the analysis of the untransduced cells in the present experiment was not indicated. Test compounds were dissolved in dimethyl sulfoxide (a final concentration, 0.2%).

Immunoblotting assay

Tat-transfected CHME5 cells (5 × 105 cells/well) or primary macrophages (1 × 106 cells/well) were treated with LPS/CHX in the presence or absence of test compounds for 2 h and then lysed (10). The supernatant of the lysates was applied to a 10% SDS polyacrylamide gel electrophoresis, followed by transfering to a nitrocellulose membrane. Protein Levels of PI3K, p-PI3K, PDK-1, p-PDK-1, Akt, p- Akt, GSK-3β, p-GSK-3β, mTOR, p-mTOR and β-actin were assayed as previously described (10). Immunodetection was carried out using an enhanced chemiluminescence detection kit.

Statistical analysis

Data are indicated as the means ± a standard deviation (S.D.) of at least three replicates. One-way variance analysis and student's t-test were used. A p-value < 0.05 was statistically significant.

RESULTS

The cytotoxic effects of gallic acid derivatives against HIV-1 Tat-transfected CHME5 cells

LPS-induced cell death in macrophages and microglial cells requires blockade of protein synthesis, whereas HIV-1-infected cells were not died by LPS/CHX (12). Therefore, to evaluate the cytotoxic effects of gallic acid derivatives against HIV-1-transfected cytoprotective macrophages, we measured the effect of the synthetized compounds (1) through (5) against Tat-transfected CHME5 cells (Fig. 2). Treatment with LPS/CHX in the presence of compound 3 eliminated Tat-transfected cytoprotective CHME5 cells most effectively, followed by compounds 1 and 2. However, treatment with LPS/CHX in the absence of gallic acid derivatives did not exhibit any cytotoxicity against Tat-transfected CHME5 cells. Next, the cytotoxic effects of compound 1 or 3 against Tat-transfected CHME5 cells in the presence and absence of LPS/CHX were examined using flow cytometric analysis (Fig. 3). Treatment with compound 1 or 3 in the presence of LPS/CHX showed the strong cytotoxicity in a dose-dependent manner. However, treatment with LPS/CHX or the test agent alone did not show any cytotoxic effects against Tat-transfected CHME5 cells.


Figure 2
The cytotoxic effects of gallic acid derivatives against cytoprotective HIV-1 Tat-transfected CHME5 cells. HIV-1 Tat-transfected CHME5 cells were treated with test compounds (0, 5, 10, and 20 µM) or miltefosine (MF, 20 µM) for 48 h. (Compound 1), gallic acid; (compound 2), methyl gallate; (compound 3), methyl 4-O-methyl gallate; (compound 4), methyl 3,4-O-dimethyl gallate; (compound 5), methyl 3,4,5-O-trimethyl gallate in the absence or presence of LPS/CHX. Cell death was determined by the trypan blue staining assay. All values are mean ± S.D. (n = 4). *,p < 0.05 compared with 0 µM treatment group.
Click for larger image


Figure 3
The cytotoxic effects of gallic acid and methyl 4-O-methyl gallate against cytoprotective HIV-1 Tat-transfected CHME5 cells. (A) Cytotoxic effects of gallic acid (compound 1) and 4-methoxy methyl gallate (compound 3) were determined using trypan blue staining assay. (B) Cytotoxic effects of gallic acid (compound 1) and 4-methoxy methyl gallate (compound 3) by the calcein AM/PI using a flow cytometer (C6 Flow Cytometer® System) and a fluorescence microscope. HIV-1 Tat-transfected CHME5 cells were treated with LPS/CHX in the absence or presence of test compounds (0, 10, and 20 µM) or miltefosine (MF, 20 µM) for 48 h. Trypsinized cells stained with calcein AM/PI were measured by a flow cytometer. All values are the mean ± S.D. (n = 4). (C) Cytotoxicity of test compounds was measured using a fluorescence microscope. Cells were stained with calcein AM/PI to distinguish between dead (red) and live (green) cells. Images (merged red and green fields) are representatives of 3 experiments conducted in duplicate. *,p < 0.05 compared with LPS/CHX treatment group.
Click for larger image

