Analysis of Peganum harmala, Melia azedarach and Morus alba extracts against six lethal human cancer cells and oxidative stress along with chemical characterization through advance Fourier Transform and Nuclear Magnetic Resonance spectroscopic methods towards green chemotherapeutic agents

Abstract

Traditional medicines implicate consumption of plant crude extracts, which may consist of extensive phytochemical diversity. Overall, the most biologically active extract of Peganum harmala (seeds) exhibited significant cytotoxic activity on Artemia salina with LC₅₀ value of 61.547 µg/mL, while P. harmala (roots) [LC₅₀ = 124.229 µg/mL] and M. azedarach (fruits) [LC₅₀ = 147.813 µg/mL] showed moderate cytotoxic potential. P. harmala (seeds) extract also showed the maximum antitumor potential with 52.278 µg/mL LC₅₀. Branches of P. harmala and Morus alba were not active in both bioassays. These outcomes were further reinforced by the levels of phenolics and flavonoids checked against gallic acid and quercetin equivalents, respectively, by standard curves. Current study aims to isolate, structurally characterize and analyze the bioactive compound from plant extracts by using chromatographic and spectrophotometric techniques. Bioactivity guided isolation of extracts led to the isolation of PH-HM-16 from ethyl acetate fraction P. harmala seeds. Chemical structure of PH-HM-16 was elucidated by ESI-MS, ¹H NMR, ¹³C NMR, HSQC and IR spectrum. The results demonstrated significant positive anticancer activities against six human cancer cell lines assessed through MTT cancer cell growth inhibition assay. PH-HM-16 was most effective against prostate cancer cell lines [IC₅₀ = 17.63 µg/mL] followed by breast cancer cell line MCF7 [IC₅₀ value of 41.81 µg/mL]. IC₅₀ value of PH-HM-16 against human myeloid leukemia cell line HL-60 and human colorectal tumor cells HCT-116 was observed as 68.77 µg/mL and 71.54 µg/mL respectively. The IC 50 value of PH-HM-16 compound was not significant against human gastric cancer SGC-7901 (111.89 µg/mL) and human lung adenocarcinoma epithelial cell line A549 (176.04 µg/mL). Isolated bioactive metabolite PH-HM-16 possesses significant antitumor potential so this could be the first step to develop an effective anticancer agent. Hence, this compound represents a promising potential to be chemically standardized or developed into pharmaceuticals for the chemoprevention and/or the treatment of certain types of cancer, especially as adjuvant phytotherapeutics in conventional chemotherapy.

Keywords: Anticancer; Flavonoids; Melia azedarach; Phenolics; Phytochemicals.

Fig. 1

Brine Shrimp Lethality Test against logarithmic concentration of the plant extracts after 12 h and 24 h of exposure. Where M.Az/F:12 = Lethality of Melia azedarach (fruit) after 12 h; M.Az/L:12 = Lethality of Melia azedarach (Leaves) after 12 h; M.Al/L:12 = Lethality of Morus alba (Leaves) after 12 h; M.Al/B:12 = Lethality of Morus alba(Branches) after 12 h; P.Ha/L:12 = Lethality of Peganum harmala (Leaves) after 12 h; P.Ha/S:12 = Lethality of Peganum harmala (Seeds) after 12 h; P.Ha/B:12 = Lethality of Peganum harmala (Branches) after 12 h; P.Ha/R:12 = Lethality of Peganum harmala (Roots) after 12 h; Vin.C:12 = Lethality of Vincristine sulphate after 12 h; Etopside:12 = Lethality of Etopside after 12 h; K2Cr2O7:12 = Lethality of K₂Cr₂O₇ (Potassium dichromate) after 12 h; M.Az/F:24 = Lethality of Melia azedarach (fruit) after 24 h; M.Az/L:24 = Lethality of Melia azedarach (Leaves) after 24 h; M.Al/L:24 = Lethality of Morus alba (Leaves) after 24 h; M.Al/B:24 = Lethality of Morus alba (Branches) after 24 h; P.Ha/L:24 = Lethality of Peganum harmala (Leaves) after 24 h; P.Ha/S:24 = Lethality of Peganum harmala (Seeds) after 24 h; P.Ha/B:24 = Lethality of Peganum harmala (Branches) after 24 h; P.Ha/R:24 = Lethality of Peganum harmala (Roots) after 24 h; Vin.C:24 = Lethality of Vincristine sulphate after 24 h; Etopside:24 = Lethality of Etopside after 24 h; K2Cr2O7:24 = Lethality of K₂Cr₂O₇ (Potassium dichromate) after 24 h.

Fig. 2

Cytotoxicity LC₅₀ and R² values determined through logarthmic regression analysis. Where M.Az/F = Melia azedarach (fruit); M.Az/L = Melia azedarach (Leaves); M.Al/L = Morus alba (Leaves); M.Al/B = Morus alba (Branches); P.Ha/L = Peganum harmala (Leaves); P.Ha/S = Peganum harmala (Seeds); P.Ha/B = Peganum harmala (Branches); P.Ha/R = Peganum harmala (Roots); Vin.C = Vincristine sulphate; Etopside = Etopside; K2Cr2O7 = K₂Cr₂O₇ (Potassium dichromate).

Fig. 3

Probit analysis of cytotoxicity determination of eight plant extracts and three standards.Where M.Az/F = Melia azedarach (fruit); M.Az/L = Melia azedarach (Leaves); M.Al/L = Morus alba (Leaves); M.Al/B = Morus alba (Branches); P.Ha/L = Peganum harmala (Leaves); P.Ha/S = Peganum harmala (Seeds); P.Ha/B = Peganum harmala (Branches); P.Ha/R = Peganum harmala (Roots); Vin.C = Vincristine sulphate; Etopside = Etopside; K2Cr2O7 = K₂Cr₂O₇ (Potassium dichromate).

Fig. 4

Percentage Inhibition of Tumor at seven different concentartions.

Fig. 5

Antitumor activiy LC₅₀ and R² value determined through logarthmic regression analysis.Where M.Az/F = Melia azedarach (fruit); M.Az/L = Melia azedarach (Leaves); M.Al/L = Morus alba (Leaves); M.Al/B = Morus alba (Branches); P.Ha/L = Peganum harmala (Leaves); P.Ha/S = Peganum harmala (Seeds); P.Ha/B = Peganum harmala (Branches); P.Ha/R = Peganum harmala (Roots); Vin.C = Vincristine sulphate; Etopside = Etopside; K2Cr2O7 = K₂Cr₂O₇ (Potassium dichromate).

Fig. 6

a) Mass spectrum, b) Infrared spectrum, c) Proton Nuclear Magnetic Resonance spectrum, d) Carbon Nuclear Magnetic Resonance e) Heteronuclear Single Quantum Coherence f) Proposed structure and 3D conformer of PH-HM-16 isolated compound.

Fig. 7

A) Percentage cell viability and B) Percentage Inhibition of PH-HM-16 against six human cancer cell lines. Where HL-60 = Human myeloid leukemia cell line; PC-3 = Prostate cancer cell line; SGC-7901 = Human gastric cancer cell line; MCF-7 = Breast cancer cell line; HCT-116 = Human colorectal cancer cell line; Lung A549 = Human lung adenocarcinoma cell line.