| Chinese sea buckthorn(Hippophae thamnoides L.subsp.sinensis Rousi),is a subspecies of Hippophae rhamnoides L..In the Chinese Pharmacopoeia,it has the functions of invigorating spleen and eliminating digestion,promoting blood circulation stasis,relieving cough and expelling phlegm and other effects.As a medicinal and edible plant,sea buckthorn is rich in nutrients and bioactive components.Sea buckthorn is used as a medicine,and the raw material that is generally developed and utilized is its fruit.The leaves are usually discarded as wastes,resulting in great waste of resources.But in recent years,it has been found that the sea buckthorn leaves also contain a large number of active ingredients,such as flavonoids,polyphenols,polysaccharides,etc.,and also rich in protein and crude fiber,while flavonoids was deemed as an important index of sea buckthorn.Compared with other parts of sea buckthorn,the researches have confirmed that the content of flavonoids was the highest in the leaves.The sea buckthorn leaves have many pharmacological activities,such as preventing cardiovascular disease,lowering blood fat,anti-inflammatory and bactericidal.The safe and multi-effect medicinal effects have great potential for development,and it is of great significance to study the comprehensive utilization of sea buckthorn resources by sea buckthorn leaves.Therefore,the following four aspects of research were carried out in this paper:(1)The chemical constituents of leaves from Chinese sea buckthorn were analyzed by UHPLC-LTQ Orbitrap MS,and the mass spectrometry was summarized to establish a rapid analysis method for its chemical constituents;(2)The extraction method of flavonoids from Chinese sea buckthorn leaves was optimized by response surface methodology;(3)Purification of flavonoids from Chinese sea buckthorn leaves was carried out by macroporous adsorption resin;(4)The cell viability of melanoma B16cells under flavonoids from Chinese sea buckthorn leaves was determined by MTT assay to evaluate whitening activity;and established insulin resistance mode on HepG2 cells to preliminarily evaluate hypoglycemic activity of flavonoids.The details are as follows:1.This study is based on UHPLC-LTQ Orbitrap MS technology to analyze the chemical constituents in sea buckthorn leaves with a good separation of all the ingredient and optimizing MS conditions for stable components cleavage fragments.By collecting domestic and foreign literatures of flavonoids in sea buckthorn leaves,it was summarized for the molecular formula of the compounds of sea buckthorn leaves,and the exact molecular weight of the compound was determined by Xcalibar 3.0 software.Therefore,the mass spectrometry cleavage rules were summarized,and the effective chemical constituents in sea buckthorn leaves were quickly analyzed and fully characterized.2.Sea buckthorn leaves are rich in flavonoids and are the material basis for their various physiological activities.Ultraviolet spectrophotometry was used to determine the content of flavonoids in sea buckthorn leaves.The conditions of Chinese sea buckthorn leaves extraction were investigated by simple factor,and the optimal extraction conditions were selected.The response surface method was used to optimize the extraction process of flavonoids from the sea buckthorn leaves.3.Screening suitable macroporous adsorption resins and optimizing their adsorption-desorption conditions by the response surface method,and provide guidance for industrial extraction of refined sea buckthorn leaves.4.The sea buckthorn leaves were enriched by optimized extraction and purification methods,and their activities were preliminarily studied.The cell viability of melanoma B16 cells under different concentrations and time of the flavonoids was determined by MTT assay to evaluate the whitening activity,and insulin-induced HepG2 cells were used to establish insulin resistance model that was used to evaluate hypoglycemic activity of the flavonoids in Chinese sea buckthorn leavesThe results and conclusion are as follows:1.The study is aimed to analyze the chemical components in leaves of Chinese sea buckthorn by using UPLC-LTQ Orbitrap MS.The analysis was performed on a Waters acquity UPLC BEH-C18 column(2.1×50 mm,1.7?m).The mobile phase consisted of acetonitrile(A)and 0.1%formic acid(B)was used as gradient elute.The flow rate was 0.4 mLˇmin-1 gradient elution and column temperature was 30?,the injection volume was 4 ul.HESI was applied and operated in negative ion mode.Through the analysis of mass spectrometry data and with the help of literature data,a total of 26 compounds were detected and most of them were flavonoids.By using UPLC-LTQ Orbitrap MS method the main chemical compounds in leaves of Chinese sea buckthorn can be rapidly and accurately identified.2.Response surface methodology(RSM)was adopted to optimize the extraction conditions of total flavonoids from leaves of Chinese sea buckthorn.Based on single-factor tests,Box-Behnken center-composite experiment was carried out with four factors,including ethanol concentration,liquid-material ratio,extraction temperature and extraction time.RSM was used to determine the effect of prime factors on the yield of flavonoids.Result showed that the optimal extraction conditions were ethanol concentration 59%,liquid-material ratio 18:1,extraction temperature 79? and extraction time 125 min.3.The purification process of flavonoids from sea buckthorn leaves with macroporous resins was established by response surface methodology.The experimental results indicated:Compared with D101,X-5,DA201,HPD600,DM-301,DM-130,NKA-9 macroporous resin,AB-8 macroporous resin had good adsorption and desorption properties to the flavonoids.The combination of single factor and response surface experiments is used to obtain the optimal process conditions for macroporous resin purification.The dynamic adsorption conditions were as follows:the loading liquid concentration was 0.8 mg/ml,the flow velocity of loading liquid was 1.8 BV/h,and the ph value of loading liquid was 4.8.The dynamic desorption conditions were as follows:the ethanol concentration was 70%in elution,the elution dosage was 4.0 BV,and the flow velocity of elution was 1 BV/h.Under these conditions,the content of flavonoids in adsorption-desorption experiment of the macroporous resin is increased by 2.4 times.4.The effects of flavonoids from sea buckthorn leaves on the cell viability of B16 cells under different concentrations and time were analyzed by MTT assay.The results showed that sea buckthorn leaves flavonoids did not inhibit cell viability of B16 cells at low concentrations,but with the increase of concentration,the flavonoids had the inhibition of B16 cells growth in a time and dose dependent manner,and these groups were significantly different from the control group(p<0.01).The hypoglycemic activity of flavonoids from sea buckthorn leaves was studied.The effects of flavonoids,metformin and insulin on HepG2 cells were analyzed by MTT assay,and the suitable concentration range was selected,which were not higher than 200?g/ml,100?g/ml,20?g/ml.The effect of the flavonoids on glucose consumption in normal HepG2 cells,the results showed that the flavonoids can increase the glucose consumption of normal HepG2 cells(except for 25 ?g/ml),and compared with the control group,the results were significantly different(p<0.05,p<0.01).The effect of the flavonoids on the glucose consumption of insulin-resistant HepG2 cells,the concentration of insulin modeled at 10?g/ml,and compared with the model group,the concentration of the flavonoids at 50?g/ml,100?g/ml,200?g/ml produced a significant effect,(p<0.05,p<0.01). |
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