Anti-HCV Tannins From Plants Traditionally Used in West Africa and Extracted With Green Solvents

Abstract

Millions of people are still infected with hepatitis C virus (HCV) nowadays. Although recent antivirals targeting HCV proteins are very efficient, they are not affordable for many people infected with this virus. Therefore, new and more accessible treatments are needed. Several Ivorian medicinal plants are traditionally used to treat "yellow malaria", a nosological category including illness characterized by symptomatic jaundice such as hepatitis. Therefore, some of these plants might be active against HCV. An ethnobotanical survey in Côte d'Ivoire allowed us to select such medicinal plants. Those were first extracted with methanol and tested for their anti-HCV activity. The most active ones were further studied to specify their IC₅₀ and to evaluate their toxicity in vitro. Greener solvents were tested to obtain extracts with similar activities. Following a phytochemical screening, tannins of the most active plants were removed before re-testing on HCV. Some of these tannins were identified by UPLC-MS and pure molecules were tested against HCV. Out of the fifteen Ivorian medicinal plants selected for their putative antiviral activities, Carapa procera DC. and Pericopsis laxiflora (Benth. ex Baker) Meeuwen were the most active against HCV (IC₅₀: 0.71 and 0.23 μg/ml respectively) and not toxic for hepatic cells. Their crude extracts were rich in polyphenols, including tannins such as procyanidins A2 which is active against HCV. The same extracts without tannin lost their anti-HCV activity. Replacing methanol by hydro-ethanolic solvent led to tannins-rich extracts with similar antiviral activities, and higher than that of aqueous extracts.

Keywords: Côte d'Ivoire; antiviral; hepatitis C; sustainable extractions; tannins; traditional medicine.

FIGURE 1

Screening of 15 Ivorian medicinal plant crude extracts for their anti-HCV activity. Each plant was extracted with methanol, and dried extracts were dissolved in DMSO (used here as negative control). Huh-7 cells were inoculated with HCV in the presence of crude extracts at 25 μg/ml, or delphinidin at 50 μM, or boceprevir at 4 µM. Infected cells were quantified 30 h post infection by immunofluorescence detection of viral E1 protein. Data are presented relative to untreated control. Data are means ± SEM of 3 experiments performed in triplicates.

FIGURE 2

Toxicity and antiviral assays of C. procera and P. laxiflora crude extracts. (A, B): Huh-7 cells were incubated with culture medium containing crude extracts at different concentrations. MTS toxicity assay was performed at 24, 48 or 72 h of incubation. (C, D): Huh-7 cells were inoculated with HCV in the presence of crude extracts at different concentrations. Infected cells were quantified 30 h post infection by immunofluorescence detection of viral E1 protein. Data are presented relative to untreated control. Data are means ± SEM of 3 experiments performed in triplicates.

FIGURE 3

Tannins are responsible for C. procera and P. laxiflora activity against HCV. Tannin removal required heating of extracts. C. procera (A) and P. laxiflora (B) methanolic extracts were tested on HCV infection after heating (tannins), and after tannins removal (w/o tannins). Quantification of infection was as described above. Data are presented relative to untreated control and are means ± SEM of 3 experiments performed in triplicates.

FIGURE 4

Effect of solvents on dried mass, total phenol and tannin yields for Carapa procera DC. and Pericopsis laxifora (Benth. ex Baker) Meeuwen. (A): Dried mass yield for C. procera with hydro-alcoholic solvents containing 0–100% alcohol. (B, C): Total phenol and tannin yields for C. procera (B) and P. laxifora (C); GAE: gallic acid equivalent; Eco: hydroalcoholic eco-extracts with 0–100% ethanol; MeOH: crude methanolic extracts.

FIGURE 5

Tannin-rich eco-extracts of C. procera and P. laxiflora are active against HCV. C. procera (A) and P. laxiflora (B) eco-extracts (50% ethanol) were tested on HCV infection before tannin removal and before heating (squares), after heating (triangles), and after tannins removal (w/o tannins). Quantification of infection was as described above. Data are presented relative to untreated control. Data are means ± SEM of 3 experiments performed in triplicates.

FIGURE 6

Anti-HCV activity of procyanidins. Huh-7 cells were inoculated with HCV in the presence of procyanidin A1 (ProA1), A2 (ProA2), B1 (ProB1), and B2 (ProB2) at 5, 25, 50 and 100 µM. Cells were fixed 30 h post-infection. Quantification of infection was as described above. Data are presented relative to untreated control and are means ± SEM of 3 experiments performed in triplicates.