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Title: Phytochemical screening and evaluation of biological activities of Acacia hydaspica (R. Parker).
Authors: Afsar, Tayyaba
Keywords: Natural Sciences
Issue Date: 2015
Abstract: The focus of current research was to evaluate pharmacological ability of Acacia hydaspica methanol extract (AHM) and its derived fractions. Qualitative screening of methanol extract demonstrated the occurrence of terpenoids, anthraquinones, coumarins, cardiac glycosides, saponins, flavonoids, tannins, phlobatannins and alkaloids. GCMS chemical fingerprinting of AHM reveals the presence of 30 different chemical constituents belonging to diverse classes, owing enormous biological activities. HPLC analysis of AHM and various fractions reveals the presence of four well known standards; catechin, gallic acid, rutin and caffeic acid in AHM in varying concentrations. Catechin and Gallic acid were detected in both AHE and AHB while myrecitin was detected only in AHE. Mineral analysis indicated the existence of various macro and micromineral elements in A. hydaspica. AHB, AHE and AHM demonstrated high level of total flavonoid and phenolic content and a noteworthy correlation with the EC50 values was determined for the quenching of DPPH radical, hydroxyl radical, hydrogen peroxide radical, ABTS radical, iron chelating, β-Carotene bleaching inhibition and anti-lipid peroxidation. Significant anti-hemolytic, antimicrobial, cytotoxic actions were demonstrated by AHM and its AHE and AHB fractions. AHM and its fraction AHE reveal marked antipyretic, anti-inflammatory, analgesic and antidepressant potential. The in vivo studies indicated that AHE fraction of A. hydaspica possessed potent chemoprotective ability against cisplatin and doxorubicin induced toxicity in rats. Treatment of rats with AHE markedly improved the serum biomarkers of organ toxicity and tissue antioxidant status by significantly ameliorating the oxidative tissues markers enzymes levels near to control. Histopathological studies of different organs verify the biochemical observations. A. hydaspica was subjected to bioassaydirected fractionation which led to the isolation of three active antioxidant, antimicrobial and cytotoxic compounds from AHE and one active compound from AHB fraction besides active isolates. The compound structures were interpreted by ESI-MS and NMR spectroscopic analysis. Three AHE compounds were identified as 7-O-galloyl catechin (GC), (+) catechin (C), methyl-gallate (MG) while catechin-3- gallate (CG) was extracted from AHB fraction. In antimicrobial testing, MG was the most active compound. Isolated compounds GC, CG and MG showed more potent activity against DPPH, hydroxyl radical, nitric oxide radical, showed highest xviii antioxidant index and FRAP (649.5±1.5 μM Fe (II)/g) potency compared to standard reference rutin and gallic acid. In vitro models of prostate (PC-3) and breast (MDA-MB-231) cancer cells were used to evaluate the anticancer potential of isolated compounds (AHCs). AHC resulted in specific effects against tested prostate and breast cancer cell lines. GC, C, CG and MG inhibit cell proliferation in PC-3 cells in a dose dependent manner, whereas CG and MG negatively affected MDA-MB-231 cell growth. Compounds induce cell death via suppressing various signal transduction pathways that regulates cell proliferation and survival. Chromatin condensation, cell shrinkage and apoptotic bodies were observed by phase contrast microscopy. Compounds significantly inhibited cell survival and colony growth in both cell lines. Staining with acridine orange, ethidium bromide, propidium iodide and DAPI demonstrated that cell death occurred at least partly through induction of apoptosis in both PC-3 and MDA-MB- 231 cells. GC, C, CG and MG repressed the expression of anti-apoptotic proteins Bcl- 2, Bcl-xL and survivin in dose and time dependent manner, which futher validate the apoptotic effect of isolated compounds. Further analysis of signaling pathways indicated that compounds treatment induced a dose and time dependent suppression of JAK2, NFκB, p-Akt (Ser473and Thr308), NFκB p65 P-Ser 529, phospho-IκB Ser32/Ser36, p-ERK1/2 (Thr202/Tyr204) in PC-3 cells. In MDA-MB-231 cells, CG and MG treatment significantly suppressed the expression of CK2α, PI3-K and JAK2. Expression of NFκB total protein and its phosphorylation at p65 P-Ser529 was significantly inhibited following treatments. Furthermore, Bcl-xL, Survivin and xIAP expression was also inhibited in MDA-MB-231 cells in a time and dose dependent manner. These findings provide strong indication that A. hydaspica compounds may be favorable therapeutic candidates against highly invasive triple negative breast cancer and androgen insensitive prostate cancer cells.
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