Method for separating and purifying Yunnan hippophae rhamnoides crude flavone

Abstract

The invention relates to the technical field of flavone separation and purification, in particular to a method for separating and purifying Yunnan sea buckthorn crude flavone. A high-speed counter-current chromatography and a preparative liquid chromatography are combined to separate and purify the Yunnan sea-buckthorn crude flavone, and the method comprises the following steps of: separating the Yunnan sea-buckthorn crude flavone by using normal hexane, ethyl acetate, methanol and water in a volume ratio of 1: 9: 1: 9 as well as ethyl acetate, n-butyl alcohol and water in a volume ratio of 4: 1: 5 as a solvent system and using the high-speed counter-current chromatography to separate the Yunnan sea-buckthorn crude flavone, the isorhamnetin-3-O-glucoside, the isoquercitrin and the rutin are obtained; and then, purifying the unseparated effluent through preparative liquid chromatography to obtain the kaempferol and the quercetin. According to the method, the high-speed counter-current chromatography and the preparative liquid chromatography are combined, so that the problems of long separation period and low purification efficiency of the traditional column chromatography are solved, and the aim of quickly separating and purifying various flavone compounds from the Yunnan sea-buckthorn crude flavone is fulfilled.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of flavonoid separation and purification, and particularly relates to a method for separating and purifying crude flavonoids from Hippophae rhamnoides Linn. var. yunnanensis. BACKGROUND

[0002] At present, there are a large number of studies on Hippophae rhamnoides at home and abroad, but the nutritional value and medicinal value of Hippophae rhamnoides cannot be fully developed and utilized. Hippophae rhamnoides contains various active ingredients, such as flavonoids, terpenes, polysaccharides and organic acids. Flavonoids have various physiological functions, such as antioxidant, anticancer, antitumor, antimicrobial and antibacterial effects. Therefore, the development and comprehensive utilization of Hippophae rhamnoides have broad application prospects.

[0003] Hippophae rhamnoides Linn. var. yunnanensis is a precious plant, which contains rich active ingredients such as flavonoids, vitamin C, organic acids and polysaccharides. With the development of extraction technology, various advanced auxiliary extraction technologies have emerged, such as microwave extraction, ultrasonic extraction method and enzyme extraction technology, which have been applied to the extraction of flavonoids. Compared with the conventional solvent extraction method, the ultrasonic extraction method can accelerate the release speed of flavonoids in plant cells; enzymes can act on the cell wall of plant cells, so that the dense structure is destroyed, which is beneficial to the dissolution of flavonoids; microwave-assisted extraction is to use microwave to selectively heat the intracellular components, which leads to changes in cell structure. Compared with the above several extraction methods, the microwave-assisted extraction method has the advantages of high extraction efficiency, energy saving and easy operation.

[0004] In recent years, the microwave-assisted extraction method has gradually become a popular research method due to its simple equipment, environmental friendliness and high efficiency in shortening the extraction period, which opens up a new perspective for the extraction of active ingredients of Hippophae rhamnoides. However, in the subsequent separation and purification of active ingredients of Hippophae rhamnoides, the existing technology still faces many challenges. Although the above advanced extraction technologies have obtained crude extracts rich in flavonoids and other active ingredients, high-purity separation and accurate identification of these components still need to break through the bottleneck of traditional methods. At present, the separation and analysis of chemical components of Hippophae rhamnoides fruits mainly adopt traditional column chromatography. This method relies on the repeated equilibrium of stationary phase and mobile phase, which leads to a long separation period, and is limited by factors such as packing performance and sample size, so the purification efficiency is low. In addition, traditional column chromatography has limited resolution for components with similar polarity in complex samples, which cannot meet the demand for preparation of high-purity monomer compounds, and further restricts the in-depth development and pharmacodynamic mechanism research of active ingredients of Hippophae rhamnoides. SUMMARY

[0005] In view of the problems in the prior art, the application provides a method for separating and purifying crude flavonoids of Hippophae rhamnoides Linn var. yunnanensis, which separates and purifies the crude flavonoids of Hippophae rhamnoides Linn var. yunnanensis by combining high-speed counter-current chromatography with preparative liquid chromatography, and identifies the monomer compounds by comparing with standard products by HPLC, LC-MS and HPLC area normalization purity analysis, so as to solve the problems of long separation period and low purification efficiency of the traditional column chromatography in the prior art, and achieve the purpose of efficiently and quickly separating and purifying various flavonoid compounds from the crude flavonoids of Hippophae rhamnoides Linn var. yunnanensis.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the application is as follows: The first object of the application is to provide a method for separating and purifying crude flavonoids of Hippophae rhamnoides Linn var. yunnanensis, which combines high-speed counter-current chromatography with preparative liquid chromatography, and comprises the following steps: The high-speed counter-current chromatography is used to separate the crude flavonoids of Hippophae rhamnoides Linn var. yunnanensis by taking n-hexane, ethyl acetate, methanol and water as the solvent system, and the volume ratio is 1:9:1:9; the effluent of 35min-44min and the effluent of 46min-60min are collected respectively; the effluent of 35min-44min and the effluent of 46min-60min are freeze-dried respectively to obtain isorhamnetin-3-O-glucoside and isoquercitrin.

[0007] The high-speed counter-current chromatography is used to separate the crude flavonoids of Hippophae rhamnoides Linn var. yunnanensis by taking ethyl acetate, n-butanol and water as the solvent system, and the volume ratio is 4:1:5; the effluent of 62min-72min is collected; and the effluent of 62min-72min is freeze-dried to obtain rutin.

