Antimicrobial activity and probable mechanisms of action of medicinal plants of Kenya: Withania somnifera, Warbugia ugandensis, Prunus africana and Plectrunthus barbatus

Abstract

Withania somnifera, Warbugia ugandensis, Prunus africana and Plectrunthus barbatus are used traditionally in Kenya for treatment of microbial infections and cancer. Information on their use is available, but scientific data on their bioactivity, safety and mechanisms of action is still scanty. A study was conducted on the effect of organic extracts of these plants on both bacterial and fungal strains, and their mechanisms of action. Extracts were evaluated through the disc diffusion assay. Bacteria and yeast test strains were cultured on Mueller-Hinton agar and on Sabouraud dextrose agar for the filamentous fungi. A 0.5 McFarland standard suspension was prepared. Sterile paper discs 6 mm in diameter impregnated with 10 µl of the test extract (100 mg/ml) were aseptically placed onto the surface of the inoculated media. Chloramphenicol (30 µg) and fluconazole (25 µg) were used as standards. Discs impregnated with dissolution medium were used as controls. Activity of the extracts was expressed according to zone of inhibition diameter. MIC was determined at 0.78-100 mg/ml. Safety studies were carried using Cell Counting Kit 8 cell proliferation assay protocol. To evaluate extracts mechanisms of action, IEC-6 cells and RT-PCR technique was employed in vitro to evaluate Interleukin 7 cytokine. Investigated plants extracts have both bactericidal and fungicidal activity. W. ugandensis is cytotoxic at IC50<50 µg/ml with MIC values of less than 0.78 mg/ml. Prunus africana shuts down expression of IL 7 mRNA at 50 µg/ml. W. somnifera has the best antimicrobial (1.5625 mg/ml), immunopotentiation (2 times IL 7 mRNA expression) and safety level (IC50>200 µg/ml). Fractions from W. ugandensis and W. somnifera too demonstrated antimicrobial activity. Mechanisms of action can largely be attributed to cytotoxicity, Gene silencing and immunopotentiation. Use of medicinal plants in traditional medicine has been justified and possible mechanisms of action demonstrated. Studies to isolate and characterize the bioactive constituents continue.

Figure 1. Antifungal activity of fractions 9, 10, 11 & 17 from dichloromethane extract of W. ugandensis against C. neoformans (A) and C. albicans (B).

They display zones of inhibition (mm) of 12, 9, 8, 6 & 14, 8, 8, 6 respectively.

Figure 2. Graphical representation of the results of IEC-6 cell proliferation/cytotoxicity assay of 4 plant extracts.

W.ugandesis (Wu), P. africana (Pa) & P. barbatus (Pb) at concentrations ranging from 0.0005–5000 µg/ml and, W. somnifera (Ws) at concentrations ranging from 0.001–10,000 µg/ml. W. somnifera (Ws), P. africana (Pa) & P. barbatus (Pb) are relatively safe for use even in dose levels exceeding 200 µg/ml. W. ugandensis has a much lower safety level below 50 µg/ml.

Figure 3. 72 hr expression of GAPDH and IL-7 on treatment with different herbs.

A&B represents GAPDH & IL 7 respectively at concentrations: WU 50 µg/ml, WS 100 µg/ml, PA 50 µg/ml, PB1 500 µg/ml & PB2 1000 µg/ml respectively. C&D represents GAPDH & IL 7 at concentrations: WU1 83.33 µg/ml, WU2 41.67 µg/ml, WU3 16.67 µg/ml, WS1 666.67 µg/ml & WS2 333.33 µg/ml respectively. IL 7 is not expressed in presence of P. africana. Doubling P. barbatus concentration does not affect expression in either direction. Abbreviations; M; marker, C; control, WU; Warbugia ugandensis, WS; Withania somnifera, PA; Prunus africana, PB; Plectrunthus barbatus.

Figure 4. Bar chart depicting the relative IL 7 mRNA expression in IEC-6 cells subjected to the 4 medicinal plants.

The assay was replicated 3 times. W. somnifera up-regulates IL-7 to approximately 2 times.

Figure 5. 72 hr expression of GAPDH and IL 7 on treatment with different combinations of the 4 plant extracts.

A & B represent GAPDH & IL 7 respectively. Combination ratios, WU/PA 1∶2, WU/PB 1∶4, WU/WS 1∶1, PA/PB 1∶2, PA/WS 2∶1, PB/WS 2∶1, PB/WS 4∶1, WU/WS/PB, 1∶1:4. P. africana was able to shut down the expression of IL 7 irrespective of the combination used.