In vitro Anti-HIV Activity of Oleanolic Acid on Infected Human Mononuclear Cells

• Abstract
• Introduction
• Materials and Methods
• Substances and virus source
• Anti-HIV activity on in vitro infected PBMC from healthy
  donors
• Anti-HIV activity on PBMC from naturally infected individuals
• Anti-HIV activity on acutely in vitro infected monocytes/ macrophages (M/M)
• p24 assay for anti-HIV actvity
• Cytotoxicity assay
• HIV-1 protease assay
• Results and Discussion
• Acknowledgements
• References

Abstract

Oleanolic acid is a triterpenoid which is quite common in nature in the form either of free acid or in triterpenoid saponin glycosides. This study describes the effect of oleanolic acid on the growth of human immunodeficiency virus-1 (HIV-1) in cultures of human peripheral mononuclear cells (PBMC) and of monocyte/macrophages (M/M). Its inhibitory activity was also evaluated on PBMC obtained from HIV-1 infected patients. Results obtained show that oleanolic acid inhibits the HIV-1 replication in all the cellular systems used (EC₅₀ values: 22.7 μM, 24.6 μM and 57.4 μM for in vitro infected PBMC, naturally infected PBMC and M/M, respectively). As regards the mechanism of action, oleanolic acid inhibits in vitro the HIV-1 protease activity.

Introduction

Oleanolic acid (3β-hydroxy-olea-12-en-28-oic acid) has long been recognised in folk medicine to be an active compound of many medicinal plants possessing anti-inflammatory, hepatoprotective, analgesic, cardiotonic, sedative and tonic effects [1], [2]. Many of these properties have been confirmed by systematic pharmacological research [3] [4] [5] [6] [7] [8].

Oleanolic acid has been noted for the inhibition of tumour promotion both in vitro and in vivo experiments [9], [10] and in Japan this compound has been recommended for skin cancer therapy [11].

In spite of a multifaceted profile of pharmacological activities, oleanolic acid is relatively non-toxic; the long-term clinical use of oleanolic acid indicates that it is therapeutically effective in the absence of apparent side effects [12].

Regarding the antiviral activity two oleanolic acid 3-hemiesters were found to exert a protection index against vaccina virus [13]. Oleanolic acid glycosides isolated from Calendula arvensis reduced vesicular stomatitis virus plaque formation [14]. A saponin mixture of six homologous oleane-type triterpenoid saponins from Maesa lanceolata exibited a virucidal effect against enveloped viruses [15]. The effect of some oleanolic acid functional groups on the antiviral activity against DNA and RNA viruses was studied by Simões et al. [16]. It has recently been reported that oleanolic acid and some structurally related triterpenoids inhibit the HIV-1 replication in acutely infected H9 cells [17]. These plant-derived compounds inhibit different stages in the replication cycle of HIV, such as virus adsorption and reverse transcription [18]. In order to extend these data, we evaluated the anti-HIV activity of oleanolic acid not only during in vitro and natural infection of PBMC, but also in monocytes/macrophages, one of the major reservoirs of HIV in vivo. The direct effect of oleanolic acid on recombinant HIV-1 protease was also studied.

Materials and Methods

Substances and virus source

Oleanolic acid (97 % pure) was purchased from Sigma (St. Louis, Mo, USA). It was dissolved in DMSO at the maximal concentration in the cultures of 0.1 % (v/v). Controls for the DMSO effect were also included in each experiment.

The HIV-1- reverse transcriptase inhibitor, 3’-azido-3’-deoxythymidine (AZT), obtained from Sigma and the HIV-1-protease inhibitor, indinavir , kindly provided by Dr. M. Andreoni (University of Tor Vergata, Rome, Italy) were used as reference of anti-HIV agents.

A primary strain of HIV-1, isolated from the plasma of an HIV-infected patient (Department of Infectious and Tropical Diseases, University of Rome ”La Sapienza”) was used as virus source. After isolation, the virus was expanded and titrated in PBMC according to standard protocols [19].