[0008] The unseparated effluent of the above-mentioned solvent system of n-hexane, ethyl acetate, methanol and water is concentrated; the concentrated unseparated effluent is twice separated by preparative liquid chromatography according to the following conditions: the chromatographic column is Agilent 5 Prep-C18 column, and the mobile phase is 0.1wt% phosphoric acid aqueous solution-acetonitrile; the effluent components are received according to the chromatographic peaks; and each effluent component is concentrated and dried to obtain kaempferol and quercetin.

[0009] Preferably, in the high-speed counter-current chromatography of the solvent system of n-hexane, ethyl acetate, methanol and water, the flow rate is 3.5mL / min-5.5mL / min, the sample injection amount is 100mg-400mg, and the rotation speed is 850rpm-1050rpm.

[0010] Preferably, in the high-speed counter-current chromatography of the solvent system of ethyl acetate, n-butanol and water, the flow rate is 3mL / min-5mL / min, the sample injection amount is 100mg-600mg, and the rotation speed is 850rpm-1050rpm.

[0011] Preferably, the detection wavelength of the high-speed counter-current chromatography and the preparative liquid chromatography is 254nm, 280nm and 330nm.

[0012] Preferably, in the high-speed counter-current chromatography, the upper phase of the solvent system is the stationary phase, and the lower phase is the mobile phase.

[0013] Preferably, in the 0.1wt% phosphoric acid aqueous solution-acetonitrile, the volume ratio of the 0.1wt% phosphoric acid aqueous solution to the acetonitrile is 60-80:20-40.

[0014] Preferably, the conditions of the second separation of the preparative liquid chromatography are as follows: gradient elution is carried out at room temperature by using the mobile phase at a flow rate of 0.8mL / min-1.0mL / min.

[0015] Preferably, the Yunnan Hippophae rhamnoides L. crude flavonoids are prepared according to the following steps: the Yunnan Hippophae rhamnoides L. powder is dissolved in an ethanol aqueous solution, and then is subjected to extraction treatment by using the ultrasonic-microwave assisted method, and after separation treatment, the Yunnan Hippophae rhamnoides L. crude flavonoids are obtained; wherein, the ethanol is used as the extraction solvent, and has low cost and good solubility to most substances.

[0016] Preferably, the mass-volume ratio of the Yunnan Hippophae rhamnoides L. to the ethanol aqueous solution is 1g:5mL-25mL, and the volume fraction of the ethanol in the ethanol aqueous solution is 40%-80%.

[0017] Preferably, the conditions of the extraction treatment are as follows: the extraction is carried out for 1min-5min under the conditions that the fixed ultrasonic power is 50W, the microwave power is 100W-500W, and the temperature is 50℃-60℃.

[0018] Compared with the prior art, the present application has the following beneficial effects: 1. The application provides a method for separating and purifying crude flavonoids of Hippophae rhamnoides Linn. var. yunnanensis, which adopts high-speed counter-current chromatography and preparative liquid chromatography to separate and purify the crude flavonoids of Hippophae rhamnoides Linn. var. yunnanensis, and the specific steps are as follows: taking n-hexane, ethyl acetate, methanol and water as the solvent system with a volume ratio of 1:9:1:9, the high-speed counter-current chromatography is used to separate the crude flavonoids of Hippophae rhamnoides Linn. var. yunnanensis, and the effluent of 35min-44min and 46min-60min is collected respectively, and the effluent of 35min-44min and 46min-60min is freeze-dried respectively to obtain isorhamnetin-3-O-glucoside and isoquercitrin; taking ethyl acetate, n-butanol and water as the solvent system with a volume ratio of 4:1:5, the high-speed counter-current chromatography is used to separate the crude flavonoids of Hippophae rhamnoides Linn. var. yunnanensis, and the effluent of 62min-72min is collected and freeze-dried to obtain rutin; the effluent of the solvent system of n-hexane, ethyl acetate, methanol and water is concentrated, and the secondary separation of the concentrated effluent is carried out by the preparative liquid chromatography with an Agilent 5Prep-C18 column and a mobile phase of 0.1wt% phosphoric acid solution-acetonitrile, and the effluent components are collected according to the chromatographic peaks, and the effluent components are concentrated and dried to obtain kaempferol and quercetin. The high-speed counter-current chromatography and the preparative liquid chromatography are combined to separate and purify the crude flavonoids of Hippophae rhamnoides Linn. var. yunnanensis, so that the problems of long separation period and low purification efficiency in the prior art are solved, and the purpose of efficiently and rapidly extracting and separating and purifying various flavonoid compounds from Hippophae rhamnoides Linn. var. yunnanensis is achieved.

[0019] 2. The ultrasonic-microwave assisted method realizes high extraction efficiency under the conditions that the volume fraction of ethanol in the ethanol aqueous solution is 60%, the solid-liquid ratio is 1g:15mL, and the extraction time is 3min, and the total flavonoid yield of Hippophae rhamnoides Linn. var. yunnanensis fruit reaches 17.992mg / g±0.49mg / g.

