Purification and partial characterization of a Myofibril-Bound Serine Protease and its endogenous inhibitor from skeletal muscle of the ostrich

The ostrich is becoming an important source of meat for humans in developed and developing countries. This study was conducted to purify and characterize myofibrilbound serine protease (MBSP) and its endogenous inhibitor (MBSPI) from skeletal muscle of the ostrich. It is well documented that MBSP is tightly bound to myofibrils and its endogenous inhibitor has been purified from the same tissue of other studied mammalian species. Literature supports an association of MBSP and its endogenous inhibitor with the degradation of myofribrillar proteins, resulting in the softening of muscle that lead to the conversion of muscle into meat with the control of the inhibitor. MBSP was successfully dissociated from washed myofibrils by 40 percent ethylene glycol at pH 8.5. Following centrifugation, MBSP was partially purified in two chromatographic steps, namely Toyopearl Super Q 650S and p-aminobenzamidine-Agarose. On the other hand, MBSPI was fractionated from the sarcoplasmic fraction using 75 percent ammonium sulfate saturation, followed by centrifugation and partially purified by three chromatographic steps, namely Toyopearl Super Q 650S, Superdex 200 and HiTrap SP HR. Ostrich MBSP was physicochemically and kinetically characterized, while MBSPI was only physicochemically characterized. Ostrich MBSP revealed an Mr of 21 kDa, cleaving synthetic fluorogenic substrates specifically at the carboxyl side of arginine residues. Optimum pH and temperature of ostrich MBSP were 8.0 and 40˚C, respectively. Kinetic parameters (Km and Vmax values) were calculated from Lineweaver-Burk plots. The characteristics of ostrich MBSP were compared to the values obtained for commercial bovine trypsin in this study, as well as that obtained for MBSP from various fish species and mouse. The results suggest that ostrich MBSP is a trypsin-like serine protease, thereby confirming the existence of MBSP in ostrich skeletal muscle. Partially purified ostrich MBSPI (Mr 17 kDa) (one form) shares 100 percent identity to myoglobin from the same species, while 2 other forms of MBSPI (Mr values of 35 and 36 kDa) exhibited high sequence identity to glyceraldehyde 3- phosphate dehydrogenase (GAPDH) (76 percent) from human and rat

Efficacy of two medical plant extracts and metformin in the prevention of diet induced fatty liver

Non‐alcoholic fatty liver diseases (NAFLD) is manifested in the absent of alcohol abuse. This disease is the major cause of liver failure and death among adults and children worldwide, including South Africa. Its increasing prevalence urges the need of therapeutic intervention. The main objectives of this study were to investigate the following: (1) The effect of 38.9% high fat diet (HFD)‐induced insulin resistance and fatty liver in male Wistar rats, (2) The efficacy of aqueous extracts from Sutherlandia frutescens leaves and Prunus africana bark and metformin in the treatment of HFDinduced insulin resistance and fatty liver. Male Wistar rats were fed on HFD (the HF group) or normal rat chow (the LF group) for 12 weeks. Even though the HFD‐fed rats had developed insulin resistance by week 12, fatty liver developed by week 16. After week 12, the HF group was divided into four groups of 6‐7 rats each and three of those groups were gavaged with either 0.125 mg P. africana extract/kg bwt/day (the HF+Pa group) or 50 mg S. frutescens extract kg bwt/day (the HF+Sf group) or 16 mg metformin/ kg bwt/day (HF+Met group), while kept on the same diet for an additional of 4 weeks, to investigate whether two medicinal plant extracts and metformin can prevent HFD to induce fatty liver or not. After 16 weeks, the liver histological images revealed that the HF group developed fatty liver in the form of both microsteatosis and macrosteatosis. Fatty liver was confirmed by significant increased liver total lipid (TL) and activities of glucose‐6‐phosphate dehydrogenase (cG6PD) and xanthine oxidase (XO), mitochondrial NADH oxidase (mNOX) and by a decrease (P5% per liver weight in all treated groups. The present study demonstrates that these two plant extracts and metformin have different glucogenic and lipogenic effects from that presented by HFD alone when compared to the LFD alone. In conclusion, metformin and P. africana extract can attenuate HFD‐induced fatty liver without changing the dietary habits. Hence S. frutescens extract is less effective in the prevention of HFD‐induced fatty liver. A change in the dietary habits is recommended to be considered during the use of these three remedies in the treatment of HFD‐induced insulin resistance and fatty liver. All three treatments enhanced antioxidant capacity, and may improve insulin resistance and fatty liver mediated by the present HFD through different mechanism of actions in the liver

Antioxidant and hepatoprotective potentials of Bauhinia variegata seeds against ferric chloride-induced lipid peroxidation in chicken liver homogenate

