Bioassay-Guided Fractionation Of Antioxidants From Crataegus -Monogyna (Common Hawthorn) And Its Role In Treating Cardiovascular Disease Using H9c2 Rat Myoblast Cardiac Cell Line
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Crataegus monogyna plants are well known for their medicinal property. Extraction and identification of compounds rich in antioxidant present in C. monogyna is the main focus of this research paper. Firstly, solid-liquid extraction of different plant parts (leaves, flowers and fruits) of C. monogyna using different solvents (aqueous, ethanol and methanol) was conducted to identify the suitable plant part and solvent type that contributed to the maximum yield of total phenolic content (TPC) and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)). Upon extraction, liquid-liquid partition using solvent with different polarities (water, n-hexane and chloroform) was conducted prior to fractionation of antioxidant compounds. Bioassay-guided fractionation of antioxidant compounds from the most antioxidant-rich plant part of C. monogyna was conducted using chromatographic methods and the antioxidant compounds were identified via Fourier transform infrared spectroscopy (FTIR) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis. Lastly, the cardiac activity of the most antioxidant-rich fraction was investigated using the H9c2 rat myoblast cardiac cell line. As results, solid-liquid extraction of different plant parts of C. monogyna using different solvents revealed that the fruit of C. monogyna extracted with ethanol had the highest TPC (0.070 mg GAE/g), antioxidant activity (DPPH 90.35% and ABTS 71.37%) and crude extract yield (0.49±0.05g). Bioassay-guided fractionation of this extract afforded twelve fractions (F1 to F12) and F9 presented the highest antioxidant values (DPPH 46.14% and ABTS 45.06%) and TPC (28±0.002 mg GAE/g). GC-MS/MS profiling of F9 revealed eleven antioxidant compounds and five major compounds as 2-(3,4 dihydroxy phenyl)-3,5,7-trihydroxychromen-4-one (19.46%), 1H-1,2,3,4-Tetrazol-5-amine,1-ethyl-N-[(1-methyl-1H-pyrrol-2-yl)methyl]-) (11.24%), benzenesulfonamide, N-[[5-(aminomethyl)-2-furanyl]methyl]- (8.21%), 4-Nonylphenol (8.01%) and 1,3,2-Dioxathiolane-4-methanol, 2-oxide (7.49%). The findings of current research conclude that the antioxidant compounds present in the most antioxidant-rich plant part of C. monogyna (fruit) are polar in nature and are majority phenols. Fraction F9 shows an effective cell viability percentage (99.5%) against H9c2 cells on a dose-dependent manner, suggesting that C. monogyna ethanol fruit extract possesses therapeutic potential against rat myoblast cells. In conclusion, C. monogyna fruit is an excellent alternative of natural antioxidant.