Extraction and purification method of total flavonoids of hippophae rhamnoides leaves

Abstract

The invention discloses a method for extracting and purifying total flavonoids of hippophae rhamnoides leaves. According to the method, an ultrasonic-assisted extraction technology is adopted, a solvent system and a material-to-liquid ratio are matched, XDA-6 macroporous adsorption resin is combined for primary purification, D101 macroporous adsorption resin is combined for secondary refining, high-selectivity separation and enrichment of the flavonoids of the hippophae rhamnoides leaves are achieved, and the purity of the finally obtained total flavone extract reaches 35.05-37.09%; the method has the beneficial effects that the prepared general flavone extract is high in purity, the conditions of the preparation process flow are mild, and the main solvents are ethanol and water, so that the method is environment-friendly; the adopted macroporous adsorption resin can be regenerated and reused for multiple times, so that the material consumption is reduced; meanwhile, the method is simple in step, low in dependence on equipment and suitable for industrial large-scale production and application; the method effectively solves the problems of insufficient research on extraction and purification of the hippophae rhamnoides leaves, resource waste and the like, provides an effective way for converting the hippophae rhamnoides leaves into high-added-value products, and improves comprehensive economic benefits of the hippophae rhamnoides

Description

Technical Field

[0001] This invention relates to the field of natural product extraction and separation technology, specifically a method for extracting and purifying total flavonoids from sea buckthorn leaves. Background Technology

[0002] Flavonoids are one of the main active components of sea buckthorn leaves. Modern pharmacological studies have shown that they possess various biological activities, including anti-inflammatory, anti-obesity, lipid-regulating, and antioxidant effects. Sea buckthorn flavonoids mainly exist as flavonoid aglycones and their glycosides with isorhamnetin, kaempferol, quercetin, and myricetin as their parent nuclei. Research indicates that the flavonoid content in sea buckthorn leaves is significantly higher than that in its fruit, and plant-derived flavonoids are naturally low in toxicity, showing broad application prospects in the food, pharmaceutical, and health product fields.

[0003] Macroporous adsorption resin technology has been widely used in the extraction and purification of natural products in recent years. However, a search revealed that current research and development on sea buckthorn flavonoids mainly focuses on sea buckthorn fruit, while research on the extraction and purification of flavonoids from sea buckthorn leaves is relatively limited. This failure to effectively utilize this abundant resource has resulted in resource waste. The utilization rate of sea buckthorn leaves is less than 10% (Sea Buckthorn Resource Development Report, 2020), with a large amount being discarded due to the lack of efficient extraction technology.

[0004] In addition, existing methods for extracting and purifying sea buckthorn flavonoids generally have several significant drawbacks: First, the extraction process is relatively complex, often involving prolonged heating and reflux or repeated extraction, resulting in high energy consumption and low efficiency; second, the purity of the final product from existing methods is generally not high, making it difficult to meet the quality requirements for developing high value-added products; finally, the process involves large solvent consumption and cumbersome purification steps, leading to high production costs and hindering its large-scale industrial application.

[0005] Therefore, in view of the above-mentioned technological gaps and defects in this field, there is an urgent need to develop a simple, low-cost, green, efficient method for extracting and purifying total flavonoids from sea buckthorn leaves that can produce high-purity products. Summary of the Invention

[0006] The purpose of this invention is to propose a method for extracting and purifying total flavonoids from sea buckthorn leaves. This method utilizes macroporous adsorption resin to extract total flavonoids from sea buckthorn leaves, overcoming the shortcomings of existing technologies in the insufficient development of sea buckthorn leaf resources and realizing the high-value utilization of sea buckthorn leaves. By employing an ultrasonic-assisted extraction and macroporous resin combined purification strategy, the operation steps are simplified to obtain a total flavonoid extract, making the preparation method suitable for large-scale production.

