Phytochemical Analysis, Biological Activities and Computational Study of Compounds Isolated from Selected Medicinal Plants of Ethiopia

Abstract

Breast cancer is a significant public health problem worldwide and the second leading cause of cancer-related death among women. Several risk factors, including environmental and genetic aspects, have been claimed to be responsible as a cause of breast cancer. Most active treatment strategies and combinations adversely affect the patient's quality of life through severe side effects. On the other hand, infectious diseases are also public health problems and a major cause of death worldwide. Increasing cases of drug resistance, unwanted side effects of existing antibiotics, and the reappearance of earlier known infections have demanded the need for new, safe and effective antimicrobial agents. In Ethiopia, over 70% of the people depend on traditional medicines in one way or another for their healthcare, including cancer and infectious diseases. More than 95% of the preparations are made from plant origin. Traditionally, Vepris dainelli (Rutaceae), Zanthoxylum chalybeum (Rutaceae), Uvaria scheffleri (Annonaceae), Clematis burgensis (Ranunculaceae), and Euphorbia schimperiana (Euphorbiaceae) are medicinal plants used for the treatment of abdominal cramps, intestinal worms, skin diseases, malaria, tuberculosis, intestinal problems, pneumonia cough, tuberculosis, asthma sore throat, and fever, and breast cancer in Ethiopia. Objectives: This study aimed to identify anticancer and antimicrobial lead compounds from selected medicinal plants of Ethiopian flora. Methods: Acid-base extraction was used to extract alkaloids from the roots of V. dainelli and Z. chalybeum. Extraction by maceration technique was used to extract the fruits of V. dainelli and the roots of U. scheffleri, C. burgensis and E. schimperiana. Essential oils were extracted by hydrodistillation. Silica gel column chromatographic technique was used to isolate compounds. Spectroscopic techniques (IR, 1D (1H, 13C, and DEPT-135) and 2D (COSY, HSQC, and HMBC) NMR, ESI-MS, HRMS, GC-MS, and LC-MS were used to elucidate structures of isolated compounds. The cytotoxicity effects of alkaloids were evaluated in vitro against MCF 7 and MDA-MB-231 human breast cancer cell lines by using MTS assay. Antimicrobial activity of isolated compounds and extracts were evaluated against S. aureus, E.coli and C. albicans via broth dilution techniques. The larvicidal activity of essential oils was determined by marinade solution, and their cytotoxicity was studied on VERO cells using an MTT assay. xxii Results: Silica gel flash column chromatographic separation of the extracts of five selected medicinal plants gave a total of 26 compounds, of which two compounds, uvarindole-F (248) and G (249), are new natural products. The cytotoxicity assay displayed alkaloids (13, 17, 55, 67, and 245) induced a significant reduction of cell growth of both breast cancer cell lines in a dose dependent manner of which chelerythrine (55) and evodiamine (245) showed the highest potency against the aggressive and metastatic MDA-MB-231 cell line (IC50= 1.73 ± 0.48 and 3.616 ± 0.51 µM), respectively, with respect to standard doxorubicin (0.23 µM). In addition, the alkaloids (55 and 245) showed the influence in the cell cycle in the MDA-MB-231 cell line by arresting some cells in the G2/M phase, preventing cells with damaged DNA from entering mitosis. On the other hand, alkaloids (13, 17 and 67) showed moderate activity against the MDA-MB-231 cell line (IC50= 69.30 ± 5.34, 55.99 ± 12.17 and 24.14±5.24 µM), respectively. Arborinine (13) and evoxanthine (17) exhibited higher cytotoxicity against MCF-7 than MDA MB-231 and showed influence the cell cycle in both cell lines by arresting some cells in the G2/M phase, preventing cells with damaged DNA from entering mitosis. Antimicrobial