Biological Evaluation, Isolation and Characterization of Compounds from Fagonia cretica

SAMREEN SALEEM
Department of Biochemistry, Quaid-i-Azam University, Islamabad

ABSTRACT
The research work presented in this thesis deals with the chemotherapeutic (anti-proliferative) and cancer chemopreventive activity of a medicinal plant from Pakistan; Fagonia cretica L. (Zygophyllaceae), based on the traditional claims for its use in folklore medicine in the treatment of different types of cancers and inflammatory
conditions. Through a bioactivity guided fractionation approach, the crude extract (FCC), resultant two organic fractions i.e. n-hexane and ethyl acetate (FCN and FCE, respectively) and an aqueous fraction (FCA) were tested for their anti-proliferative and cancer chemopreventive activity by employing different standard bioassays. In this investigation, it was demonstrated that F. cretica extract and fractions have anti-proliferative activity towards human breast cancer cells and it can exert cancer chemopreventive effects via NFκB inhibition, iNOS inhibition and QR induction. The most potent fraction was ethyl acetate (FCE) fraction followed by n-hexane fraction (FCN) in this study. Six compounds in total (FCEE1, FCEK2, FCEK3, FCEM4, FCEN5 and FCNC6) were isolated from potent fractions of this medicinal plant using repeated flash column chromatography. Structural elucidation was carried out through a series of spectroscopic experiments (1-D and 2-D NMR). Among these six compounds, one compound (FCEE1) is a triterpene acid, two compounds (FCEK2 and FCEN5) belong to the triterpene saponin class, one compound FCEM4 belongs to steroidal saponin class and two compounds (FCEK3 and FCNC6) belong to phytosterol class of chemical compounds. All the isolated compounds have known structures but these are isolated from this plant species for the first time. All the compounds were evaluated for their anti-proliferative activity against breast cancer cell lines (MCF-7 and MDA-MB-231), non-tumorigenic breast epithelial cell line (MCF-10A) and cervical cancer cell line (HeLa) by using sulforhodamine B (SRB) assay. Cancer chemopreventive potential of compounds was also estimated in different bioassays i.e. TNF-α activated nuclear factor-kappa B (NFκB) assay, inhibition of lipopolysaccharide (LPS)-activated nitric oxide (NO) production in murine macrophage RAW 264.7 cells (iNOS) assay, Aromatase inhibition assay and Quinone reductase 1 (QR1) induction assay. A serial dilution of eight concentrations (0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25,50 μ M) for each compound was tested. Purified compounds were able to reduce cell viability significantly in two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231). This activity was markedly reduced in normal mammary epithelial cells (MCF-10A) thus indicating selective toxicity between malignant and normal cells. Among compounds, in NFκB inhibition assay, FCEE1 showed maximum inhibition (IC 50, 5.26μM) while in iNOS inhibition assay, FCEM4 demonstrated best activity (IC 50, 0.38μM). For QR1 induction, FCNC6 was proved to be best compound (CD, 17.07μM). None of the compounds were active in inhibiting aromatase enzyme. Compound FCEE1, a triterpene acid was the most potent compound in this study followed by FCEM4. Elucidation and characterization of the cytotoxic mechanism was undertaken by cell cycle status, apoptosis and expression of transcription factors and their targets i.e. p53, p21, Bax, γ-H2AX and FOXO3a. Pure compound treatment was able to induce a time and dose-dependent decrease on cell viability of two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231), while demonstrating a markedly decreased cytotoxic effect of triterpene acid (FCEE1) and derivatives i.e. triterpene saponins (FCEK2 and FCEN5) on normal mammary epithelial cells (MCF-10A). Cell cycle arrest (expressed by sub-G1 peak) and apoptosis was induced in both MCF-7 and MDA-MB-231 cells. Overall all the purified compounds were more effective against estrogen receptor negative breast cancer (MDA-MB-231) cells. Loss of breast cancer cell viability is associated with induction of DNA double strand breaks which were detected in both cell lines by pure compounds’ treatment. In MCF-7 cells, expression of p53 by FCEE1 seems to induce upregulation of the CDK inhibitor, p21, and pro-apoptotic Bax. In current study, pure compounds’ treatment to human breast cancer cells induced double strand breaks to DNA associated with activation of DNA repair protein γ-H2AX. Compound FCEM4 treatment was able to induce FOXO3a expression in both MCF-7 and MDA-MB-231 cells. This suggests that activation of FOXO3a in the absence of functional p53 plays a vital role in induction of cytotoxicity. The results of this study suggest that FCEE1 is cytotoxic through p53 dependent as well as independent pathways while FCEM4 uses only p53 independent pathway.