Background: Iron accumulation in the liver is customary in pathological conditions associated with oxidative stress. Iron is also an essential element for organisms at its physiological levels. This study was aimed at investigating the antioxidant and hepatoprotective potential of acetone and methanol defatted seed extracts of Bauhinia variegata Linn (Leguminosae) against ferric chloride (FeCl3)-induced lipid peroxidation in chicken liver in vitro.Materials and Methods: Antioxidant capacity and hepatoprotective potential of acetone and methanol defatted seed extracts of B. variegata were evaluated using the established in vitro models, FeCl3-induced lipid peroxidation in chicken liver homogenate and thin layer chromatography-2,2-diphenyl-1-picrylhydrazyl (TLC-DPPH). Ascorbic acid, and acetone and methanol seed extracts of milk thistle (Silybum marianum) were used as antioxidant and hepatoprotective controls.Results: An overall of 80% acetone and absolute methanol defatted seed extracts of B. variegata [B. variegata acetone extract (BVAc) and B. variegata methanol extract (BVMe)] revealed to possess antioxidant and free radical scavenging capacity, which can protect the liver from FeCl3-induced lipid peroxidation in a concentration-dependent manner when compared with controls, ascorbic acid and extracts of milk thistle. At concentrations of 250 μg/mL and 300 μg/mL, thiobarbituric acid reactive species (TBARS) inhibitions were 80.5% and 97.6% for BVAc; and 84% and 98.7% for milk thistle acetone extract (MSAc). At concentration of 300 μg/mL, BVMe and milk thistle methanol extract (MSMe) TBARS inhibitions were 89.0% and 93.8%, respectively. These findings may confirm the presence of antioxidant compounds with hepatoprotective potential in both defatted seed extracts of B. variegata.Conclusion: The findings of this study suggest acetone and methanol extracts of B. variegata defatted seeds may serve as good sources of natural antioxidant and hepatoprotective agents.Keywords: Antioxidant, Bauhinia variegata, hepatoprotective, lipid peroxidation, seed

ANTIOXIDANT AND HEPATOPROTECTIVE POTENTIALS OF BAUHINIA VARIEGATA SEEDS AGAINST FERRIC CHLORIDE-INDUCED LIPID PEROXIDATION IN CHICKEN LIVER HOMOGENATE

Background: Iron accumulation in the liver is customary in pathological conditions associated with oxidative stress. Iron is also an essential element for organisms at its physiological levels. This study was aimed at investigating the antioxidant and hepatoprotective potential of acetone and methanol defatted seed extracts of Bauhinia variegata Linn (Leguminosae) against ferric chloride (FeCl3)-induced lipid peroxidation in chicken liver in vitro. Materials and Methods: Antioxidant capacity and hepatoprotective potential of acetone and methanol defatted seed extracts of B. variegata were evaluated using the established in vitro models, FeCl3-induced lipid peroxidation in chicken liver homogenate and thin layer chromatography-2,2-diphenyl-1-picrylhydrazyl (TLC-DPPH). Ascorbic acid, and acetone and methanol seed extracts of milk thistle (Silybum marianum) were used as antioxidant and hepatoprotective controls. Results: An overall of 80% acetone and absolute methanol defatted seed extracts of B. variegata [B. variegata acetone extract (BVAc) and B. variegata methanol extract (BVMe)] revealed to possess antioxidant and free radical scavenging capacity, which can protect the liver from FeCl3-induced lipid peroxidation in a concentration-dependent manner when compared with controls, ascorbic acid and extracts of milk thistle. At concentrations of 250 µg/mL and 300 µg/mL, thiobarbituric acid reactive species (TBARS) inhibitions were 80.5% and 97.6% for BVAc; and 84% and 98.7% for milk thistle acetone extract (MSAc). At concentration of 300 µg/mL, BVMe and milk thistle methanol extract (MSMe) TBARS inhibitions were 89.0% and 93.8%, respectively. These findings may confirm the presence of antioxidant compounds with hepatoprotective potential in both defatted seed extracts of B. variegata. Conclusion: The findings of this study suggest acetone and methanol extracts of B. variegata defatted seeds may serve as good sources of natural antioxidant and hepatoprotective agents

Antibacterial activity and in situ efficacy of Bidens pilosa Linn and Dichrostachys cinerea Wight et Arn extracts against common diarrhoea-causing waterborne bacteria

Abstract Background Bidens pilosa and Dichrostachys cinerea extracts were investigated for the antibacterial properties against waterborne diarrhoeagenic bacteria. Methods The plant materials were extracted using the direct and serial exhaustive methods using solvents of varying polarities, namely, hexane, dichloromethane, ethyl acetate, acetone and methanol. Qualitative phytochemical analysis and quantitative determination of total phenolic content of the leaf powders of the two plants were tested. The antioxidant activities of the plants were determined using the 2, 2-diphenyl-1-picrylhydrazyl method. The toxic effect of the extracts on C2C12 muscle cell line were assessed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method and the antibacterial activity was determined using the serial microbroth dilution. Results Methanol leaf extracts both plants had the highest yield in both direct and serial exhaustive extraction methods. Phytochemical profiling of the extracts displayed the presence of various secondary metabolites. The Benzene: ethanol: ammonia hydroxide solvent system showed a good resolution of chemical compounds in plant extracts from both plants. Most antioxidant compounds observed were developed in chloroform: ethyl acetate: formic acid and ethyl acetate: methanol: water solvent systems. All the bacterial species tested were sensitive to the effect of different extracts of both plant species, with E. coli being less sensitive to the effect of the extracts from D. cinerea. Following the simulated gastric fluid (SGF) treatment, a decrease in the antibacterial potency of the extracts was observed. No extract was toxic to the C2C12 muscle cell line. Conclusion The presence of the secondary metabolites and nontoxic effect of the two plants tested may affirm the medicinal value of these leaf extracts. Our results suggest that B. pilosa and D. cinerea contain constituents with antioxidant and antimicrobial activities, which could be used in the treatment of diarrhoea in a case where untreated surface water is used