assay of Chelerythrine (55) isolated from Z. chaleybeum, dichloromethane extract and isolated Uvarindole-F (248) from roots of U. scheffleri displayed potential antibacterial activity against S. aureus (MIC =12.5, 6.25 and 12.5 µg/mL), respectively, compared with gentamicin (5 µg/mL against S. aureus). Chelreythrine (55) and uvarindole-F (248) showed an antifungal effect on C. albicans (50 µg/mL and 12.5), respectively, compared with fluconazole (8 µg/mL against C. albicans). In silico molecular docking analysis of isolated compounds against E. coli DNA gyrase B and against human topoisomerase IIα revealed a promising scoring pose (lowest energy) with a value ranging from -7.5 to -5.5 Kcal/mol and -6.4 to -5.3 Kcal/mol, compared to ciprofloxacin (-7.2 Kcal/mol) and vosaroxin (-6.2 Kcal/mol). Among the studied compounds, chelerythrine (55, -6.4 Kcal/mol), 6-hydroxydihydrochelerythrine (62, -6.3 Kcal/mol), dihydrocherythrine (67, -6.3 Kcal/mol) and evodimaine (245, -6.3 Kcal/mol)) showed higher lowest binding energy, interactions with human topoisomerase IIα than vosaroxin (-6.2 Kcal/mol) whereas interactions with E. coli DNA gyrase B, chelerythrine (55,-7.4 Kcal/mol), dihydrocherythrine (67, -7.5 Kcal/mol) and uvarindole-F (248, -7.3 Kcal/mol) showed higher lowest binding energy with respect to ciprofloxacin (-7.2 Kcal/mol). In addition, ADMET studies showed the highest drug likeness properties of the isolated compounds, except compound 250, suggesting these compounds can act as a drug and exhibit remarkable biological activities. DFT calculations indicated that studied compounds showed the lowest gap energy and were chemically reactive. Essential oils of U. scheffleri roots, Z. chalybeum and V. dainelli fruits were extracted by hydrodistillation to afford 0.5, 2.0, and 2.7 % (v/w) yields, respectively. Gas Chromatography Mass Spectrometry analysis of essential oils revealed a total of 58, 18 and 20 chemical compositions, representing 86.3, 99.6, and 98.8 % of the total oil contents, respectively. Tricyclo[5.3.0.0(3, 9)] decane was identified to be the principal constituent in essential oils of Z. chalybeum (82.8%) and V. dainelli (69.8%), reported herein for the first time from both species and could be considered as a marker constituent for Ethiopian species. The larvicidal activity of the essential oils in marinated solutions was tested in vitro against Anisakis L3 larvae isolated from blue whiting fish (Micromesistius poutassou) for the first time, achieving 100% mortality after 7 h at 0.5 and 1% essential oils concentrations of U. scheffleri and Z. chalybeum, and at 5% concentration after 5 and 3 h, respectively. Similarly, 100% mortality was observed after 7 and 5 h at 1 and 5% concentrations, respectively, for V. dainelli. The cytotoxicity study of essential oils on VERO cells displayed moderate toxicity with half-maximal inhibitory concentration values of 65.46 µg/mL, 83.88 µg/mL, and 96.82 µg/mL, respectively. Conclusions: The results obtained from molecular docking, drug-likeness properties, ADMET, and DFT analysis are in good agreement with in vitro experimental results obtained for compounds. Hence, compounds 13, 17, 55, 67 and 245 may serve as a lead molecules that could be developed into potent topoisomerase IIα inhibitors against human breast cancer whereas compounds 55 and 248 may serve as a lead molecules that could be developed into potent E. coli DNA gyrase B inhibitors. The findings of this study support the traditional uses of the root decoction of U. scheffleri and Z. calybeum for the treatment of infectious diseases caused by gram-positive bacteria strains such as pneumonia and sore throat. The weak cytotoxicity of the essential oils at low concentrations increases the possibility of developing effective and safe botanical larvicides. However, these results should be further supported by in vivo studies.