The cytotoxic effects of gallic acid (compound 1) and methyl 4-O-methyl gallate (compound 3) in HIV-1-D3-infected human macrophages

To confirm the cytotoxic effects of compounds 1 and 3 against HIV-1-infected macrophages, HIV-1-D3 was transfected into the primary human macrophages, and then the cytotoxicity of which was measured by Trypan blue staining and PI staining with FACS using Live/Dead assay kit (Fig. 4). Treatment with compound 1 or 3 in the presence LPS/CHX showed the strong cytotoxicity in a dose-dependent manner. Compound 3 showed cytotoxic effect against the HIV-1-D3-infected primary human macrophages more potently than gallic acid (compound 1). However, compound 1 or 3 at a dose of 20 µM did not show the cytotoxicity (< 5%) under the experimental condition.


Figure 4
Cytotoxic effects of gallic acid and methyl 4-O-methyl gallate against HIV-1-D3-infected human primary macrophages. (A) Cytotoxic effects of gallic acid (compound 1) and 4-methoxy methyl gallate (compound 3) were determined using trypan blue staining assay. (B) HIV-1-D3-infected primary macrophages were treated with LPS/CHX in the absence or presence of gallic acid (compound 1) or methyl 4-O-methyl gallate (compound 3) (0, 5, 10, and 20 µM). Cytotoxic effects of gallic acid (compound 1) and methyl 4-O-methyl gallate (compound 3) by PI/FACS assay. Cells stained with PI were examined by a flow cytometer. All values are mean ± S.D. (n = 4). (C) Cytotoxicity of compounds was determined using a fluorescence microscope. Cells were stained with PI to distinguish between dead (red) and live (green) cells. Images (merged red and green fields) are representatives of 3 experiments conducted in duplicate. *,p < 0.05 compared with LPS/CHX treatment group.
Click for larger image

To investigate the cytotoxic mechanism of compounds 1 and 3 in HIV-1-D3-infected human macrophages, their effects on the PI3K/Akt cell survival signaling pathway were measured (Fig. 5). Treatment with LPS/CHX significantly increased the phosphorylation of PI3K, PDK1, Akt, GSK-3β, and mTOR. However, compounds 1 and 3 inhibited LPS/CHX-induced phosphorylation of PI3K, PDK1, Akt, GSK-3β, and mTOR.


Figure 5
Effects of gallic acid and 4-methoxy methyl gallate on the phosphorylation of PI3K, PDK1, Akt, GSK3β, mTOR and β-actin in LPS/CHX-stimulated HIV-1-D3-infected human primary macrophages. (A) Effects of gallic acid (compound 1). (B) Effect of methyl 4-O-methyl gallate (compound 3). HIV-1-D3-transfected primary macrophages were treated with LPS/CHX in the absence or presence of gallic acid (compound 1), methyl 4-O-methyl gallate (compound 3) (0, 5, 10, and 20 µM) for 120 min. Proteins were measured by immunoblotting. #,p < 0.05 compared with normal control; *,p < 0.05 compared with LPS/CHX treatment group.
Click for larger image

DISCUSSION

HIV-1-infected human macrophages, including microglial cells, extend their lifespans by activating the PI3K/Akt cell survival signaling pathway (4) and transforming these cells into HIV-1 reservoirs, which persistently release the HIV-1 virus (13). Moreover, HIV-1-infected microglia and macrophages secrete nitric oxide and toxic viral proteins such as Tat and gp120, which establish the cytotoxic environment around the HIV-1-infected cells (4). The excessive release of NO and toxic viral proteins from these HIV-1-infected cells can cause neuron cell death, resulting in HIV-1-associated neurodegenerative diseases (14). Therefore, in order to diminish neuronal cell death and HIV-1-associated neurodegenerative diseases, it needs to eliminate HIV-1-infected macrophages.