CONCLUSION
F. cretica is a herbaceous plant which is commonly used in Pakistan as a treatment for a variety of ailments especially against breast cancer. In this investigation, it was demonstrated that crude extract and fractions of F. cretica have anti-proliferative activity towards human breast cancer cells and it can exert cancer chemopreventive effects via NFκB inhibition, iNOS inhibition and QR induction. The most potent fraction was ethyl acetate (FCE) fraction in this study. Six compounds (a triterpene acid, three saponins and two phytosterols) were isolated randomly from the potent fractions of the plant with the help of normal and reverse phase flash column chromatography. All these compounds have known structures but these are isolated from this plant species for the first time. All the compounds were evaluated for their anti-proliferative and cancer chemopreventive potential. Compound FCEE1, a triterpene acid was the most potent compound in this study followed by FCEM4. In NFκB inhibition assay, FCEE1 showed maximum inhibition while in iNOS inhibition assay, FCEM4 demonstrated best activity. For QR1 induction, FCNC6 was best compound. None of the compounds were active in inhibiting aromatase enzyme. Pure compound treatment was able to induce a time and dose-dependent decrease in cell viability of two phenotypically different breast cancer cell lines (MCF-7 and MDA-MB-231) while demonstrating a markedly decreased cytotoxic effect of triterpene acid and derivatives i.e. triterpene saponins on normal mammary epithelial cells (MCF-10A). Cell cycle arrest and apoptosis was induced in both MCF-7 and MDA-MB-231 cells. Loss of cell viability is associated with induction of DNA double strand breaks which was detected in both cell lines. In MCF-7 cells, expression of p53 by FCEE1 may have induced upregulation of the CDK inhibitor, p21, and pro-apoptotic Bax. In current study, pure compounds’ treatment to human breast cancer cells induced double strand breaks to DNA detected by expression of protein Ȗ-H2AX. FCEM4 treatment was able to induce FOXO3a expression in both MCF-7 and MDA-MB-231 cells. This suggests that activation of FOXO3a in the absence of functional p53 plays a vital role in induction of cytotoxicity. From the results described in this study, it is obvious that FCEE1 is cytotoxic through p53 dependent and it becomes effective in short time while FCEM4 uses both p53 dependent and independent pathways though p53 dependent pathway induction is slower than in case of FCEE1.

In summary, results from the present study are in agreement with the ethno-botanical or traditional use of F. cretica against cancer growth. The current research work has established that F. cretica is a rich source of glycosides and certainly possesses significant anticancer activity against breast cancer cells. Most of the triterpene glycosides induce apoptosis in tumor cells; as evident from the present results, so they are preferable drugs for the treatment of cancer, because the elimination of tumor cells by apoptosis is helpful in lowering the side-effects in patients by avoiding necrosis. In addition, this research work also emphasizes the specific activity of the glycoside from F. indica against hormone independent breast cancer cells (MDA-MB-231), reported to be more aggressive and difficult to treat due to growing resistance to available chemotherapy. Overall, the identification and characterization of isolated compounds from medicinal plants in the treatment of cancer, the very common “plague” of modern times, justify both the evolutional knowledge coming from pharmacognosy and its historical roots in ancient herbal medicine, as well as the tremendous possibility of a natural product-based drug discovery approach.

Source: http://prr.hec.gov.pk/jspui/bitstream/123456789/7138/1/Samreen_Saleem_Biochemistry_QAU_2015.pdf