| Oxidation is a metabolic process that provides energy for essential cell activities. In normal physiological condition, oxygen derived free radicals are produced in this process, such as O2-·,·OH and so on, which were commonly known as reactive oxygen species (ROS). In our living systems, varieties of antioxidants play an important role in combating ROS, such as the effect of oxidoreductase and non-enzymatic compounds. However, in pathology state, the defense systems would become over-whelmed during a metabolic derangement due to an imbalance caused by excessive generation of ROS or a diminished antioxidant capacity. Excessive ROS are harmful for cells, which lead to various impairments, such as cancer, aging, cirrhosis, rheumatoid arthritis, diabetes, as well as neurodegenerative diseases. A large number of studies have shown that taking some antioxidants or free radical scavengers reasonably could effectively reduce the damage to the body and prevent such the happening of the diseases. In the last century, some synthetic antioxidants such as butylated hydroxytoluene(BHT), propyl gallate(PG), butylated hydroxyanisole(BHA), and tertiary butylhydroquinone(TBHQ) were widely used in food industry, but its safety was called into question, while natural source of antioxidants tend to be favored for its wide source, high security, strong antioxidant activity. Therefore, screening of natural antioxidants has important realism meaning.This paper gave a general review of the general situation of resource, chemical constitutes and pharmacological actions of flavonoids of persimmon leaves. The purified flavonoids was subjected to identify the presence and content of quercetin, and kaempferol. We also studied the antioxidant ability, free radical scavenging capacity of flavonoids of persimmon leaves (PLF) and ethyl acetate fraction (EAF), as well as the hepatoprotective effect of EAF.The experiment was divided into four parts:1. Extraction and Fractionation of flavonoids of persimmon leaves:Samples powder is 80 g, the concentration of ethanol is 70%, the extraction temperature is 85℃, the ratio of solid to liquid is 1:15, the same procedure was repeated two times, the extraction time were 4 h and 2 h, respectively. Then extracts were combined and centrifugated at 6000 r/min for 10min. The supernatants were evaporated to form a syrup at 50℃. The syrup was partitioned with equal volume of petroleum, chloroform, ethyl acetate and n-butanol, ranging from low polar to hight polar, for three times, respectively. The petroleum fraction was deserted and other fractions were then removed under reduced pressure and dried in oven at 50℃. The next, extraction yield, flavonoid content and purity of chloroform fraction, ethyl acetate fraction, n-butanol fraction and surplus fractions was determined.2. EAF with the highest purity (81.06%) was subjected to HPLC to identify the presence and content of quercetin, and kaempferol. The high-performance liquid chromatographic system equipped fitted with a SinoChrom ODS-BP analytical column (250mm×4.6 mm I.D,5μm particle size). The gradient mobile phase consists of (A) MeOH and (B) 0.4% phosphoric acid with the flow rate of 1mL/min. The elution program involved a linear gradient from 35 to 60% A in B for 30 min, detection was done at wavelength of 360 nm and the column temperature was room temperature and the injection system used was a 20μL sample loop.3. Comparative antioxidant activity of PLF and EAF from persimmon leaves. The antioxidant activity of PLF and EAF were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH·) radical, NBT-NADH-PMS system, Fenton system assays, the reducing power and lipid peroxidation were also measured.4. Protective effects of EAF against tetrachloride-induced hepatotoxicity in mice. Mice were administered intragastrically with EAF at different doses per day for 7 days, one time a day, and three hour after the final treatment, the mice were treated with 0.1%CCl4 [(20 mg/kg body weight, intraperitoneally (i.p.) dissolved in corn oil (0.1%, v/v)]. Then, the therapeutic effects of EAF on the model were evaluated by the activity determination of alanine aminotransferas (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP) in serum, superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and the content of malondialdehyde (MDA) in liver, and the changes of hepatic pathohistology.The main results were as follows:1. Ethanol extract was prepared from persimmon leaves (80 g) and the obtained dried extract was 18.67g (PLF), so the extraction yield is 23.34% and the content of flavonoids was 4.96%, the purity of flavonoids was 22.17%. After liquid-liquid partition of PLF, the sequence of extraction yields of fractions were in the decreasing order of SF (10.53%)> BF (3.00%)> CF (2.94%)> EAF (1.53%), the content of flavonoids were in the order of EAF (1.24%)> BF (1.02%)> SF (1.00%)> CF (0.39%). The most interesting outcome is the purity of flavonoids, which was in the order of EAF (81.06%)> BF (34.10%)> CF (13.10%)> SF (9.46%).2. The linearity was in the range of 0.04-0.60μg (n= 6) for quercetin and kaempferol, and all the correlation coefficients were 0.9981 (n= 6). The average recoveries (n= 6) for quercetin and kaempferol were 99.47%(RSD= 0.903%) and 102.7%(RSD= 0.469%), respectively. The content of quercetin and kaempferol in EAF was 97.52±2.30 mg/g and 103.6±3.73 mg/g, respectively.3. Comparative antioxidant activity of PLF and EAF. The results showed that EAF after purification had markedly antioxidant ability several antioxidant methods in vitro than PLF. The IC50 of PLF on scavenging DPPH·, O2-·,as well as·OH was 15.9μg/mL,59.0μg/mL,8.35μg/mL, respectively. In addition, similar results with free radical scavenging were showed in reducing power and inhibition of lipid peroxidation generally. EAF exhibited good inhibition activities against the lipid peroxidation (IC50 was 82.6μg/mL).4. EAF has potential to exert curative effects against liver injury. The results indicated that different doses of EAF significantly lowered ALT, AST, AKP levels in serum and MDA level in liver along with the restoring of hepatic enzymes like SOD, GPx, and CAT against CCl4 treated mice. The pathological changes were also significantly improved in the mice of treating group. |
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