[0020] 3. The protective effects of the two flavonoid compounds of isoquercitrin and isorhamnetin-3-O-β-D-glucoside on HepG2 cell oxidative damage are studied. By establishing a low-density lipoprotein-induced HepG2 model, the contents of SOD, MDA, GSH-PX, BCA, TG, TC, HDL-C and LDL-C in cells are determined, and it is found that isoquercitrin and isorhamnetin-3-O-β-D-glucoside can reduce the generation of MDA and ROS in HepG2, inhibit the generation of oxidative stress, and also reduce the contents of TG, TC and LDL-C in HepG2, and reduce the accumulation of lipids in HepG2. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1The figure is the result of the influence of the volume fraction of ethanol in the ethanol aqueous solution, the ratio of material to liquid and the extraction time on the total yield of the crude flavonoids of Hippophae rhamnoides Linn. in Yunnan. Wherein, a is the volume fraction of ethanol in the ethanol aqueous solution, b is the ratio of material to liquid, and c is the extraction time.

[0022] Figure 2 The figure is the result of the HSCCC separation of the ethyl acetate component.

[0023] Figure 3 The figure is the result of the HSCCC separation of the n-butanol component.

[0024] Figure 4 The figure is the HPLC of the five flavone monomers. Wherein, A is isorhamnetin-3-O-β-D-glucoside, B is isoquercitrin, C is kaempferol, D is quercetin, and E is rutin.

[0025] Figure 5 The figure is the mass spectrum of the five flavone monomers. Wherein, A is isorhamnetin-3-O-β-D-glucoside, B is isoquercitrin, C is kaempferol, D is quercetin, and E is rutin.

[0026] Figure 6 The figure is the result of the influence of isoquercitrin and isorhamnetin-3-O-β-D-glucoside on the proliferation rate of HepG2 cells. Wherein, A is isoquercitrin, and B is isorhamnetin-3-O-β-D-glucoside.

[0027] Figure 7 The figure is the influence of isoquercitrin and isorhamnetin-3-O-β-D-glucoside on the contents of TC, TG, HDL-C and LDL-C in HepG2 cells induced by low-density lipoprotein. Wherein, A is TC, B is TG, C is HDL-C, and D is LDL-C.

[0028] Figure 8 The figure is the result of the determination of the contents of SOD, MDA and GSH-Px in HepG2 cells. Wherein, A is SOD, B is MDA, and C is GSH-Px.

[0029] Figure 9 The figure is the result of the determination of ROS in HepG2 cells. DETAILED DESCRIPTION

[0030] The technical solutions of the present application will be described clearly and completely below in combination with the data in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application. The following experimental methods and detection methods are all conventional methods unless otherwise specified; the following reagents and raw materials are all commercially available reagents and raw materials unless otherwise specified.

[0031] In existing technologies, traditional column chromatography relies on a linear separation mechanism between a single stationary phase and a mobile phase, which has drawbacks such as high solvent consumption and low separation efficiency, resulting in long extraction cycles and high purification costs for flavonoids from sea buckthorn in Yunnan.

[0032] To address the problems of low separation efficiency and long purification cycle in existing technologies, this invention combines ultrasonic-microwave assisted extraction with high-speed countercurrent chromatography-preparative liquid chromatography to construct a multi-stage solvent system gradient separation strategy, thus overcoming these issues.

[0033] The technical solution of the present invention will be further explained and illustrated below with examples, as detailed below: (1) Extraction of crude flavonoids from sea buckthorn in Yunnan: Weigh 0.5g of Yunnan sea buckthorn, add 20mL of 40% ethanol aqueous solution, and extract using ultrasonic-microwave assisted extraction at 50℃ and 300W for 2min to obtain the extract. After cooling the extract to room temperature, centrifuge at 8000r / min for 10min, collect the supernatant and make up to volume to obtain flavonoids, thus obtaining crude flavonoids from Yunnan sea buckthorn.

[0034] Based on the above extraction method, the volume fraction of ethanol in the aqueous ethanol solution, the extraction time, and the solid-liquid ratio were screened. The volume fractions of the aqueous ethanol solution were 50%, 60%, 70%, and 80%, respectively; the extraction times were 1 min, 3 min, 4 min, and 5 min, respectively; and the solid-liquid ratios were 1 g: 5 mL, 1 g: 10 mL, 1 g: 15 mL, and 1 g: 20 mL, respectively.

[0035] Based on the single-factor experiments, extraction time (A), ethanol volume fraction in ethanol-water solution (B), and solid-liquid ratio (C) were selected as optimization factors. A three-factor, three-level Box-Behnken response surface design was carried out using Design-Expert 13 software. The specific factor levels are shown in Table 1.

[0036] Table 1 Experimental Design Table for Factor Response Surface Analysis Depend on Figure 1 Figure a shows that when the ethanol volume fraction in the aqueous solution is 40%–50%, the yield of crude flavonoids from *Hippophae rhamnoides* gradually increases, and when the ethanol volume fraction is 50%–60%, the yield rapidly increases to its maximum value. When the ethanol volume fraction in the aqueous solution is between 60% and 80%, the total yield of crude flavonoids from *Hippophae rhamnoides* decreases rapidly. Therefore, ethanol volume fractions of 50%, 60%, and 70% were chosen for subsequent experiments.Figure 1 Figure b in the table shows that the extraction rate of crude flavonoids from Yunnan Hippophae rhamnoides L. rapidly increased with the gradual increase of the ratio of material to liquid. The maximum value was reached when the ratio of material to liquid was 1g:15mL. Therefore, the optimal ratio of material to liquid (1g:10mL, 1g:15mL, 1g:20mL) was selected for subsequent optimization experiments. Figure 1 Figure c in the table shows that the extraction rate of crude flavonoids from Yunnan Hippophae rhamnoides L. first increased and then decreased. The extraction rate of crude flavonoids from Yunnan Hippophae rhamnoides L. rapidly increased when the extraction time was in the range of 1min~3min, and decreased when the extraction time was in the range of 3min~5min. The optimal extraction time (2min, 3min, 4min) was selected for subsequent optimization experiments.

