In vitro anticancer and toxicological evaluation of Gunnera perpensa L. isolated compounds

Abstract

INTRODUCTION: The leading cause of mortality worldwide is cancer. According to estimates, 11.5 million people will die from cancer by 2030. A painful cancer fact is that this disease kills more people than malaria, tuberculosis, and AIDS combined. Breast cells are where breast cancer first develops. When it comes to cancers affecting women, research suggests that breast cancer ranks second. Unfortunately, modern therapies like targeted therapy, radiation, and chemotherapy are toxic, often barbaric, and usually ineffective. There is thus an obvious need for alternative treatments and therapies against breast cancer. The identification of new lead compounds for cancer treatment may be accomplished by the use of natural medicines, including medicinal plants. The creation of different anticancer drugs has been influenced by plant-derived compounds, which include vinblastine, vincristine, the camptothecin derivatives, topotecan and irinotecan, etoposide, and paclitaxel. It has been discovered that some medicinal plants possess phytochemicals that have biological activity. The presence of secondary metabolites can indicate the biologically active molecules that are known to be anticancer, antimicrobial, anti-oxidant, anti-inflammatory, antifungal, antiviral, and anthelmintic compounds. In addition to treating parasitic diseases, kidney problems, urinary complaints, and sexually transmitted infections, herbal remedies containing Gunnera perpensa have been used to induce labour and ease menstrual pain. Certain chemical compounds extracted from G. perpensa were reported to be from the class of phytochemical constituents known as triterpenes. Triterpenoids have been demonstrated to possess a wide range of medicinal uses, including fighting cancer, malaria, and pain. AIM: This study aimed to separate natural compounds from G. perpensa and test how harmful, selective, and poisonous they were to cells. In addition, the phytochemicals, together with their total phenolic content (TPC) and total flavonoid content (TFC), were evaluated. METHODOLOGY: Phytochemical testing was performed on crude aqueous and methanol (MeOH) extracts. The Folin-Ciocalteu colorimetric technique, using Gallic acid as the standard, was used to determine the TPC. The TFC was quantified using a colorimetric technique with aluminium chloride, using quercetin as the standard. To obtain the best results from thin-layer chromatography (TLC) for separating the compounds of the active extracts, different mobile phases were created. The preparation and fractionation of the MeOH extract was done through vacuum liquid chromatography (VLC). Purified compounds were obtained through preparative thin-layer chromatography (PTLC). Structural elucidation of the pure compound was facilitated by the use of nuclear magnetic resonance (NMR). The in vitro anticancer and toxicological effects of crude extracts, active fractions, and pure compounds were tested on MDA-MB-231 and MCF-7 cell lines and non-cancerous Vero cells. The cells were grown using standard methods and cytotoxicity and clonogenic tests were performed using standard methods. In each experiment, cells were exposed to dimethyl sulphoxide (vehicle control). Calculating the selectivity index was done using the Vero normal cell line. The data that was acquired was evaluated statistically. GraphPad Prism 5 was the tool employed to analyse data and figure out the IC50. RESULTS: The phytochemical testing showed that there are terpenoids, alkaloids, flavonoids, reducing sugars, tannins, and phenols which may function inhibitors. The TPC obtained were 17.11±0.036 mg GAE/g and 16.02±0.012 mg GAE/g for MeOH and water (H2O) extracts respectively. Similarly, the TFC values obtained were 10.33±0.01 mg QE/g and 19.69±0.05 mg QE/g for MeOH and H2O extracts respectively. Six major sub-fractions concentrated from VLC were obtained. It was possible to isolate two pure compounds, one of which crystallised. MR-A was identified as methyl lespedezate, whereas MR-B was determined to be Z-venusol. Its chemical formula is (7,8-Dihydroxy-6-(hydroxymethyl)-3-[(Z)-(4-hydroxyphenyl) methylidene]-tetrahydro-4aH-pyrano (2, 3-b) (1, 4) dioxin-2-one] and the molecular formula C15H16O8. Incorporating 1H and 13C NMR spectroscopy into the process improved our understanding of the chemical structure. The compounds' names were confirmed by adding standards to some of them and matching the results from ultraviolet data, tandem mass spectrometry, and high-resolution mass spectrometry with data that had already been published. Following MTT-based in vitro cytotoxic screening the results of the extract, fractions, and isolated compounds of G. perpensa against the tested cancer cells displayed moderate to low cytotoxicity. The compound Z-venusol (MR-B) had the best selectivity, suggesting that it might be a safe chemotherapeutic option. The in vitro toxicity of Compound MR-B showed weak cytotoxic effects. The diluted MCF-7 cells responded differently to G. perpensa extracts depending on the concentration of the extracts. This means that a lower dose rate leads to more cell survival. The findings suggest that this compound may exhibit enhanced efficacy in conjunction with other compounds found in the crude extract of G. perpensa due to synergism. CONCLUSION: The findings provide evidence that G. perpensa isolated compounds reduce the survival of MCF-7 breast cancer cells in a way that depends on the concentration, and with apparently low toxicity.