RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: There have been different methods proposed to prevent the sexual transmission of HIV-1 and many of them have centered on the use of anti-retrovirals as microbicides. Given that a large section of the African population still relies on herbal medicine, Lobostemon trigonus (L. trigonus), a traditionally used medicinal plant in South Africa to treat HIV-1 was further investigated for its potential as a natural microbicide to prevent the sexual transmission of HIV-1. METHODS: The aerial parts of L. trigonus were oven-dried at 80 °C, ground, extracted with boiling water for 30 min and then filtered. The aqueous extract produced was then bioassayed using different HIV-1 inhibition assays. The active components were purified and chemically profiled using ultra-performance liquid chromatography/quadrupole time-of flight mass spectrometry (UPLC-qTOF-MS). The mechanism of HIV-1 inhibition was determined by fusion arrest assay and time of addition assay. Molecular modelling and molecular dynamic simulations, using Schrödinger, were used to better understand the molecule's mechanism of entry inhibition by evaluating their docking affinity and stability against the gp120 of HIV-1. RESULTS: The aqueous extract of this plant had a broad spectrum of activity against different subtypes of the virus; neutralizing subtype A, B and C in the TZM-bl cells, with IC50 values ranging from 0.10 to 7.21 µg/mL. The extract was also inhibitory to the virus induced cytopathic effects in CEM-SS cells with an EC50 of 8.9 µg/mL. In addition, it inhibited infection in peripheral blood mononuclear cells (PBMC) and macrophages with IC50 values of 0.97 and 4.4 µg/mL, respectively. In the presence of vaginal and seminal simulants, and in human semen it retained its inhibitory activity albeit with a decrease in efficiency, by about 3-fold. Studies of the mode of action suggested that the extract blocked HIV-1 attachment to target cells. No toxicity was observed when the Lactobacilli strains, L. acidophilus, L. jensenii, and L. crispatus that populate the female genital tract were cultured in the presence of L. trigonus extract. UPLC-qTOF-MS analyses of the purified fraction of the extract, confirmed the presence of six compounds of which four were identified as rosmarinic acid, salvianolic acids B and C and lithospermic acid. The additional molecular dynamic simulations provided further insight into the entry inhibitory characteristics of salvianolic acid B against the HIV-1 gp120, with a stable pose being found within the CD4 binding site. CONCLUSION: The data suggests that the inhibitory effect of L. trigonus may be due to the presence of organic acids which are known to possess anti-HIV-1 properties. The molecules salvianolic acids B and C have been identified for the first time in L. trigonus species. Our study also showed that the L. trigonus extract blocked HIV-1 attachment to target cells, and that it has a broad spectrum of activity against different subtypes of the virus; thus, justifying further investigation as a HIV-1 microbicide.
