Antiproliferative, anti-inflammatory and antioxidant activities of leaf extracts, fractions and isolated compounds from Ptaeroxylon obliquum

Abstract

Inflammation is a primordial response to protect the body against infection or trauma from outside or inside the body, such as harmful stimuli, viruses, bacteria and chemical exposures. This response is aimed at restoring damaged tissue to its normal physiological function. In the process of inflammation, macrophages act as the body's first line of defence, coordinating the inflammatory response by releasing a number of mediators. However, excessive or unregulated mediator production can induce chronic inflammation, which is the foundation of many unpleasant human diseases, including rheumatoid arthritis and cancer. Cancer is a leading cause of death not just in South Africa but also worldwide. The disease is an abnormal cell formation brought on by multiple gene expression alterations that result in an unbalanced ratio of cell proliferation and death. Oncogenes are activated and tumour suppressors are deactivated by DNA mutations that cause cancer to begin. The host metabolism and cell structure must change for cancer to proliferate. It is widely acknowledged that the inflammation microenvironment plays a role in tumorigenesis. Numerous studies have shown a connection between chronic inflammation and cancer formation as well as the influence of tumour-induced inflammation on tumour growth. Significant pathways between inflammation and the growth or metastasis of tumours have been discovered. In the inflammatory microenvironment, elevated levels of ROS, cytokines, prostaglandins, nuclear factor kB (NF-kB), and microRNAs affect angiogenesis, DNA mutation rates, cell death and cellular senescence. Numerous cancers release cytokines like IL-6, which have been associated with metastasis, carcinogenesis and inflammation. CD8+ T cells release anti-inflammatory cytokines including IL-10, which have anti-inflammatory and anti-tumorigenic characteristics as they reduce levels of IL-6 and TNF-α. Chronic inflammation and other factors like oxidative stress are the cause of more than 25% of malignancies. Free radicals, such as reactive oxygen/nitrogen species (ROS/RNS), are created by epithelial and inflammatory cells during chronic inflammation. Oxidative stress occurs when the balance of ROS production to ROS detoxification favours an increase in ROS levels. Unregulated ROS/RNS generation during inflammation damages DNA in organs and promotes the development of cancer. Given that chronic inflammation is connected to more than one-fifth of cancer incidence, research into chemopreventive drugs or preparations against inflammation-related carcinogenesis is urgently needed. Ptaeroxylon obliquum (Rutaceae), commonly known as sneezewood, was selected based on its traditional uses against inflammatory disorders such as fevers, arthritis, and rheumatism and work done on this species in the Phytomedicine Programme relating to other applications. Extracts, fractions and purified compounds from the leaves of the tree were evaluated for anti-proliferative, antioxidant and antiinflammatory activities in this thesis. Column chromatography was used to fractionate the active extracts based on bioactivity, and NMR and UPLC-MS analyses were used to identify and characterize the isolated compounds. Using a colorimetric tetrazolium bromide test, antiproliferative activity was evaluated against normal Vero cells as well as a number of cancerous human cells, including lung adenocarcinoma (A549), human breast cancer (MCF-7), hepatocarcinoma (HepG2) and human cervical cancer cells (HeLa). The 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 2, 2-diphenyl- 1-picrylhydrazyl (DPPH) tests were used to measure the free radical scavenging activity. The nitric oxide (NO) inhibition assay in lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages, soybean 15-lipoxygenase (15-LOX) inhibitory assay and cyclooxygenase (COX)-2 enzyme activity utilizing an ELISA method were used to determine the anti-inflammatory activity. Additionally, the effects of the extracts and isolated compounds on the production of the anti-inflammatory cytokine (IL-10) and pro-inflammatory cytokines (IL-1) and TNF-α were evaluated using ELISA kits in this study. Using mouse ELISA kits, the effects of the substances on the levels of caspase-3 and TNF- were determined and quantified in HepG2 and HeLa cancer cells. The alterations in HepG2 and HeLa cell morphology were observed using light microscopy. The secondary metabolite composition of the four P. obliquum acetone leaf extracts showed some geographical variation, as shown by the UPLC-MS chromatograms and the positive mode of the ESI. Using silica gel open column chromatography, obliquumol, O-methylalloptaeroxylin, and a combination of lupeol and β-amyrin were isolated from the chloroform fraction. Acetone extracts were relatively toxic to the HepG2 cells with IC50 values from 8 to 200 μg/mL, but they were less toxic to the other cell lines, with selectivity index values as high as 14. At concentrations evaluated against all cell lines, aqueous extracts and fractions has low toxicity (IC50 > 100 μg/mL). Isolated compounds had IC50 values against HepG2 and HeLa cells ranging from 52 to 539 μg/mL and 189 to 247 μg/mL, respectively. The cytotoxicity of the acetone extracts was confirmed by light microscopy, which revealed alterations in the morphology of HepG2 and HeLa cells. P. obliquum extracts showed selective cytotoxicity against cancer cell lines, rendering them potential sources for development for use in anticancer therapy.