Anti-HIV activity on in vitro infected PBMC from healthy
donors

PBMC from healthy donors were isolated by density gradient centrifugation, using Histopaque 1077 (Sigma). After centrifugation, cells were suspended in RPMI 1640 complete medium (10 % fetal calf serum, penicillin G 50 U/ml, streptomycin 50 mg/ml and L-glutamine 2mM) at a concentration of 1 × 10⁶ cells/ml and were incubated in presence of phytohemagglutinin (PHA, Sigma), 5 μg/ml, for 72 h at 37 °C in an atmosphere enriched with 5 % CO₂ using T175 Falcon flasks (Becton Dickinson, Meylan, France). Cells were centrifugated, resuspended in 1 ml of complete medium and were infected with the HIV-1 clinical isolate by adding 500 TCID50/10⁶ PBMC. After an incubation of 2 h at 37 °C, 5 % CO₂, the cells were adjusted at final concentrations of 1 × 10⁶ cells/ml in complete medium supplemented with 5 U/ml IL-2 (Sigma, St.Louis, Mo) and seeded in 48-well plates (Becton Dickinson, Meylan, France). Infected PBMC were cultivated for seven days with or without various concentrations (10, 20, 40, 80 μM) of oleanolic acid. AZT (3.7 μM) was used as a well-established reference compound. After three days medium and substances were replaced by exchanging 50 % of the culture supernatants. After seven days supernatants were collected and stored until p24 antigen detection. To evaluate the virucidal effect of oleanolic acid, the HIV clinical strain was pre-incubated for 1 hour with different concentrations of the plant-derived compound before the in vitro infection of PBMC.

Anti-HIV activity on PBMC from naturally infected individuals

Five untreated HIV1-infected patients with high plasma viral load levels (> 360,000 copies/ml) were selected. PBMC were obtained from those patients as previously described. Cells were plated in 96-well plates at a concentration of 1 × 10⁵ cells/well and were stimulated with PHA (5 μg/ml). PBMC were cultivated for seven days in the presence or absence of different concentrations of oleanolic acid (10, 20, 40, 80 μM). AZT (3.7 μM) was used as reference compound. Cell supernatant was collected at day seven and viral production was determined by HIV-1 p24 antigen ELISA.

Anti-HIV activity on acutely in vitro infected monocytes/ macrophages (M/M)

PBMC were obtained from healthy HIV-1 negative donors as described above. Cells (3 × 10⁶ cells/well) were plated on biocoat 24-well plates (Becton Dickinson, Meylan, France), in complete RPMI supplemented by 1000 U/ml GM-CSF (Sigma) and cultured for 72 h, at 37 °C in an atmosphere enriched with 5 % CO₂. Non-adherent cells were carefully removed by washing with warmed RPMI. Cellular morphology and non-specific esterase staining showed a monolayer of adherent cells > 95 % of M/M which were suspended in complete medium. Cells were infected with 300 TCID50/well of the viral stock for 2 h, at 37 °C, 5 % CO₂. After viral challenge M/M were washed to remove excess virus and incubated in complete LPS-supplemented (10 ng/ml) medium with various concentration of oleanolic acid. AZT (3.7 μM) was used as reference compound.

Every 4 days 0.5 ml of supernatant was collected and replaced with fresh complete medium with or without oleanolic acid. The cells were cultured for 18 days and viral production was detected by HIV-1 p24 antigen ELISA after 3, 7, 11, 14 and 18 days of incubation.

p24 assay for anti-HIV actvity

Viral replication was detected by measuring HIV-1 p24 production in cell supernatants using an ELISA test (Innogenetics N.V., Belgium). The results were expressed as mean of three different experiments. The EC₅₀ was determined by linear regression of the percentage of HIV-1 p24 production (compared with untreated controls) versus the drug concentration according to Tallarida and Murray [20].

Cytotoxicity assay

Oleanolic acid cytotoxicity was evaluated in parallel with the different experiments. The crystal violet method was used to test the effect of oleanolic acid at different concentrations on PBMC from HIV+ and healthy subjects and on M/M.