[0037] Based on the results of single-factor experiments, a Box-behnken experiment was designed, as shown in Table 2. The extraction rate of crude flavonoids from Yunnan Hippophae rhamnoides L. was the highest (18.23mg / g) when the extraction time was 3min, the volume fraction of ethanol in the aqueous ethanol solution was 60%, and the ratio of material to liquid was 1g:15mL.

[0038] Table 2 Experimental design and results of response surface optimization of total flavonoids from Yunnan Hippophae rhamnoides L. a. Model establishment and significance detection: The quadratic multinomial regression equation of the extraction rate of crude flavonoids from Yunnan Hippophae rhamnoides L. (Y) with respect to the extraction time (A), the concentration of aqueous ethanol solution (B), and the ratio of material to liquid (C) was: .

[0039] Table 3 Regression analysis results of the model of total extraction rate of flavonoid compounds and regression coefficients Note: P<0.01 is extremely significant, represented by **, P<0.05 is significant, represented by *, P>0.05 is not significant, represented by ns, and - represents no.

[0040] As shown in Table 3, the p value of the model was less than 0.01, the difference was extremely significant, the p value of the loss of fitting was 0.1109, which was greater than 0.05 (not significant), indicating that there was no loss of fitting factor, the model fitting effect was good, the predicted value deviated from the true value was small, fully reflected the actual situation, and had statistical significance. The model correction coefficient R 2 Ad j =0.9782, indicating that 97.8% of the cases could be explained by the regression equation; the coefficient of variation CV was 2.79%, indicating that the model had good reproducibility, high reliability and accuracy.

[0041] Table 4 Model correlation analysis results of the experiment Note: P<0.01 is extremely significant, represented by **, P<0.05 is significant, represented by *, P>0.05 is not significant, represented by ns.

[0042] It is concluded from Table 4 that the first-order terms A, C are extremely significant, and B is significant; the second-order terms A 2 , B 2 , C 2 are extremely significant; the interaction terms AB, BC are extremely significant, and AC is not significant. The test results show that the change of the response value is relatively complex, the influence of each factor on the extraction rate of the crude flavonoids of Yunnan Hippophae rhamnoides is a quadratic relationship rather than a simple linear relationship, and there is an interaction between the three factors. The F values of the coefficient terms of each factor are arranged in descending order, and the factors that have a significant influence on the extraction rate of the crude flavonoids of Yunnan Hippophae rhamnoides are arranged in descending order of significance as follows: C>A>B.

[0043] On the premise of the maximum yield of the crude flavonoids of Yunnan Hippophae rhamnoides, the optimal extraction process of the crude flavonoids of Yunnan Hippophae rhamnoides is determined in the Design-expert 11 software as follows: the extraction time is 3.195, the volume fraction of ethanol in the aqueous ethanol solution is 59.941%, and the solid-liquid ratio is 1g:13.965mL. Considering the operation problem, the optimal process is determined as follows: the extraction time is 3min, the volume fraction of ethanol in the aqueous ethanol solution is 60%, and the solid-liquid ratio is 1g:15mL. Under the conditions, three repeated experiments are carried out, and the average yield of the crude flavonoids of Yunnan Hippophae rhamnoides is 17.992mg / g±0.498mg / g, which is close to the theoretical value 18.262mg / g.

[0044] Under the process conditions of the extraction time being 3min, the volume fraction of ethanol in the aqueous ethanol solution being 60%, and the solid-liquid ratio being 1g:15mL, the crude flavonoids of Yunnan Hippophae rhamnoides are extracted from Yunnan Hippophae rhamnoides, and the crude flavonoids of Yunnan Hippophae rhamnoides obtained under the process are separated and purified.

[0045] (2) The crude flavonoids of Yunnan Hippophae rhamnoides are separated and purified: a. HPLC optimization results: In the HPLC optimization process, water and acetonitrile are first used for gradient elution, it is found that the compounds have a tailing phenomenon, and finally 0.1% phosphoric acid is added for peak shape optimization as shown in Table 5, and the detection wavelength is 254nm.

[0046] Table 5 HPLC chromatographic analysis condition table b. Selection of two-phase solvent system: The preliminary screening and investigation are carried out through pre-experiment, wherein the separation of ethyl acetate component is carried out under the condition that n-hexane-ethyl acetate-methanol-water has good separation effect on two target compounds of Yunnan Hippophae rhamnoides Linn, and then the solvent system is further optimized, and three ratios of n-hexane-ethyl acetate-methanol-water solvent system are determined, and the results are shown in Table 6.

[0047] Table 6: Screening results of ethyl acetate component flavonoid compound solvent system According to Table 6, the K value of the flavonoid compound in 2# is 0.42 and 0.2, which leads to too fast peak time in HSCCC and poor separation between peaks. The K value of the flavonoid compound in 3# is also small, which leads to too fast peak time and poor separation. The K value and the alpha value of the flavonoid compound in 1# are moderate, and the solvent system is layered quickly, and the n-hexane-ethyl acetate-methanol-water (1:9:1:9) solvent system is selected for separating the ethyl acetate component after actual measurement.

