Study On Extraction,Isolation And Biological Activity Of Flavonoids From Acacia Mearnsii Leaves

Acacia mearnsii Leaves lack suitable processing methods and are typically discarded in large quantities,then deposited as waste and harbor pests and diseases.Hence,developing strategies to utilize A.mearnsii leaves is important for economic value and environmental benefits.This thesis systematically investigated the optimal extraction conditions and purification methods of the flavonoids from A.mearnsii Leaves,and then the antioxidant,hypoglycemic and antimicrobial activities of the flavonoids from A.mearnsii leaves were explored to provide basic data for the development and utilization of A.mearnsii leaves.The main research results are as follows:（1）By analysis of chemical composition,it was determined that A.mearnsii Leaves contained amino acids,proteins,sugars and glycosides,organic acids,phenols and tannins,flavonoids,anthraquinones,cardiac glycosides,steroids,alkaloids,coumarins,lactones,and without saponins,volatile oils.The total polyphenol content of A.mearnsii leaves was 23.35%,the total flavonoid content was 2.10%,the proanthocyanidin content was 0.20%and the alkaloid content was 1.32%as detected by the soxhlet extraction method.The extraction solvents of total flavonoids from A.mearnsii leaves were studied by ultrasound-assisted extraction method,and the best extraction solvent was 80%methanol.Through the single factor combined with the response surface test,it was concluded that the influence degree of each factor on the yield of total flavonoids was in the following order:solid-liquid ratio>ultrasonic temperature>ultrasonic time,and the interaction between solid-liquid ratio and ultrasonic temperature was good.In addition,the optimal process conditions for ultrasound-assisted extraction of total flavonoids from A.mearnsii Leaves were as follows:80%methanol,solid-liquid ratio 1:41（g:mL）,ultrasonic time 78 min,ultrasonic temperature 57℃,the total flavonoids yield was 1.91%.When ultrasonic extraction times were two times,the yield of total flavonoids reached 2.05%,and the extraction efficiency was more than 90%.（2）The extracts of A.mearnsii leaves were crudely fractionated by solvent partition to obtain the petroleum ether layer（L1）,dichloromethane layer（L2）,ethyl acetate layer（L3）and water layer（L4）in turn.The L3 with good biological activity was separated by AB-8 macroporous adsorbent resin and eluted by ethanol solution with different concentrations to obtain fractions Fr1～Fr7.The easily separated components Fr3（20%ethanol elution）and Fr4（30%ethanol elution）were purified by Sephadex LH-20 and eluted with different concentrations of methanol solution to obtain fractions BFr1～BFr5.The BFr3 fraction（40%methanol elution）,which was easily separated and had the highest flavonoid content（51.8%）,was subjected to a final preparative high performance liquid chromatograph（Prep-HPLC）purification.Compound W3 with 98.4%purity and compounds W1 and W2 with>95%purity were obtained in yields of 7.3,1.8 and 0.6 mg/g crude extracts,respectively.The isolated products were structurally characterized by HPLC,ultraviolet-visible absorption spectrometry,mass spectrometry,nuclear magnetic resonance technology.Compound W1 was deduced to be myricetin-3-O-β-D-glucoside,compound W2 was myricetin-3-O-arabinoside and compound W3 was myricetin-3-O-α-L-rhamnoside（myricitrin）.（3）In vitro antioxidant test revealed that L4 had significant scavenging ability against ABTS radicals,but showed moderate antioxidant activity against DPPH radicals and in the FRAP method,while L1 and L2 showed weak antioxidant activity in all three assays.At the concentration of 0.32mg/mL,the scavenging ability of the three flavonoids and L3 against DPPH radicals was comparable to that of vitamin C（control）.At the concentration of 0.08 mg/mL,the three flavonoids and L3 had significant scavenging ability（close to 100%）against ABTS radicals.In addition,the three flavonoids and L3 have strong Fe³⁺reducing ability and are potential antioxidants.In vitro hypoglycemic activity study revealed that L3,L4 and compound W2 showed moderate inhibitory activity againstα-glucosidase andα-amylase,while L1 and L2 showed weaker inhibitory activity againstα-glucosidase andα-amylase.The IC50 values of compounds W1,W3 and acarbose（control）were 0.104,0.074 and 0.102 mg/mL forα-glucosidase and 0.286,0.176 and 0.204 mg/mL forα-amylase,respectively.It can be seen that compounds W1 and W3 have good hypoglycemic activity and are potential type Ⅱ diabetes inhibitors.In the antimicrobial activity experiment,the four extracts and compound W3 did not significantly inhibit in vitro F.graminearum,S.sclerotiorum,Botryosphaeria dothidea and in vitro Botryosphaeria dothidea at the sample concentration of 50mg/L,but the four extracts showed some inhibition effect on in vitro Phytophthora infestans.The antibacterial activity of nanocellulose gel loaded with azoxystrobin,loaded with azoxystrobin and compound W3 was compared.It was found that compound W3 can promote the inhibitory effect of azoxystrobin against the above F.graminearum,S.sclerotiorum,Botryosphaeria dothidea and Phytophthora infestans.In conclusion,significant amounts of discarded A.mearnsii leaves are rich in flavonoids,which can be used as a source of myricitrin,myricetin-3-O-β-D-glucoside and myricetin-3-O-arabinoside,thus providing more adequate material for the production of antioxidants and type II diabetes inhibitors and microbial inhibitors.This thesis is important to promote the rational utilization of the world’s A.mearnsii resources.