Role of Chitosan-Loaded Solanine Glycoalkaloid from Solanum scabrum Mill. Leaf Extract as Anti-Inflammatory and In Vitro Anticancer Agents

Abstract

Objectives: Solanum scabrum Mill. commonly "African nightshade" or "huckleberry" is a plant, whose leaves are used by tribes in Nigeria and Cameroon for making the popular "Kombi" and "Njama Njama" soups, respectively. This study aimed to evaluate the anti-inflammatory and anticancer activities of the leaf crude methanol extract from S. scabrum.

Materials and methods: Fractions of the plant were tested for anti-inflammatory potential and in vitro anticancer activity on MCF-7 and HMVII cell lines by carrageenan-induced oedema in mice, and cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, transwell migration and invasion assays, and apoptosis study by flow cytometry, respectively.

Results: Bioguided isolation yielded a white crystalline compound 3-nitro dibenzofuran (C₁₂H₇NO₃, m/z; 213.19 g/mol, m.p.; 181.49 °C). ¹H-NMR showed seven signals at δ (ppm) 2.8-4.3 consisting of two doublets and five singlets, while ¹³C-NMR revealed twelve carbons, which are majorly methyl carbons at δ (ppm) between 120 and 195. All tested samples demonstrated dose-dependent anti-inflammatory activity in carrageenan-induced mice. The isolated compound, i.e. solanine, and chitosan-loaded drugs showed significant inhibitory activity on the cell lines with inhibitory concentration 50 (IC₅₀) values of 8.52, 0.82, and 22.1 μg/mL, respectively on MCF-7 cell line and 4.54, 0.08, and 12.1 μg/mL, respectively, on HMVII cell line, while doxorubicin (adriamycin) positive control, had IC₅₀ values of 0.02 and 0.06 μg/mL, respectively, on MCF-7 and HMVII cancer cells. Selectivity index of solanine was the lowest in the study, hence, it lacks the ability to differentiate between cancerous and normal cell Vero E6 cell lines. Chitosan-loaded drugs quicken early apoptosis and sustained late apoptosis in cells with much improved selective indices.

Conclusion: The results obtained from this study further affirmed the use of chitosan nanoparticles as carriers for anticancer drugs.

Keywords: Solanum scabrum; anti-inflammatory; anticancer; chitosan; glycoalkaloid; solanine.

Figure 1

NMR and MS spectra and HPLC chromatogram of the isolated compound from Solanum scabrum leaf; a; ¹H-NMR with seven protons between 2.81-4.33 δ (ppm), b; ¹³C-NMR with twelve carbon atoms, c; MS with m/z 213 and molecular peak ion 139 at 62.89% peak area for 3-nitrodibenzofuran, d; HPLC chromatogram showed 19.11 min with isocratic elution. CDCl₃ was used as a solvent for NMR analysis. MS: Mass spectrometry, HPLC: High-performance liquid chromatography

Figure 2

SEM morphology of chitosan loaded with crude Solanum scabrum extract (CE3), solanine (CS2), and doxorubicin (CDr4). Glutaraldehyde was used as cross-linking agent 3000x. SEM: Scanning electron microscope

Figure 3

Anti-inflammatory activity of Solanum scabrum extracts and chitosan NPs loaded extracts; a; (carrageenan-induced paw oedema), b; (effects on protein denaturation). Results are means ± SD. The values of p<0.05 were statistically significant. HF: Hexane fraction, EF: Ethyl acetate fraction, BF: Butanol fraction, CE3: Chitosan-loaded crude extract batch code 3, CS2: Chitosan-loaded solanine batch code 2, S: Solanine, SD: Standard deviation, NPs: Nanoparticles

Figure 4

Transwell migration and invasion assay of solanine (S) and chitosan-loaded solanine batch code 2 (CS2) as viewed using a hemocytometer. Results are mean ± SD for three replicate readings. Cellular migration and invasion were much more pronounced with CS2 treatment when compared to cells exposed to solanine alone. S: Solanine, CS2: Chitosan-loaded solanine batch code 2, SD: Standard deviation, Dox: Doxorubicin

Figure 5

Fluorescence images of MCF-7 cancer cells after treatment with CS2 and solanine (S) with various concentrations (12.5, 25, 40, and 50 μg/mL) at 12, 48 and 72 hours. Apoptotic cellular death and necrosis were detected by staining with propidium iodide and analysed with flow cytometry. Results are mean ± SD for three consecutive readings. p<0.05 was taken as a significance different. Ea: Early apoptosis, La: Late apoptosis, Cn: Clear nuclear, N: Necrosis of cells, Ln: Late necrosis, S: Solanine, SD: Standard deviation