Study On Extraction And Biological Activity Of Flavonoid From Amomum Villosum

Amomum is the dried fruit of Zingiberaceae Amomum villosum Lour,Amomum villosum Lour.var.xanthioides T.L.Wuet Senjen and Amomum longiligulare T.L.Wu.According to the different producing areas,it can be divided into three types.Amomum villosum Lour is produced in Guangdong Province of China,Amomum villosum Lour.var.xanthioides T.L.Wuet Senjen and Amomum longiligulare T.L.Wu.are produced in Xishuangbanna and Yunnan.Amomum villosum Lour is a genuine medicinal material in Yangchun City,Guangdong Province.It is one of the eight important Chinese herbal medicines in Guangdong Province and one of the four southern medicines in China.Amomum villosum is rich in a variety of active ingredients,including flavonoids,polysaccharides,volatile oil and terpenes.It has antioxidant,anticancer,antibacterial,hypoglycemic and other biological activities.Amomum villosum resources are very rich in China.As a traditional Chinese medicine,Amomum villosum has high medicinal value and edible value.It is of great significance to study these active ingredients for new drug development and health food.In recent years,the use of volatile oil,polysaccharides,flavonoids and other functional components in Amomum villosum has become a research hotspot.However,the extraction rate of flavonoids in Amomum villosum is not high,and the pharmacological effects of antioxidant and anticancer are unclear.In this paper,the extraction conditions and stability of flavonoids from Amomum villosum were determined.The antibacterial,antioxidant and anticancer activities of flavonoids from Amomum villosum were studied.The main research results are as follows:（1）The optimum conditions of ultrasonic-microwave synergistic extraction of total flavonoids from Amomum villosum were as follows:the optimum conditions of ultrasonic-microwave synergistic extraction of total flavonoids from Amomum villosum were as follows:microwave power 300 W,ethanol concentration 50%,extraction time 30 min,solid-liquid ratio 1:29（g:m L）.Under these conditions,the yield of total flavonoids from Amomum villosum was 45.87±0.12 mg/g,and the content of total flavonoids increased to85.54±0.78 mg/g after purification,and the purity increased by 1.8 times.（2）Study on the stability of total flavonoids from Amomum villosum:The stability of total flavonoids from Amomum villosum.was studied from six aspects:light,temperature,p H,redox agent,food additives and metal ions.The results showed that the stability of total flavonoids from Amomum villosum.was relatively stable under the conditions of temperature25°C-70°C,p H 5-7 and darkness.The stability of total flavonoids from Amomum villosum was decreased by redox agent and some metal cations.0.2%and above chitosan and 0.15%CMC could maintain the stability of total flavonoids from Amomum villosum.Therefore,the preservation conditions of total flavonoids in Amomum villosum should be room temperature,weak acid,light avoidance,and avoid contact with redox agents,Fe²⁺,Fe³⁺,etc.（3）Study on the biological activity of total flavonoids from Amomum villosum:The free radical scavenging ability and cell antioxidant activity of total flavonoids from Amomum villosum were studied by in vitro chemical method and in vitro cell method.The results showed that total flavonoids had strong scavenging ability on DPPH·and ABTS free radicals,and the total reducing power was similar to that of vitamin C at the same concentration.Hep G2 cells were induced by 850μM H₂O₂to construct a cell oxidative damage model.In vitro cell antioxidant experiments were carried out at three concentrations of total flavonoids from Amomum villosum（100,300 and 500 mg/m L）.The results showed that total flavonoids from Amomum villosum could significantly improve the survival rate of oxidative damage Hep G2cells and the activity of superoxide dismutase in cell culture medium,and significantly reduce the activity of lactate dehydrogenase and the content of malondialdehyde,thereby protecting the damage of Hep G2 cells caused by oxidative stress.At the same time,by measuring the minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC/MFC）,it was found that the antibacterial ability was Staphylococcus aureus>Escherichia coli>Penicillium>Aspergillus niger.Staphylococcus aureus with the best inhibitory effect was selected to determine its bactericidal curve.The results showed that the number of colonies decreased by 3 lg values after 10 h treatment with MIC and 2MIC.（4）The anti-tumor activity of total flavonoids from Amomum villosum was studied by fluorescence inverted microscope and Hoechst 33342 staining.The results showed that the number of MGC 803 gastric cancer cells treated with total flavonoids from Amomum villosum decreased,cytoplasm retraction and fluorescence intensity increased,indicating that total flavonoids from Amomum villosum can promote the apoptosis of MGC 803 gastric cancer cells.MTT assay was used to detect the inhibitory effect of total flavonoids from Amomum villosum on MGC 803 gastric cancer cells.The results showed that the inhibitory effect of total flavonoids from Amomum villosum on MGC 803 gastric cancer cells was time-dependent and dose-dependent.Using Annexin V-FITC/PI double staining method,the results of flow cytometry analysis showed that the apoptosis rate of total flavonoids in Amomum villosum increased with the increase of concentration.When the concentration was 300μg/m L,the apoptosis rate was 28.89±0.75%（P<0.01）,indicating that total flavonoids in Amomum villosum may inhibit cell proliferation by promoting apoptosis of MGC 803 cells.