Extraction Of Total Flavonoids From Elderberry Leaves And Its Anti-Osteoporosis Effect

Sambucus Williamsii Hance is a Lonicerae elderberry plant,which is called Shujinshu,Maniaosao,Jiegucao,etc in Chinese.It is distributed in temperate and subtropical regions and has more than 20 species.The elderberry has strong adaptability and is not strict with the climate.It grows in Northeast,Southwest and North China,etc.Elderberry is widely used in promoting blood circulation,relieving phlegm and blood stasis,setting a broken bone,reducing swelling and relieving pain,etc.Modern pharmacology shows that elderberry is rich in chemical active ingredients,including phenolic acids,flavonoids,triterpenoids,lignins,sterols,etc.,which can be used for anti-bacterial,anti-inflammatory,heatclearing and detoxification,wind-dispelling and dampness-removing,swelling-relieving and pain-relieving.Elderberry is also used as a formulation,plaster,honey pill,decoction,etc.,in clinical production,which are widely used to treat fractures,bruises and osteoporosis.The aim of this study was to optimize the extraction process of flavonoids from elderberry leaves and investigate the purification methods,and then explore the anti-oxidation effect and anti-osteoporosis mechanism of total flavonoids from elderberry leaves.The total flavonoids were extracted by ethanol method.The extraction time,extraction temperature,liquid-to-material ratio,and ethanol volume fraction were analyzed by single factor level,and the best extraction scheme was determined by Box-Behnken test.In static adsorption experiments,the crude flavonoids of elderberry leaves were purified by AB-8 macroporous adsorption resin,D4020 macroporous adsorption resin,D101 macroporous adsorption resin,NKA-9 macroporous adsorption resin,S8 macroporous adsorption resin,and screened out the optimal purification resin.In dynamic adsorption experiments,the optimal conditions were obtained by screening six conditions including sample volume,sample loading rate,sample solution concentration,sample solution pH,eluent dosage,and eluent volume fraction.The anti-oxidation experiment in vivo was carried out by ethanol-induced mouse model of oxidative damage.The total flavonoids in the leaves of elderberry given by gavage to the mice were designed: low(80.5mg/kg),medium(161mg/kg),high(322mg/kg)and positive control VC.Malondialdehyde(MDA),reduced glutathione(GSH),and superoxide dismutase(SOD)in serum and liver of mice were determined.The protective effect of elderberry leaves flavonoids on oxidative damage of liver cells induced by alcohol was investigated by pathological analysis of liver tissue.In the anti-osteoporosis study,retinoic acid and streptozotocin were used to establish a mouse osteoporosis model.The total flavonoids in the leaves of elderberry given by gavage to the mice were designed: low(80.5mg/kg),medium(161mg/kg),high(322mg/kg)and positive control Vitamin D calcium tablets.Mouse calcium,phosphorus,ACP,ALP,TRAP,BGP content were detected and mouse femur pathology sections were made to discuss the anti-osteoporosis mechanism of elderberry leaf flavonoids.The test results were as follows:1.According to the Box-Behnken analysis in Design-Expert8.06 software,the order of influence of ethanol volume fraction(A),liquid-to-liquid ratio(B)and temperature(C)on the total flavonoids of the sapwood leaves was C> A> B.The optimum extraction conditions were as follows: extraction temperature 70 ° C,extraction time 60 min,ethanol volume fraction 70%,liquid-to-material ratio 70:1(mL: g),and the average yield of the three parallel tests was 7.101%.2.Through the static adsorption and desorption experiments of five macroporous adsorption resins,it was found that the purification effect of total flavonoids from elderberry leaves with AB-8 macroporous adsorption resin was best.The single factor screening test by dynamic adsorption of macroporous resin showed that the optimal conditions for dynamic desorption of total flavonoids from elderberry leaves were 50% ethanol eluent volume 40 mL,flow rate 1.5 mL/min,and sample solution concentration 1.2 mg/mL.The yield of total flavonoids in the purified elderberry leaves was 15.43 mg/g,which was 73% higher than that before purification.3.The results of antioxidant tests showed that the high and middle doses of total flavonoids from elderberry leaves significantly increased SOD and GSH levels(P<0.01),decreased MDA content(P<0.01),and eliminated free radicals in serum and liver,then achieve good anti-oxidation effect in the body.The pathological results of liver tissue showed that the low,medium and high doses of total flavonoids from elderberry leaves could effectively reduce the fat content of liver cells in mice and significantly improve the lysis of liver cells,indicating that the total flavonoids from elderberry leaves could reduce the accumulation of fat in liver tissue of mice,and alleviate the oxidative damage of alcohol on liver cells.Some papers showed that oxidative stress was one of the important causes of osteoporosis.This study showed that the total flavonoids of elderberry leaves had a good anti-oxidation effect,so it could prevent the occurrence of osteoporosis.4.The results of anti-osteoporosis test showed that in the model of osteoporosis in mice induced by retinoic acid,total flavonoids from elderberry leaves could significantly increase calcium and ALP levels in serum,decrease P,ACP,TRAP and BGP levels in serum,showing that elderberry leaves flavonoids could have a good therapeutic effect on osteoporosis caused by retinoic acid.In the study of streptozotocin-induced osteoporosis model in diabetic mice,flavonoids in elderberry leaves can significantly increase calcium,ALP levels of serum,decrease P,ACP,TRAP,BGP levels,thereby playing an anti-osteoporosis effect.Through the effects of the two different osteoporosis models of the total flavonoids of elderberry leaves,it could be found that the total flavonoids in the leaves of elderberry could have a good therapeutic effect on two kinds of osteoporosis,especially for the treatment of retinoic acid.Therefore,it was speculated that its anti-osteoporosis principle may be a substitute for estrogen,which plays a good role in anti-osteoporosis,but its mechanism remains to be further studied.