Gallic acid and its methyl ester are known to have anti-inflammatory (15), anti-oxidant (16), and anti-tumor properties (17, 18). Moreover, Ahn et al. proposed that the galloyl moiety plays a major role in inhibiting these compounds' 3'-processing of HIV-1 integrase of these compounds (19). Rivero-Buceta et al. reported that the 2,3,4- trihydroxybenzoyl moiety has better antiviral properties against HIV-1 than the galloyl (3,4,5-trihydroxybenzoyl) moiety that is present in natural green tea catechins (20). Moreover, methyl gallate inhibited HIV-1 reverse transcriptase, integrase, and viral replication activities (21, 22, 23). Liu et al. reported that tea polyphenols including gallic acid could inhibit entry of HIV-1 into target cells by blocking envelope-mediated membrane fusion (24). However, the cytotoxic effects of gallic acid derivatives against HIV-1-infected macrophages have not been studied. In a previous study, we found that 5,7-dihydroxyl-6-methoxyflavonoids, such as oroxylin A and tectorigenin, were shown to exhibit the cytotoxic activity against HIV-1-D3-infected macrophages. The eliminating activity was structurally dependent on the number of hydroxyl groups of flavonoids at the 5-, 6-, and 7-positions and methylation of the 6-hydroxyl group (10).

In the present study, we synthesized gallic acid derivatives and compared their cell toxicity and PI3K/Akt-inhibitory activities in HIV-1-D3-infected macrophages, which are the long-term cell survival phenotypes against LPS/CHX stress by activating PI3K/Akt signaling pathway. Of these compounds, methyl 4-O-methyl gallate (compound 3) exhibited the most potent cytotoxic effect. However, we should investigate how of gallic acid derivatives to inhibit the PI3K/Akt signaling pathway in HIV-1-infected macrophages in near future. These studies raised the possibility that the methyl 4-O-methyl gallate can be beneficial for AIDS patients. These findings suggest that methyl 4-O-methyl gallate (compound 3) is a potent PI3K inhibitor and delivers anti-HIV-1 effects by decrease the survival of HIV-1 infected macrophages.

Notes

This study was supported by a grant from World Class University Program through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology (R33-2008-000-10018-0). We also thank Prof. Baek Kim (Emory University, Atlanta, GA, USA) for his assistance.