HIV-1 protease assay

HIV-1 protease was obtained from Sigma. The assay was performed in tubes containing 25 ng of HIV-1 protease and 5 mg of Azocoll (Sigma), an aspecific protease substrate, in 0.5 ml of phosphate buffered solution (PBS) at pH 7.4 and incubated overnight at 37 °C. Tubes were then spun at 14 000 g for 5 minutes and the absorbance of the supernatant measured at 540 nm. The proteolytic activity was measured in the presence of different concentrations of oleanolic acid (10, 20, 40, 80 μM). Indinavir was used as reference substance. The results were expressed as percentage of inhibition with respect to the control without oleanolic acid.

Results and Discussion

When PBMC isolated from healthy donors and infected with HIV-1 strain were incubated with oleanolic acid at concentrations of 10, 20, 40 and 80 μM a dose-dependent inhibition of HIV-1 replication was observed (Fig. [1]). At the maximal concentration used the inhibition of HIV-1 replication was 70 %. Estimation of the EC₅₀ gave a value of 22.7 μM.

Experiments carried out using the PBMC of five untreated HIV-infected patients gave similar results. At all concentrations studied oleanolic acid inhibited the production of HIV-1 p24 antigen (Fig. [2]). At the concentration of 80 μM there was a 60 % inhibition of viral production.

When the EC₅₀ was estimated it indicated a value of 24.6 μM. The suppressive effect of AZT on p24 production in PBMC from healthy donors and HIV-infected patients was 70 % and 78 %, respectively, at the concentration (3.7 μM). Moreover, oleanolic acid exhibited a statistically significant antiviral activity in infected M/M at the concentrations of 40, 60 and 80 μM (Fig. [3]). At these doses a decrease in virus production was detectable starting from day 11 and reached its peak 18 days after the infection. At this time oleanolic acid exhibited an EC₅₀ value of 57.4 μM, the suppressive effect of AZT in this cellular system was 62 % at the concentration of 3.7 μM. No virucidal effect of oleanolic acid was detected (data not shown).

When oleanolic acid was tested for its effect on HIV-1 protease a dose-dependent inhibition of proteolytic activity was observed. This suppressive effect was 43 % at 10 μM and reached the value of 90 % at 80 μM. Indinavir inhibited the HIV-1 protease activity almost completely at a concentration of 100 nM.

Oleanolic acid toxicity in both M/M and PBMC was assessed in parallel with the above experiments. No evidence of cell killing was found up to 160 μM; at this concentration a 20 % inhibition of cell viability was observed in all cellular systems.

Since HIV has been identified as the aetiologic agent of AIDS, numerous compounds have been evaluated for their inhibitory effects on HIV replication. Over 15 anti-HIV-drugs and their combinations are available for clinical use and provide benefits in terms of mortality and morbidity among HIV infected patients. One of the major obstacles to long-term treatment is the remarkable HIV mutation capacity that causes the appearance of HIV-resistant strains. Acute and long-term toxicity is another crucial point of clinical managment of antiretroviral therapy. For this reason there is an ongoing need for new, structurally different anti-HIV agents which are less toxic and cheap enough to be accessible to all AIDS patients.

In recent years many compounds of plant origin have been identified as inhibitors of HIV-replication at different stages of its viral cycle [18].

It has been found that among these compounds, several terpenes exhibit an antiretroviral activity; the most interesting being glycyrrhizin, a triterpenoid saponin of Glycyrrhizia glabra. This compound delayed the progression to AIDS when used in HIV-infected patients [21]. Moreover in vitro tests on the murine AIDS model suggest that glycyrrhizin is effective in preventing the progression of the disease [22].

In the present work oleanolic acid was found to inhibit HIV-1 replication in fresh PHA-activated in vitro HIV-1 infected PBMC from healthy donors. The inhibitory effect was seen also on PHA-activated PBMC from HIV-1 infected patients.

Results obtained in these cellular systems, acute and chronically infected PBMC, gave similar results. In fact the EC₅₀ values of oleanolic acid were 22.7 and 24.6 μM in PBMC from healthy donors and AIDS patients, respectively.