[0048] According to the polarity rule of the two-phase solvent system and the polarity of the n-butanol component, the application selects a solvent system with larger polarity, and the separation degree of the target compounds B-1, B-2, B-3 and B-4 in the system is calculated through experiment, and the calculation results are shown in Table 7. Four ratios are screened, which are 3:2:5, 3.5:1.5:5, 5:1.5:5 and 4:1:5. In the four solvent systems, the K value of ethyl acetate-n-butanol-water (4:1:5) is between 0.5 and 2, but the alpha of compounds B-1 and B-2 is not greater than 1.5, and the compounds are not separated, and it is actually measured that the compounds B-1 and B-2 are not separated, but according to the peak situation, they can still be purified.

[0049] Table 7: Screening results of n-butanol component flavonoid compound solvent system c. HSCCC separation condition optimization: The n-butanol component: for the sample amount, too much sample amount will lead to instrument separation overload phenomenon, and thus reduce the purity. Therefore, the maximum sample amount should be kept at about 300 mg. For the flow rate of the mobile phase, under the condition that other parameters are unchanged, with the increase of the flow rate of the high-speed counter-current chromatography mobile phase, the overall peak time is delayed, which leads to poor separation effect and thus reduces the purity. Therefore, the flow rate of the mobile phase is selected as 3 mL / min.

[0050] The ethyl acetate component: under the condition that the rotation speed is 950 r / min, the flow rate is 3.5 mL / min, and the sample amount is 400 mg, the high-speed counter-current chromatography has good separation effect on the ethyl acetate component.

[0051] d. High speed counter-current chromatography flavonoid monomer separation results: Ethyl acetate component flavonoid monomer separation and preliminary qualitative analysis: the fraction was collected at 35 min~44 min to obtain E-1; the fraction was collected at 46 min~60 min to obtain E-2; after the separation of the first two compounds, the non-separated components were freeze-dried for preparation of liquid chromatography. HPLC was used to detect the peak of the two components at the wavelength of 254 nm and 330 nm. The results showed that the compounds E-1 and E-2 were separated well in the system of n-hexane-ethyl acetate-ethanol-water (1:9:1:9); and the HPLC detection of the two compounds was basically single peak. Further, LC-MS was used to preliminarily analyze the two compounds, E-1 was isorhamnetin-3-O-glucoside, and E-2 was isoquercitrin, as shown in Figure 2 .

[0052] n-Butanol component flavonoid monomer separation and preliminary qualitative analysis: the fraction was collected at 40 min~50 min to obtain B-1; the fraction was collected at 46 min~60 min to obtain B-2; the fraction was collected at 62 min~72 min to obtain B-3; the fraction was collected at 80 min~100 min to obtain B-4. When the baseline was 0, the stationary phase, mobile phase and collected waste liquid in the machine were rotary evaporated and freeze-dried for preparation of liquid chromatography. HPLC was used to detect the purity of the four separated substances at the wavelength of 254 nm and 330 nm by area normalization method. The results showed that the compounds B-1, B-2, B-3 and B-4 were separated well in the system of ethyl acetate-n-butanol-water (4:1:5), the purity of B-2, B-3 and B-4 was more than 90%, and the purity of B-1 was not high. LC-MS was used to preliminarily analyze the compounds B-1, B-2, B-3 and B-4, B-3 was rutin, B-4 was not identified, and B-1 and B-2 were non-flavonoid compounds, as shown in Figure 3 .

[0053] d. Preparation of liquid chromatography separation condition optimization: The crude polysaccharide of Yunnan Hippophae rhamnoides L. was dissolved in pure methanol, the injection volume was selected from 0 μL to 900 μL, the flow rate was 10 mL / min, and the elution gradient was shown in Table 8.

[0054] Table 8 Prep-HPLC chromatographic analysis condition table e. Purification of high speed counter-current flavonoid monomers and non-separated components by preparation liquid chromatography: The four kinds of flavone monomer components obtained by HSCCC were further purified by Prep-HPLC. Two kinds of flavonoids E-3 and E-4 were purified from the ethyl acetate component stationary phase and waste liquid of HSCCC by Prep-HPLC.

[0055] (3) Qualitative analysis of flavone monomers: Five kinds of flavone monomers were obtained by preparative liquid separation and purification, and were qualitatively analyzed by HPLC comparison with standard samples and LC-MS. Figure 4 and Figure 6 It is concluded that compound E-1 has a molecular quasi-ion peak of 479.11758, and its molecular formula is C 22 H 22 O 12 Further comparison with standard samples by HPLC determines that the compound is isorhamnetin-3-O-glucoside. Compound E-2 has a molecular quasi-ion peak [M-H] - of 465.10193, and its molecular formula is C 21 H 20 O 12 Further comparison with standard samples by HPLC determines that the compound is isoquercitrin. Compound E-3 has a molecular quasi-ion peak [M-H] - of 285.04031, and its molecular formula is C 15 H 11 O6, and further comparison with standard samples by HPLC determines that the compound is kaempferol. Compound E-4 has a molecular quasi-ion peak of, and its molecular formula is C 15 H 10 O7, and further comparison with standard samples by HPLC determines that the compound is quercetin. Compound B-1 has a molecular quasi-ion peak [M-H] - of 609.14630, and its molecular formula is C 27 H 30 O 16 Further comparison with standard samples by HPLC determines that the compound is rutin.

