Study On Active Components And Its Product Of Hippophae Rhamnosides L. Leaves

Hippophae rhamnosides L.Leaves is a very precious medicinal plant with many effects and functions,which has a great effect on ecological construction and human health.However,there are relatively few studies on its by-product Hippophae rhamnosides L.leaves.This article has carried out research on flavonoids and polysaccharides in its main ingredients and their products,the adsorption resin was used to purify the flavonoids from Hippophae rhamnosides L.Leaves and optimal purification process was selected.And the biological activities before and after purification were compared;at the same time,the structural characterization and biological activity of Hippophae rhamnosides L.leaves polysaccharides were studied;finally,the brewing process of Hippophae rhamnosides L.leaves tea was explored.The main research conclusions are as follows:1、The flavonoids of Hippophae rhamnosides L.leaves were extracted by alcohol.Under the conditions of 70%ethanol and 70℃,reflux with a material-to-liquid ratio of 1:10 for 2 hours,and then reflux with a material-to-liquid ratio of 1:8 for 1.5 hours,the extraction rate of flavonoids is 6.47±0.18%,and the purity of flavonoids is 12.90±0.1%.The flavonoids were purified by AB-8 macroporous adsorption resin,and the content was measured by ultraviolet method.The adsorption rate,desorption rate and recovery rate were investigated;Based on single factor experiment and analytic hierarchy process,Box-Bohnken Design was used to study the effects of ethanol concentration,sample loading concentration and elution flow rate on the purification of flavonoids.The optimal purification process was obtained:ethanol concentration of 82.95%,sample loading concentration of 0.09 mg/m L,elution flow rate of 1.71 m L/min,the purity of the obtained flavonoids is 75.17±1.6%.2、Hippophae rhamnosides L.leaves polysaccharides were extracted by water,the extraction temperature was 90℃,the material-to-liquid ratio was 1:60 g/m L,and the extraction time was 4 h.The extraction rate of polysaccharide is 8.06±0.16%,and the purity of crude polysaccharide is about 16.85±0.22%.After decolorization by cotton-like DEAE,the purity was42.06±0.55%.The obtained crude polysaccharide was separated with DEAE-52,and eluted with distilled water,0.1,0.2,0.3,0.4 and 0.5 mg/m L Na Cl solution at a flow rate of 1 m L/min to get HRLLP-1,HRLLP-2,HRLLP-3,HRLLP-4,HRLLP-5,HRLLP-6 six polysaccharide components.Using infrared,high-efficiency liquid to analyze the structure of monosaccharides,infrared spectroscopy showed that each component contained-OH,-CH₂groups,HRLLP-1,HRLLP-2,HRLLP-4,HRLLP-5,and HRLLP-6 contained uronic acid structure,and it could be seen that the uronic acid content of each component was different.Monosaccharides were composed of fucose,glucosamine,ribose,xylose,mannose,galacturonic acid,glucuronic acid,rhamnose,arabinose,glucose and galactose,of which glucose accounted for the largest proportion.The tested monosaccharide composition is richer.Its molecular weight ranged from1.77×10⁵Da to 6.0×10⁵Da.Congo red experiments showed that HRLLP-2,HRLLP-4,HRLLP-5,and HRLLP-6 had a triple-helical conformation,while HRLLP-1 and HRLLP-3 did not have a triple-helical structure.3、Study on antioxidant activity of flavonoids from Hippophae rhamnosides L.Leaves before and after purification.The results were the determination of the ability to scavenge DPPH free radicals:IC₅₀before purification=0.016±0.0003 mg/m L,IC₅₀after purification=0.002±0.0001 mg/m L;determination of hydroxyl radical scavenging ability:IC₅₀before purification=1.501±0.0089 mg/m L,IC₅₀after purification=1.131±0.0077 mg/m L;the absorbance of reducing force increased with the increase of sample concentration.The results showed that the antioxidant activity of the purified samples was higher than that before purification.4、The research on the antioxidant properties and immune activities of the crude polysaccharides from Hipopphae rhamnoides L.leaves,showed that it had scavenging effects on DPPH free radicals,ABTS free radicals and hydroxyl free radicals.The IC₅₀of DPPH was0.0034±0.0002 mg/m L,the IC₅₀of ABTS was 0.0032±0.0001 mg/m L,and the IC₅₀of hydroxyl radical was 5.3007±0.012 mg/m L;The immune activity of the six fractions after separation was detected by the proliferation ability,phagocytic ability and NO secretion of RAW264.7 cells by polysaccharides.Among them,HRLLP-1,HRLLP-2,HRLLP-3,HRLLP-4,and HRLLP-5 at low concentrations（25μg/m L）had the largest proliferation of macrophages to130.86%,127.08%,123.69%,135.08%,131.72%,respectively;at a concentration of 50μg/m L,HRLLP-2 had the strongest effect on phagocytosis of neutral red by macrophages,and its phagocytic index was 3.50;at a concentration of 800μg/m L,the maximum amount of NO secretion by HRLLP-6 was 5.34±0.68μmol/L.The results showed that the six HRLLPs fractions all showed different degrees of immunomodulatory activity.5、The research and development product is Hipopphae rhamnoides L.leaves tea,and the relationship between its brewing process and activity was investigated.The results showed that the active ingredients were polysaccharide 15.56±0.012%,flavonoids 2.51±0.25%,caffeine3.06±0.31%,and tea polyphenols 18.45±0.72%,the optimal brewing process of Hipopphae rhamnoides L.tea was as follows:brewing time 6 min,brewing temperature 80℃,solid-liquid ratio 1:49（g/m L）.At the same time,HRLL tea had the highest antioxidant activity,and the scavenging rate of DPPH free radical was 83.25±1.75%.The research results in this paper showed that Hipopphae rhamnoides L.leaves are a highly effective antioxidant and immunomodulator,and its related product Hipopphae rhamnoides L.tea also has good biological activity,which can be further applied to the food and pharmaceutical industries to provide for the efficient utilization of seabuckthorn and further research.