Oleanolic acid was also effective in inhibiting viral replication on M/M infected with HIV-1. The inhibitory effect was visible after 11 days of culture indicating inhibition at the late stage of the virus cycle. The EC₅₀ value was two-fold higher with respect to the PBMC system ( EC₅₀ after 18 days = 57.4 μM). These results appear particularly interesting because of the peculiarity of HIV infection in macrophages. In fact M/M are important target cells for HIV-1, that can replicate over a long period of time without any cytopathic effect. Because of their resistance to the cytopathic effect of HIV, M/M are considered the most important viral reservoir in the body and a crucial target for a successful therapeutic regimen. Whether oleanolic acid inhibits the release of the virus remains to be addressed. The inhibition of some other viruses, like vaccinia and vescicular stomatitis virus, indicate a non-specific mechanism for the antiviral activity [18]. In agreement with Ma et al. [23] our results showed that this compound inhibits in vitro HIV-1 protease activity, thus suggesting a direct action on HIV aspartyl-protease. Considering the crucial role of the HIV-1 protease inhibitors in the High Active Antiretroviral Therapy (HAART) and the need of new less toxic agents the activity of oleanolic acid appears promising and worthy of further study.

Acknowledgements

Dr. Lucia Battinelli was supported by the ”Enrico and Enrica Sovena Foundation” (Italy).

References

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Lucia Battinelli

Dipartimento di Farmacologia delle Sostanze Naturali e

Fisiologia Generale

Università ”La Sapienza”

