Lamiaceae plant extracts and isolated compounds demonstrate activity against HIV/AIDS

Abstract

Background: HIV/AIDS remains a major health concern worldwide and the number of people infected in Sub-Saharan Africa continues to increase. This despite increased awareness and availability of HIV drugs in most countries. The success of current HIV-1 drugs is overshadowed by the emergence of drug resistant viral strains and the adverse side-effects they may cause. It is these limitations and many more that drives the continuous search for better HIV treatments. Research into drug discovery and development using natural products is becoming better established. With natural products, there are endless opportunities for discovering novel compounds which either ends up as final drugs or as backbones of drug leads. Methods: In this thesis, sixteen Lamiaceae (mint) plants were investigated for inhibitory properties against HIV-1 as well as for beneficial immune enhancing effects. This family of plants is commonly used in traditional medicine preparations for the treatment of various ailments including those that are virus induced. Cytotoxicity of the plant material was determined using tetrazolium dyes and the results subsequently confirmed with flow cytometry and real-time cell analysis. Direct enzyme assays were used to determine the inhibitory properties of the extracts and isolated compounds against HIV-1 protease (PR), reverse transcriptase (RT) and integrase (IN). The effect of the plant materials was also evaluated in an in vitro model of chronic and latent infection by measuring HIV-1 p24 protein secretion of an infected cell line (U1) following treatment. Most HIV-infected individuals only seek treatment during the chronic stages of disease and latent reservoirs of the virus perpetuate treatment. The immune modulating properties were determined by quantitating the effects of plant extracts/compounds on Th1/2/17 cytokine production in human mononuclear cells using the cytometric bead array technology. Finally, anti-oxidant and anti-inflammatory properties were also assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide colorimetric assays respectively. Results and discussion: The 50% cytotoxic concentration (CC50) of the extracts was between 4.2 and 100 μg/ml. Of the sixteen, extracts from six plants (Ocimum labiatum, Ocimum serratum, Plectranthus barbatus, Plectranthus neochilus, Salvia apiana and Stachys byzantina) were active against HIV-1. Four plants (P. neochilus, O. serratum, S. apiana and S. byzantina) demonstrated moderate inhibitory properties against HIV-1 PR, RT and IN (40-49%) and three of these plants (O. serratum, S. apiana and S. byzantina) significantly (p<0.05) suppressed HIV-1 replication in U1 cells. The most exciting data was obtained from extracts of P. barbatus and O. labiatum which demonstrated inhibition classified as good (>50%) against HIV-1 PR (IC50s 62 ±0.2 and 49.8 ±0.4 μg/ml), reduced the production of pro-inflammatory cytokines at non-cytotoxic concentrations and demonstrated strong antioxidant properties (IC50 values 13 ±0.8 and 15.8 ±0.3 μg/ml). O. labiatum extract also suppressed HIV-1 expression in U1 cells, significantly (p<0.05). In addition, one of the extracts (P. ciliatus) had anti-cancer potential with CC50 values <10 μg/ml. O. labiatum extract was purified to yield a chlorophyll derivative, pheophytin-a (phy-a); triterpene isomers (3-hydroxy-4,6a,6b,11,12,14b-hexamethyl- 1,2,3,4,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-octadecahydropicene-4,8adicarboxylic acid), amyrin and a labdane diterpenoid (labda-8(17),12E,14-triene-2R,18- diol). Phy-a inhibited HIV-1 PR with an IC50 value of 44.4 ±1.5 μg/ml. The triterpenes activated latent HIV-1 (a serious obstacle in the eradication of the virus) while the diterpenoid reduced the production of pro-inflammatory cytokines. These activities were observed at non-toxic concentrations of these compounds. There is an ongoing search for novel compounds that are able to activate latent HIV-1 to use in conjunction with HAART. If the triterpenes were to progress to clinical use, their use would be in activating latent virus for eradication by existing treatments. Conclusion: The findings presented in this thesis provide some scientific explanation for the anecdotal success of some Lamiaceae plants used traditionally to manage HIV/AIDS. The findings also conform to recommendations by the scientific community regarding the validation of the beneficial effects of plant products used traditionally.