Research On Extraction,purification And Antioxidant Activities Of Flavonoids From Honeysuckle Leaves

Lonicera japonica Thunb.is one of the most commonly used Chinese herbal medicines,it has anti-bacterial anti-inflammatory,heat-clearing and detoxifying,hepatoprotective cholagogue and other functions.Honeysuckle leaf is a by-product of honeysuckle,it is high yield,cheap and easy to collect,but not fully exploited.Honeysuckle leaves is rich in a variety of bioactive ingredients,which the highest content of flavonoids.Honeysuckle leaf flavonoids have a great activity on antioxidant,anticancer and antibacterial,therefore,the discussion on the utilization method of honeysuckle leaves has certain social and economic benefit.In this paper,the extraction,purification and antioxidant of flavonoids from Honeysuckle leaves were studied.The conclusions are as follows:?1?Firstly,the ultrasonic assisted extraction of flavonoids from honeysuckle leaves by ethanol was studied.The ethanol volume fraction,extraction temperature,liquid to material ratio and ultrasonic power were selected as the factors,the extraction rate of total flavonoids was taken as index.Optimization of ultrasonic-assisted extraction of flavonoids from honeysuckle leaves by simultaneous rotary combination design was studied.The results showed that the highest yield of flavonoids of 15.67%was achieved when the extraction was carried out with an ultrasonic power of 250 W using 60% ethanol as the extraction solvent at a solvent/solid ratio of 65:1?mL/g?at a temperature of 46?.?2?The purification characteristics and technology of flavonoids from honeysuckle leaves were studied.14 kinds of maeroporous resin were evaluated to carry out static and dynamic adsorption-desorption experiments and the NKA-? was selected as the ideal adsorbent.We concluded that the adsorption mechanism indicated that the process was better explained by pseudo-second-order kinetics;the fitting degree of adsorption isotherm and Langmuir and Freundlich function curve was very high;the optimum technology parameters are sample concentration of 1.5 mg/mL,sample pH of 3.0,flow rate of 2.0 BV/h,75 % ethanol for elution,elution velocity of1.0 BV/h.Under these conditions,the purity of flavonoids from Lonicera japonica Thunb.Leaves was increased 3.24 times to 78.74% compared with that before optimization.The purification process can remove most of the organic acids in the leaves by NKA-? resin,effectively enrich and purify the flavonoids from Lonicerajaponica Thunb.Leaves.?3?The antioxidant activity in vitro of purified flavonoids from honeysuckle leaves was studied.The total reducing power and the scavenging ability of hydroxyl radical,superoxide anion radical and DPPH free radicals were determined by using ascorbic acid as the positive control.Antioxidant tests showed that the honeysuckle leaf flavonoids possessed strong antioxidant ability,the purified flavonoids scavenging hydroxyl radical,superoxide anion free radical,DPPH radical capacity and reducing power were higher than that of the crude product,and it was equivalent to the ascorbic acid under the sufficient concentration.?4?To study the antioxidant capacity of flavonoids from Lonicera japonica Thunb.leaves and the protective effects of ginger oleoresin on alleviating oxidative damage induced by H₂O₂ in RAW 264.7 cells.RAW264.7 cells were cultured in vitro,and were injured by H₂O₂,the cell viability was measured by methyl thiazolyl tetrazolium?MTT?,the contents of malondialdehyde?MDA?,glutathione?GSH?,superoxide dismutase?SOD?and lactate dehydrogenase?LDH?in cell and cell culture were determined by kit method.Results showed that flavonoids from Lonicera japonica Thunb.leaves can protect H₂O₂-induced toxicity in RAW 264.7 cells by a dose-dependent manner,reduced the content of MDA in cell and cell culture medium;improved the activity of SOD and GSH content and increased intracellular LDH activity.?5?To study the effect of Honeysuckle leave flavonoids on antioxidation system in aging mice.The model of aging mice was established by subcutaneous injection of Dgalactose in the neck and back,the mice were fed with different doses of honeysuckle leave flavonoids.The changes of body weight and the indexes of liver,brain,kidney,spleen and heart were observed.Determination of total antioxidant capacity?T-AOC?,superoxide dismutase?SOD?,glutathione peroxidase?GSH-Px?,catalase?CAT?activity and malondialdehyde?MAD?,glutathione?GSH?content in serum and liver,brain,kidney and heart tissues.Results: Compared with the control group,the body weight and organ index of mice in model group decreased significantly,the serum and organs of T-AOC,SOD,GSH-Px,CAT activity and GSH content were decreased,MDA content increased,and most of the differences were significant?P < 0.05?,which indicated that the aging mice model was constructed successful.Compared with the model group,the body weight and organ index of each dose group of flavonoids wereincreased,T-AOC,SOD,GSH-Px,CAT activity and GSH content had different degrees of increase in serum and organ tissues,MDA content decreased,the high dose group had the most significant effect.We can concluded that Honeysuckle leave flavonoids can effectively improve the antioxidant capacity of aging mice,its antioxidant mechanism may be related to increasing the activity of antioxidant enzymes and the content of reducing protective substances,and reducing the content of lipid peroxide.