[0056] (4) Protective effect of flavone monomers on oxidative damage of HepG2 cells: The protective effect of isoquercitrin (compound A) and isorhamnetin-3-O-β-D-glucoside (compound B) on oxidative damage of HepG2 cells in vitro was studied as follows: Isoquercitrin: fully dissolved in 200 μL of DMSO, with a mass concentration of 100 mg / mL; isorhamnetin-3-O-β-D-glucoside: fully dissolved in 200 μL of DMSO, with a mass concentration of 100 mg / mL.

[0057] 1) CCK-8 method for detecting cell viability: Take HepG2 cells in the logarithmic growth phase, with good growth state, 3x103 / well in a cell culture 96-well plate, 37℃, 5% CO2 incubator overnight. (Add 200 μL of sterile PBS to the cell well around the hole)

[0058] Calculation formula: cell proliferation rate (%) = (treated group absorbance-blank group absorbance) / (control group absorbance-blank group absorbance) 2) Low-density lipoprotein-induced HepG2 model construction: a. According to the standard reference procedure, add different mass concentrations of drugs: isoquercitrin 1 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL; isorhamnetin-3-O-β-D-glucoside 1 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL; blank group; control group; incubate the culture plate in the incubator for 24 h, then add 10 μL of CCK-8 to each well, set 3 replicate wells for each concentration, 37℃ incubate for 2 h, detect cell toxicity by CCK-8 method, and measure the absorbance of each well by enzyme label instrument OD 450.

[0059] b. After determining the cell modeling concentration, culture the cells according to 1), and treat the cells according to the following grouping: ① normal control group, ② model group, ③ model+compound A, ④ model+compound B.

[0060] Action time: 50 μg / mL low-density lipoprotein induction for 24 h, 20 μg / mL compound treatment for 24 h. Measure the contents of TG, TC, HDL-C, LDL-C and BCA in the cells.

[0061] The TC determination method strictly follows the operation method in the detection kit to calculate the total cholesterol content in HepG2. First, take out the prepared cell suspension and centrifuge at 1000 r / min for 10 min, and reserve the cell precipitate. Add 0.3 mL of 0.1 mol / mL, pH=7.2 phosphate buffer for homogenization, and take the supernatant for detection after ultrasonic crushing under ice water bath condition.

[0062] The TG determination method strictly follows the operation method in the detection kit to calculate the triglyceride content in HepG2. First, take out the prepared cell suspension and centrifuge at 1000 r / min for 10 min, and reserve the cell precipitate. Add 0.3 mL of 0.1 mol / mL, pH=7.2 phosphate buffer for homogenization, and take the supernatant for detection after ultrasonic crushing under ice water bath condition.

[0063] The HDL-C determination method is strictly in accordance with the operation method of the detection kit, and the high-density lipoprotein cholesterol content in HepG2 is calculated. The prepared cell suspension is taken out, centrifuged at 1000 r / min for 10 min, and the cell precipitate is reserved; washed with 0.1 mol / L, pH=7.2 phosphate buffer solution for 2 times, and centrifuged at 1000 r / min for 10 min; then 0.3 mL of 0.1 mol / L, pH=7.2 phosphate buffer solution is added for homogenization, and the supernatant is detected after ultrasonic crushing under ice water bath condition.

[0064] The LDL-C determination method is strictly in accordance with the operation method of the detection kit, and the low-density lipoprotein cholesterol content in HepG2 is calculated. The prepared cell suspension is taken out, centrifuged at 1000 r / min for 10 min, and the cell precipitate is reserved; washed with 0.1 mol / L, pH=7.2 phosphate buffer solution for 2 times, and centrifuged at 1000 r / min for 10 min, and the supernatant is discarded, and the cell precipitate is reserved, 0.3 mL of 0.1 mol / L, pH=7.2 phosphate buffer solution is added for homogenization, and the supernatant is detected after ultrasonic crushing under ice water bath condition.

[0065] The BCA determination method is strictly in accordance with the operation method of the detection kit, and the protein content in HepG2 is calculated. Specifically, the protein standard is completely dissolved in PBS solution, 20 μL is taken and diluted to 100 μL, so that the final concentration is 1 mg / mL. The sample is also diluted with PBS solution. According to the number of samples, 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1) are prepared to prepare appropriate amount of BCA working solution, and the working solution is effective on the same day.

[0066] The contents of SOD, MDA, GSH-PX and BCA in HepG2 are determined by using the kit.

[0067] The SOD determination method is strictly in accordance with the operation method of the detection kit. First, the buffer solution is mixed at a ratio of 1:200 to prepare the substrate application liquid; then the enzyme diluent is mixed at a ratio of 1:10 to prepare the enzyme working solution. According to the normal control, model group and three drug groups, the measured homogenate supernatant, distilled water and enzyme working solution are added according to the kit operation method, mixed well, incubated at 37°C for 20 min, and read at 450 nm by enzyme label instrument.

[0068] MDA determination method is strictly in accordance with the operation method of the detection kit, according to the experimental operation table after sample operation, cover on the centrifugal tube cover (in the cover with a needle on a small hole), vortex mixer mixing, 95 ℃ above water bath 40 min, remove after water cooling, 4000 r / min centrifugation 10 min, 532 nm, accurate suction each tube supernatant 0.2 mL into the 96 well plate, enzyme labeled instrument determination of each hole absorbance.

