Bioassy Guided Isolation and Characterization of Secondary Metabolites Rumex Hastatus D. Don and their Anticancer Activities

Abstract

The inquisitive nature of human compelled the nature to bestow myriads of resources and commodities to man. Since yonks ago, the human have been fully dependent on the earth’s ecosystem. Shortly, among the most productive natural sources, the extraordinary role of plants is prettily inevitable. Besides providing the food, shelter and living, the plants also provide health ameliorating entities known as drugs or medicine. The drugs from the plant sources are literally known as herbal medicine. Based on the very importance of herbal medicine, one of the scantly explored herb i.e., Rumex hastatus, which belongs to family Polygonaceae has been investigated over here in the terms of phytochemistry and anticancer aspects. As a lot of knowledge has been transferred from the previous era to the modern world via ethnobiology. So the R. hastatus has also been explored based on the ethnomedicine (traditional medicine) to authenticate its purported traditional uses especially for anticancer purposes. In the current study, different samples of R. hastatus have been investigated i.e., the crude sample, the solvent fractions of crude sample, the essential oil and the pure compounds. The crude sample was collected via maceration process while the isolation of essential oil was carried out following hydrodistillation via Clevenger apparatus. The fractions of different solvents have been obtained using successive solvent extraction procedure via separating funnel. Similarly, the compounds were obtained via bioassay guided isolation procedure through gravity columns with the help of thin layer chromatography. The compounds obtained were purified with pen/pencil column or small diameter gravity columns. The pure compounds obtained were identified with various techniques i.e., Mass spectroscopy, carbon NMR and proton NMR. Four compounds i.e., SA-1, SA-2, SA-3 and SA-4 were properly identified and structurally elucidated. Three compounds were in sufficient quantities, which were subjected to various anticancer assays. The anticancer assays were based on mechanistic approach. The assays performed were MTT assay, Comet assay, CAM assay, antitumor assay, anti-mutagenic assay, apoptosis assay, antithrombin assay, intercalation assay and antioxidant assay. Before these assays the phytochemical screening was carried out for the detection of different types of secondary metabolites in the samples. The screening of samples was also performed to figure out the presence of heavy metals and their amounts were figured out. The samples were also tested for acute toxicity via mice animal model. The investigations revealed that two compounds SA-1 and SA-2 were highly active against cancer cells. The SA-3 was found to be the least active compound against cancer. If we go to the detail of the test compounds, the SA-1 was found to have 46% antiangiogenic, 85% antimutagenic, 75% antioxidant, 61% antithrombin, 41% antitumor, 50% apoptosis, 42% necrosis, 67% cytotoxicity by MTT, 118/200 comet score and significant intercalation to DNA at the highest test concentration. The SA-2 has revealed 41% antiangiogenic, 70% antimutagenic, 55% antioxidant, 53% antithrombin, 37% antitumor, 61% apoptosis, 22% necrosis, 53% cytotoxicity my MTT, 91/200 comet score and moderate intercalation to DNA. The acute toxicity assay produce no visible anomaly, toxicity or lethality, while the heavy metals analysis revealed the presence of heavy metals within the permissible limits as per WHO guidelines. Based on the results of anticancer activities with various mechanisms, it may be deduced that the SA-1 was quite active against cancer and mechanistically we can say that SA-1 is strongly antimutagenic i.e., it quenches the the mutagens or carcinogens. Secondly we can see that the SA-1 has a significant antioxidant potential so it also inhibit the free radicals. Thirdly, it has also been demonstrated to cause cytotoxicity significantly. Similarly, In regards to the possible mechanistism, the SA-1 have the ability to bind with the genetic material, to quench the mutagens, to inhibit the free radicals, to inhibit the thrombin, to cause cytoxicity, to cause programmed cell death and necrosis, to break down the DNA. The SA-1 also moderately inhibit the neovascularization i.e., new blood vessels formation and inhibit the tumor. So the SA-1 has been found to possess highest potential for quenching the mutagens and inhibit the mutagenesis while the lowest potential has been found against tumorigenesis. Similarly, the SA-2 has also been demonstrated with moderate anticancer potential with the highest potential against mutagenesis and lowest potential to induce necrosis. Both of the compounds SA-1 and SA-2 have been verified in the current investigational studies to combat cancer especially the cervical and colon cancer with no toxicity profile. So further work should be performed by the dosage form designers for these compounds to hit the market.