P.le Aldo Moro, 5

00185 Rome

Italy

Phone: +39-06-4991-2903

Fax: +39-06-4991-2480

Email: lucia.battinelli@uniroma1.it

References

• 1 Wang B, Jiang Z H. Studies on oleanolic acid.  Chinese Pharmaceutical Journal. 1992;  27 393-7
  PubMedSearch in Google Scholar
• 2 Liu J. Pharmacology of oleanolic acid and ursolic acid.  Journal of Ethnopharmacology. 1995;  49 57-68
  CrossrefPubMedSearch in Google Scholar
• 3 Liu Z, Wang D, Wang S, Ha S, Li P. Oleanolic acid in decreasing hyperglycemia.  Chinese Pharmaceutical Journal. 1994;  29 725-6
  PubMedSearch in Google Scholar
• 4 Ma X H, Zhao Y C, Yin L, Han D W, Ji C X. Studies on the effect of oleanolic acid on experimental liver injury.  Acta Pharmaceutica Sinica. 1982;  17 93-7
  PubMedSearch in Google Scholar
• 5 Jeong H G. Inhibition of cytochrome P450 2E1 expression by oleanolic acid: hepatoprotective effects against carbon tetrachloride-induced hepatic injury.  Toxicology Letters. 1999;  105 215-22
  CrossrefPubMedSearch in Google Scholar
• 6 Liu J, Liu Y P, Parkinson A, Klaassen C D. Effect of oleanolic acid on hepatic toxicant-activating and detoxifying systems in mice.  Journal of Pharmacology and Experimental Therapeutics. 1995;  275 768-74
  PubMedSearch in Google Scholar
• 7 Zhang L Z, Li X F. Study on the mechanism of oleanolic acid against experimental liver injury in rats.  Traditional Medicine and Clinical Pharmacology. 1992;  8 24-6
  PubMedSearch in Google Scholar
• 8 Singh G B, Singh S, Bani S, Gupta B D, Banerjee S K. Anti-inflammatory activity of oleanolic acid in rats and mice.  Journal of Pharmacy and Pharmacology. 1992;  44 456-8
  PubMedSearch in Google Scholar
• 9 Ohigashi H, Takamura H, Koshimizu K, Tokuda H, Ito Y. Search for possible antitumor promoters by inhibition of 12-o-tetradecanoylphorbol-13-acetate-induced Epstein-Barr virus activation: ursolic acid and oleanolic acid from an anti-inflammatory Chinese medicinal plant, Glechoma hederaceae L.  Cancer Letters. 1986;  30 143-51
  CrossrefPubMedSearch in Google Scholar
• 10 Oguro T, Liu J, Klaassen C D, Yoshida T. Inhibition effect of oleanolic acid on 12-O-tetradecanoylphorbol-13-acetate-induced gene expression in mouse skin.  Toxicological Science. 1998;  45 88-93
  PubMedSearch in Google Scholar
• 11 Muto Y, Ninomiya M, Fujiki H. Present status research on cancer chemoprevention in Japan.  Japanese Journal of Clinical Oncology. 1990;  20 219-24
  PubMedSearch in Google Scholar
• 12 Xu L Z, Wan Z X. The effect of oleanolic acid on acute hepatites (70 cases).  Human Medicine. 1980;  7 50-2
  PubMedSearch in Google Scholar
• 13 Serra C, Lampis G, Pompei R, Pinza M. Antiviral activity of new triterpenic derivatives.  Pharmacological Research. 1994;  29 359-66
  CrossrefPubMedSearch in Google Scholar
• 14 De Tommasi N, Conti C, Stein M L, Pizza C. Structure and in vitro antiviral activity of triterpenoid saponins from Calendula arvensis .  Planta Medica. 1991;  57 250-3
  PubMedSearch in Google Scholar
• 15 Sindambiwe J B, Calomme M, Geerts S, Pieters L, Vlietinck A J, Vanden Berghe D A. Evaluation of biological activities of triterpenoid saponins from Maesa lanceolata .  Journal of Natural Products. 1998;  61 585-90
  CrossrefPubMedSearch in Google Scholar
• 16 Simões C MO, Amoros M, Schenkel E P, Shin-Kim J S, Rücker G, Girre L. Preliminary studies of antiviral activity of triterpenoid saponins: relationship between their chemical structure and antiviral activity.  Planta Medica. 1990;  56 652-3
  PubMedSearch in Google Scholar
• 17 Kashiwada Y, Wang H K, Nagao T, Kitanaka S, Yasuda I, Fujioka T, Yamagishi T, Cosentino L M, Kozuka M, Okabe H, Ikeshiro Y, Hu C Q, Yeh E, Lee K H. Anti-AIDS agents 30. Anti-HIV activity of oleanolic acid, pomolic acid and structurally related triterpenoids.  Journal Natural Products. 1998;  61 1090-5
  PubMedSearch in Google Scholar
• 18 Vlietinck A J, De Bruyne T, Apers S, Pieters L A. Plant-derived leading compounds for chemotherapy of human immunodeficiency virus (HIV) infection.  Planta Medica. 1998;  64 97-109
  PubMedSearch in Google Scholar
• 19 Perno C F, Yarchoan R. Current protocols in immunology. Vol. 3, New York; John Wiley & Sons 1993: 12.4.1-.11
  Search in Google Scholar
• 20 Tallarida R, Murray R B. Manual of Pharmacologic Calculation with Computer Programs. 2nd Edn, Springer Verlag New York; 1981
  Search in Google Scholar
• 21 Hattori T, Ikematsu S, Koito A, Matsushita S, Maeda Y, Hada M, Fujimaki M, Takatsuki K. Preliminary evidence for inhibitory effect of glycyrrhizin on HIV replication in patients with AIDS.  Antiviral Research. 1989;  11 255-61
  CrossrefPubMedSearch in Google Scholar
• 22 Ito M, Nakashima H, Baba M, Pauwels R, De Clercq E, Shigeta S, Yamamoto N. Inhibitory effect of glycyrrhizin on the in vitro infectivity and cytopathic activity of the human immunodeficiency virus [HIV (HTLV-III/LAV)].  Antiviral Research. 1987;  7 127-37
  CrossrefPubMedSearch in Google Scholar
• 23 Ma C, Nakamura N, Miyashiro H, Hattori M, Shimotohno K. Inhibitory effects of costituents from Cynomorium songaricum and related triterpene derivatives on HIV-1 protease.  Chemical and Pharmacological Bulletin. 1999;  477 141-5
  PubMedSearch in Google Scholar

Lucia Battinelli

Dipartimento di Farmacologia delle Sostanze Naturali e

Fisiologia Generale

Università ”La Sapienza”

P.le Aldo Moro, 5

00185 Rome

Italy

Phone: +39-06-4991-2903

Fax: +39-06-4991-2480

Email: lucia.battinelli@uniroma1.it