[0069] GSH-Px determination method is strictly in accordance with the operation method of the detection kit, can refer to the experimental method of Kang Meijuan

[94] , specific operation: a. standard solvent application liquid configuration: stock solution: distilled water = 1:9, namely 10 times dilution to prepare application liquid, take immediately, stored at 4 ℃; B. 1 mmoL / L GSH standard solution preparation: before determination, GSH standard powder was added to the standard solution application liquid 10 mL, fully dissolved, after preparation, stored at 4 ℃ for 2 weeks; C. take 0.2 mL, 1 mmoL / L GSH solution, add GSH standard solution application liquid 10 mL, namely 20 μmoL / L GSH standard solution; D. according to the requirements of the kit, mixed at room temperature for 15 min, then use 1 cm aperture colorimetric cup at 412 nm to measure the OD value of the test tube, and adjust to zero with distilled water.

[0070] BCA determination method is strictly in accordance with the operation method of the detection kit, specific steps: a. preparation of appropriate amount of BCA working solution, fully mixed, in the standard hole of 96 well plate, respectively, 0 μL, 4 μL, 8 μL, 12 μL, 16 μL and 20 μL of standard substance, then add the dilution of standard substance solution, so that it reaches 20 μL; B. the PBS diluted protein sample, diluted standard protein were added to the 96 well plate, each hole added sample total volume 20 μL; C. the A and B liquid of BCA kit were mixed according to the ratio of 50:1, added to the 96 well plate, each hole added 200 μL, attention not to produce air bubble, so as to avoid affecting the reaction; D. 37 ℃ incubation for 13 min, according to the actual color termination reaction, enzyme labeled instrument determination of OD568.

[0071] ROS was determined according to the method specified in the reactive oxygen species detection kit. The specific experimental steps are as follows: a. The cells cultured in (1) were grouped and treated according to the constructed cell model, and after 24 h of treatment, the cells were collected by trypsin digestion, centrifuged at 1200 rpm for 5 min, the supernatant was taken out, and resuspended in PBS; b. The cells were washed once with PBS at a speed of 1200 rpm for 5 min; c. DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 to make the final concentration 10 μmoL / L; d. 1 mL of diluted DCFH-DA was added; e. Incubate in a 37°C incubator for 20 min, stir every 3 min, wash the cells with serum-free medium three times, centrifuge at 1200 rpm for 5 min, remove the supernatant, and resuspend in PBS for flow cytometry detection.

[0072] (5) Experimental results: To ensure the accuracy of the cell experiment results and avoid the interference of cell toxicity caused by improper drug concentration, the CCK-8 experiment was designed in this study. This experiment will be aimed at cells treated with isoquercitrin and isorhamnetin-3-O-β-D-glucoside at different concentrations for activity evaluation, in order to accurately select the most suitable modeling concentration and drug concentration.

[0073] After treating cells with isoquercitrin at a mass concentration of 1 μg / mL to 100 μg / mL for 24 h, it was found through CCK-8 detection that within the range of 1 μg / mL to 40 μg / mL mass concentration, isoquercitrin had no significant effect on cell growth. However, when the mass concentration of isoquercitrin increased to 60 μg / mL, the cell viability decreased to 88.13%, indicating that high-dose isoquercitrin caused damage or even death to cells. Therefore, the selected mass concentration of isoquercitrin for drug administration was 20 μg / mL.

[0074] The cells were treated with isorhamnetin-3-O-β-D-glucoside at a mass concentration of 1 μg / mL to 100 μg / mL for 24 h. The results of CCK-8 detection showed that within the range of 1 μg / mL to 100 μg / mL mass concentration, isorhamnetin-3-O-β-D-glucoside had no significant effect on cell growth. Notably, when the mass concentration of isorhamnetin-3-O-β-D-glucoside reached 100 μg / mL, the cell viability was still as high as 93.03%. Therefore, the selected concentration of isorhamnetin-3-O-β-D-glucoside for actual drug administration was 20 μg / mL.

[0075] Isoquercitrin and isorhamnetin-3-O-β-D-glucoside at a concentration of 20 μg / mL were used to establish a low-density lipoprotein-induced HepG2 model. The results showed that Figure 6The results showed that the cell proliferation rate in the model group was significantly lower than that in the normal control group, indicating that the corresponding treatment had an inhibitory effect on cell proliferation. Compared with the model group + compound combination, the model group + compound combination significantly promoted cell proliferation, with compound A increasing the cell proliferation rate by 3.8% and compound B increasing the cell proliferation rate by 5.19%.

[0076] Effects of isoquercitrin and isorhamnetin-3-O-β-D-glucoside on low-density lipoprotein-induced lipid metabolism in HepG2 cells: Depend on Figure 7 The results showed that, compared with the normal control group, the levels of TC, TG, and LDL-C in LDL-induced HepG2 cells were significantly increased, while the level of HDL-C was significantly decreased. The results indicated that the model + compound combination significantly reduced the levels of TC, TG, and LDL-C in LDL-induced HepG2 cells, with TC values ​​decreasing by 36.5% and 53.35%, TG values ​​decreasing by 31.25% and 40.625%, and LDL-C levels decreasing by 24.43% and 37.12%, respectively. Compared with the model group, the model + compound combination significantly increased intracellular HDL-C by 30% and 39.2%, respectively, and reduced lipid accumulation in cells.

[0077] Effects of isoquercitrin and isorhamnetin-3-O-β-D-glucoside on low-density lipoprotein-induced oxidative damage in HepG2 cells: like Figure 8 As shown in Figure A, the SOD activity and GSH-Px content of HepG2 were significantly reduced, while after drug treatment, SOD activity and GSH-Px content increased, indicating that the drug inhibits oxidative stress by reducing ROS and increasing the expression of antioxidant enzymes. Furthermore, studies have shown that increasing the activity of glutathione peroxidase, catalase, and SOD can reduce ROS production; see [reference needed]. Figure 8 Figures B and C in the diagram.