References
1. Chugh P, Bradel-Tretheway B, Monteiro-Filho CM, Planelles V, Maggirwar SB, Dewhurst S, et al. Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy. Retrovirology 2008;5:11.
2. Faried A, Kurnia D, Faried LS, Usman N, Miyazaki T, Kato H, et al. Anticancer effects of gallic acid isolated from Indonesian herbal medicine, Phaleria macrocarpa (Scheff.) Boerl, on human cancer cell lines. Int J Oncol 2007;30:605–613.
3. Lucas A, Kim Y, Rivera-Pabon O, Chae S, Kim DH, Kim B. Targeting the PI3K/Akt cell survival pathway to induce cell death of HIV-1 infected macrophages with alkylphospholipid compounds. PLoS One 2010:5.
4. Chugh P, Fan S, Planelles V, Maggirwar SB, Dewhurst S, Kim B. Infection of human immunodeficiency virus and intracellular viral Tat protein exert a pro-survival effect in a human microglial cell line. J Mol Biol 2007;366:67–81.
5. Kim Y, Hollenbaugh JA, Kim DH, Kim B. Novel PI3K/ Akt inhibitors screened by the cytoprotective function of human immunodeficiency virus type 1 Tat. PLoS One 2011;6:e21781.
6. Brown A, Zhang H, Lopez P, Pardo CA, Gartner S. In vitro modeling of the HIV-macrophage reservoir. J Leukoc Biol 2006;80:1127–1135.
7. Schrier RD, McCutchan JA, Venable JC, Nelson JA, Wiley CA. T-cell-induced expression of human immunodeficiency virus in macrophages. J Virol 1990;64:3280–3288.
8. Cosenza MA, Zhao ML, Lee SC. HIV-1 expression protects macrophages and microglia from apoptotic death. Neuropathol Appl Neurobiol 2004;30:478–490.
9. Irish BP, Khan ZK, Jain P, Nonnemacher MR, Pirrone V, Rahman S, et al. Molecular Mechanisms of Neurodegenerative Diseases Induced by Human Retroviruses: A Review. Am J Infect Dis 2009;5:231–258.
10. Jeong JJ, Kim DH. 5,7-Dihydroxy-6-Methoxy-Flavonoids Eliminate HIV-1 D3-transfected Cytoprotective Macrophages by Inhibiting the PI3K/Akt Signaling Pathway. Phytother Res. 2015
11. Jeong JJ, Kim B, Kim DH. Ginsenoside Rb1 eliminates HIV-1 (D3)-transduced cytoprotective human macrophages by inhibiting the AKT pathway. J Med Food 2014;17:849–854.
12. Suzuki T, Kobayashi M, Isatsu K, Nishihara T, Aiuchi T, Nakaya K, et al. Mechanisms involved in apoptosis of human macrophages induced by lipopolysaccharide from Actinobacillus actinomycetemcomitans in the presence of cycloheximide. Infect Immun 2004;72:1856.
13. Aquaro S, Bagnarelli P, Guenci T, De Luca A, Clementi M, Balestra E, et al. Long-term survival and virus production in human primary macrophages infected by human immunodeficiency virus. J Med Virol 2002;68:479–488.
14. Mattson MP, Haughey NJ, Nath A. Cell death in HIV dementia. Cell Death Differ 2005;12 Suppl 1:893–904.
15. Kroes BH, van den Berg AJ, Quarles van, van Dijk H, Labadie RP. Anti-inflammatory activity of gallic acid. Planta Med 1992;58:499–504.
16. Hsieh TJ, Liu TZ, Chia YC, Chern CL, Lu FJ, Chuang MC, et al. Protective effect of methyl gallate from Toona sinensis (Meliaceae) against hydrogen peroxideinduced oxidative stress and DNA damage in MDCK cells. Food Chem Toxicol 2004;42:843–850.
17. Gras G, Kaul M. Molecular mechanisms of neuroinvasion by monocytes-macrophages in HIV-1 infection. Retrovirology 2010;7:30.
18. Kim TW, Paveen S, Lee YH, Lee YS. Comparison of Cytotoxic Effects of Pentagalloylglucose, Gallic Acid, and its Derivatives Against Human Cancer MCF-7 and MDA MB-231 Cells. Bull Korean Chem Soc 2014;35:987–988.
19. Ahn MJ, Kim CY, Lee JS, Kim TG, Kim SH, Lee CK, et al. Inhibition of HIV-1 integrase by galloyl glucoses from Terminalia chebula and flavonol glycoside gallates from Euphorbia pekinensis. Planta Med 2002;68:457–459.
20. Rivero-Buceta E, Carrero P, Doyaguez EG, Madrona A, Quesada E, Camarasa MJ, et al. Linear and branched alkyl-esters and amides of gallic acid and other (mono-, di- and tri-) hydroxy benzoyl derivatives as promising anti-HCV inhibitors. Eur J Med Chem 2015;92:656–671.
21. Kratz JM, Andrighetti-Frohner CR, Kolling DJ, Leal PC, Cirne-Santos CC, Yunes RA, et al. Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate. Mem Inst Oswaldo Cruz 2008;103:437–442.
22. Modi M, Goel T, Das T, Malik S, Suri S, Rawat AK, et al. Ellagic acid & gallic acid from Lagerstroemia speciosa L. inhibit HIV-1 infection through inhibition of HIV-1 protease & reverse transcriptase activity. Indian J Med Res 2013;137:540–548.
23. Wang CR, Zhou R, Ng TB, Wong JH, Qiao WT, Liu F. First report on isolation of methyl gallate with antioxidant, anti-HIV-1 and HIV-1 enzyme inhibitory activities from a mushroom (Pholiota adiposa). Environ Toxicol Pharmacol 2014;37:626–637.
24. Liu S, Lu H, Zhao Q, He Y, Niu J, Debnath AK, et al. Theaflavin derivatives in black tea and catechin derivatives in green tea inhibit HIV-1 entry by targeting gp41. Biochim Biophys Acta 2005;1723:270–281.