Ethnopharmacological exploration and isolation of HIV-1 latency-reversing agents from Sudanese medicinal plants

Abstract

HIV-1 infection remains a major health challenge, especially in resource-limited settings such as Sudan, where traditional medicine is widely practiced for managing infectious diseases, including HIV/AIDS. In this study, we selected ten Sudanese medicinal plants traditionally used to treat immune-related and infectious diseases. The selection was based on ethnobotanical reports and local knowledge of HIV/AIDS-related treatments. Crude extracts were prepared using either absolute methanol or 50% ethanol via maceration, resulting in a total of 20 extracts. The extracts were then screened for HIV-1 latency reversal using a luciferase reporter assay in TZM-bl cells. The 50% ethanolic extract of G. kraussiana showed the highest LTR activation (EC₅₀ = 3.75 μg/mL) with no significant cytotoxicity observed. Bioactivity-guided fractionation of the Gnidia kraussiana extract led to the isolation of gnidilatidin, a daphnane-type diterpenoid, using ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS). Gnidilatidin demonstrated potent latency-reversing activity (EC₅₀ = 5.49 nM in J-Lat 10.6 cells) and downregulated CD4 and CXCR4, suggesting enhanced inhibition of HIV-1 entry. This study supports the ethnopharmacological relevance of G. kraussiana and validates its traditional use. It also identifies gnidilatidin as a promising lead compound for HIV-1 latency-reversal-based strategies. Further studies are needed to optimize its pharmacological profile and further elucidate its therapeutic potential, particularly as part of an optimized combination regimen with combination antiretroviral therapy (cART).

Keywords: HIV-1/AIDS; Sudanese medicinal plants; Sudanese traditional healers; anti-HIV drugs; latency-reversing agents.

FIGURE 1

Extracts of Sudanese medicinal plants induce HIV-1 LTR transcriptional activity. (A) Cytotoxicity of extracts. TZM-bl cells were treated with 100 μg/mL of each extract. (B) Transcriptional activity from HIV-1 LTR induced by methanolic extracts. TZM-bl cells were treated with 100 μg/mL of each extract. (C) Transcriptional activity from HIV-1 LTR induced by 50% ethanolic extracts. TZM-bl cells were treated with 100 μg/mL of each extract. (D) Transcriptional activity from HIV-1 LTR induced by both methanolic and 50% ethanolic extracts at 1 μg/mL. (E) Dose-response curve and calculated EC₅₀ value for the 50% ethanolic extract of Gnidia kraussiana. For all panels, each extract was dissolved in DMSO and prepared to a final concentration of 0.5% DMSO in the culture medium. Control corresponds to 0.5% DMSO. M.P, Maerua pseudopetalosa; G.V, Grewia villosa; A.S, Anticharis senegalensis; L.A, Leptadenia arborea; C.N, Catunaregam nilotica; J.C, Jatropha curcas L.; L.P, Leptadenia pyrotechnica; F.C, Fagonia cretica L.; G.C, Gnidia chrysantha; G.K, Gnidia kraussiana. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (GraphPad Prism). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each value represents the mean ± S.D of three experiments.

FIGURE 2

Estimation of active compounds in Gnidia kraussiana, G.K. (A) Transcriptional activity from HIV-1 LTR induced by fractionated samples. Samples of each fraction were evaluated using TZM-bl cells by dilution as indicated. Control corresponds to 0.5% DMSO. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test in GraphPad Prism. *P < 0.05, ****P < 0.0001. Each value represents the mean ± S.D of three experiments. (B) The Total Ion Chromatograms (TICs) of the 50% ethanolic extract and fractions 1–4 shows the overall chemical profile of the extract. (C) Extracted Ion Chromatograms (EICs) obtained from UHPLC-HRMS analysis of (i) 50% ethanolic extract of G. kraussiana, (ii) active fraction 4, and (iii) commercially available gnidilatidin standard. The peak of interest showed an experimental m/z value of 649.3005, closely matcTIChing the theoretical value of 649.3007, confirming the identity as gnidilatidin. (D) Chemical structure of proposed compound identified using dictionary of natural products online database.

FIGURE 3

Effects of gnidilatidin in J-Lat 10.6 cells.(A) Cytotoxicity of gnidilatidin. J-Lat 10.6 cells were treated with various concentrations of gnidilatidin for 24 h, and cytotoxicity was assessed by flow cytometry. (B) Dose-dependent latency-reversing activity of gnidilatidin. J-Lat 10.6 cells were treated with gnidilatidin or prostratin for 24 h, and the percentage of GFP-positive cells was measured by flow cytometry. Control corresponds to 0.5% DMSO. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (GraphPad Prism). ****P < 0.0001; ns, not significant. Each value represents the mean ± S.D of three experiments.

FIGURE 4

Analysis of expression levels of HIV-1 entry receptors. The surface expression levels of HIV-1 entry receptors of (A) TZM-bl cells and (B) Jurkat cells were analyzed by flow cytometry. Cells were treated with gnidilatidin for 48 h. Control corresponds to 0.5% DMSO. Statistical analysis was performed by unpaired t-test (GraphPad Prism) **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Each value represents the mean ± S.D of three experiments.