[0078] like Figure 9As shown, the effect of isoquercitrin, isorhamnetin-3-O-beta-D-glucoside on low density lipoprotein-induced oxidative stress in HepG2 cells was studied by determining the ROS level. Compared with the control group, the fluorescence intensity in the model+compound combination group was significantly increased in low density lipoprotein-induced HepG2, and the generation of ROS was increased; compared with the model group, the model+compound combination reduced the ROS level in low density lipoprotein-induced liver cancer cells, helped to alleviate oxidative stress, and regulated the related antioxidant defense response. In addition, the accumulation of ROS induces the generation of MDA, thereby causing lipid peroxidation and oxidative damage. Both SOD and GSH-Px are involved in the process of scavenging peroxide, further proving that the two flavones can effectively prevent the imbalance of the redox state of cells by reducing the generation of ROS, the content of MDA, and up-regulating the activity of GSH-Px and SOD.

[0079] It should be noted that when the present application involves a numerical range, it should be understood that both endpoints of each numerical range and any number between the two endpoints can be selected. Since the same step method and examples are used, in order to prevent repetition, the present application describes the preferred examples.

[0080] Although the preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to be interpreted as including all the preferred embodiments and all changes and modifications falling within the scope of the present application.

Claims

1. A method for separating and purifying crude flavonoids from Hippophae rhamnoides L. subsp. sinensis (Rousi) Chow, characterized in that, The crude flavonoids from sea buckthorn in Yunnan were separated and purified using a combination of high-speed countercurrent chromatography and preparative liquid chromatography, including the following steps: Using hexane, ethyl acetate, methanol, and water as solvents in a volume ratio of 1:9:1:9, crude flavonoids from sea buckthorn in Yunnan were separated by high-speed countercurrent chromatography. The eluents from 35 min to 44 min and from 46 min to 60 min were collected, and the eluents from 35 min to 44 min and from 46 min to 60 min were freeze-dried to obtain isorhamnetin-3-O-glucoside and isoquercitrin. Using ethyl acetate, n-butanol, and water as solvents in a volume ratio of 4:1:5, crude flavonoids from sea buckthorn in Yunnan were separated by high-speed countercurrent chromatography. The eluent from 62 min to 72 min was collected and freeze-dried to obtain rutin. The unseparated eluent, prepared using hexane, ethyl acetate, methanol, and water as solvents, was concentrated. Then, under preparative liquid chromatography conditions (Agilent 5 Prep-C18 column, 0.1 wt% phosphoric acid aqueous solution-acetonitrile), the concentrated eluent was subjected to secondary separation. The eluent components were collected according to chromatographic peaks, and each component was concentrated and dried to obtain kaempferol and quercetin.

2. The method for separating and purifying crude flavonoids from Hippophae rhamnoides Linn. according to claim 1, characterized in that, In high-speed countercurrent chromatography using hexane, ethyl acetate, methanol, and water as solvent systems, the flow rate is 3.5 mL / min to 5.5 mL / min, the injection volume is 100 mg to 400 mg, and the rotation speed is 850 rpm to 1050 rpm.

3. The method according to claim 1, characterized in that, In high-speed countercurrent chromatography using ethyl acetate, n-butanol, and water as solvent systems, the flow rate is 3 mL / min to 5 mL / min, the injection volume is 100 mg to 600 mg, and the rotation speed is 850 rpm to 1050 rpm.

4. The method according to claim 1, characterized in that, The detection wavelengths for high-speed countercurrent chromatography and preparative liquid chromatography are 254 nm, 280 nm, and 330 nm.

5. The method according to claim 1, characterized in that, In high-speed countercurrent chromatography, the upper phase of the solvent system is the stationary phase, and the lower phase is the mobile phase.

6. The method according to claim 1, wherein the crude flavonoids are separated and purified from Hippophae rhamnoides L. ssp. sinensis (Rousi) Cz. et E. Y. C. Chen. In the 0.1wt% phosphoric acid aqueous solution-acetonitrile mixture, the volume ratio of the 0.1wt% phosphoric acid aqueous solution to acetonitrile is 60~80:20~40.

7. The method for separating and purifying crude flavonoids from Yunnan sea buckthorn according to claim 1, characterized in that, The conditions for secondary separation by preparative liquid chromatography are as follows: elution is performed at room temperature using a mobile phase at a flow rate of 0.8 mL / min to 1.0 mL / min.

8. The method for separating and purifying crude flavonoids from Yunnan sea buckthorn according to claim 1, characterized in that, Yunnan sea buckthorn crude flavonoids were prepared according to the following steps: Yunnan sea buckthorn powder was dissolved in an ethanol aqueous solution, and then extracted using an ultrasonic-microwave assisted method. After separation, Yunnan sea buckthorn crude flavonoids were obtained.

9. The method for separating and purifying crude flavonoids from Yunnan sea buckthorn according to claim 1, characterized in that, The mass-to-volume ratio of Yunnan sea buckthorn to ethanol aqueous solution is 1g:5mL~25mL, and the volume fraction of ethanol in the ethanol aqueous solution is 40%~80%.

10. The method for separating and purifying crude flavonoids from Yunnan sea buckthorn according to claim 1, characterized in that, The extraction conditions were as follows: extraction for 1 min to 5 min under fixed ultrasonic power of 50 W, microwave power of 100 W to 500 W, and temperature of 50 °C to 60 °C.

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