[0007] To achieve the above objectives, the present invention provides a method for extracting and purifying total flavonoids from sea buckthorn leaves, characterized by comprising the following steps: (1) Pulverize the dried leaves of sea buckthorn to obtain sea buckthorn leaf powder; (2) Add the powder obtained in step (1) to an ethanol aqueous solution with a volume fraction of 50%-70% at a material-to-liquid ratio of 1:(20-40), extract it by ultrasonic extraction at a power of 400-600W for 20-30 minutes, filter it, concentrate the filtrate to remove ethanol, and obtain crude extract. (3) Dilute the crude extract to 2-5 mg / ml and load it onto the macroporous resin chromatography column of the purified crude extract. The specific surface area of ​​the macroporous resin of the purified crude extract is 450-600 m² / g and the average pore size is 10-15 nm. First, wash with water to remove impurities, and then elute with a gradient of 10%-60% ethanol solution. The elution flow rate is 0.5-2 BV / h and the elution volume is 2-4 BV. (4) Collect the eluent from step (3), concentrate the eluent to remove ethanol, freeze dry to obtain a first-purified product; (5) Dissolve the first-purified product to a concentration of 2-5 mg / ml and load it onto a macroporous resin chromatography column for purifying the first-purified product. The macroporous resin for purifying the first-purified product has a specific surface area ≥700 m² / g and an average pore size of 8.5-11 nm. The loading flow rate is 1-3 BV / h. After loading, elute with 2-4 BV of water and then elute with 2-4 BV of 10%-60% ethanol. Concentrate the eluent to remove the ethanol and freeze-dry to obtain the purified total flavonoid extract of sea buckthorn leaves. The purity of the purified total flavonoid extract of sea buckthorn leaves is 35.05-37.09%.

[0008] 2. The method as described in claim 1, characterized in that: the sea buckthorn in step (1) is Chinese sea buckthorn, which is a seed source from Zhangxian County, Gansu Province; the moisture content of the dried sea buckthorn leaves is 5%-8%, and the dried sea buckthorn leaves are crushed and passed through a 40-60 mesh sieve.

[0009] The filtrate in step (2) is concentrated to remove ethanol by using a rotary evaporator to concentrate the filtrate to remove ethanol. The concentration temperature is 40-45℃ and the vacuum degree is -0.1-0.08 MPa. The eluent in step (4) is concentrated to remove ethanol by using a rotary evaporator to concentrate the eluent to remove ethanol. The concentration temperature is 35-40℃, the vacuum degree is -0.1-0.08 MPa, the freezing temperature is -70--90℃, and the dryness is 100 Pa. The eluent in step (5) is concentrated to remove ethanol by using a rotary evaporator to concentrate the eluent to remove ethanol. The concentration temperature is 35-40℃, the vacuum degree is -0.10-0.08 MPa, the freezing temperature is -70--90℃, and the dryness is 100 Pa.

[0010] The macroporous resin in the crude extract in step (3) is macroporous resin XDA-6; before loading the macroporous resin XDA-6 into the chromatography column in step (3), it is activated. The activation step is to soak it in anhydrous ethanol solution for 24h to 48h for activation, and to continuously rinse it with distilled water until the eluent is clear. Finally, the activated adsorbent that has been rinsed clean is placed in an oven to dry for later use.

[0011] In step (3), the diameter-to-height ratio of the chromatography column is 1:(10-20), the chromatography column is 2×30cm-4×50cm, and the packing volume is 60-300ml.

[0012] The macroporous resin used to purify the first-stage purified product in step (5) is D101 macroporous resin. Before loading the D101 macroporous resin into the chromatography column in step (5), it is activated by soaking it in anhydrous ethanol solution for 24-48 hours, rinsing it continuously with distilled water until the eluent is clear, and finally drying the cleaned and activated adsorbent in an oven for later use.

[0013] In step (5), the diameter-to-height ratio of the chromatography column is 1:(10-20), the chromatography column is 2×30cm-4×50cm, and the packing volume is 60-300ml.

[0014] The total flavonoid extract obtained by the method is used in the preparation of lipid-lowering drugs, wherein the lipid-lowering activity includes reducing intracellular total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL-C) levels, and increasing high-density lipoprotein (HDL-C) levels.

[0015] The present invention discloses a method for extracting and purifying total flavonoids from sea buckthorn leaves. The beneficial effects are as follows: This method employs ultrasonic-assisted extraction technology with specific power, combined with an optimized solvent system and material-to-liquid ratio, shortening the extraction time and increasing the flavonoid dissolution rate. Primary purification is achieved using the specific properties of XDA-6 macroporous adsorption resin, followed by secondary purification using the specific properties of D101 macroporous adsorption resin. This results in highly selective separation and enrichment of flavonoids from sea buckthorn leaves, ultimately obtaining a total flavonoid extract with a stable purity of over 35%. The high-purity total flavonoid extract prepared by this method exhibits a synergistic ratio of active ingredients. In vitro cell model verification has shown that at a concentration of 150 μg / mL, it can significantly reduce intracellular total cholesterol, triglycerides, and low-density lipoprotein cholesterol levels. This invention not only improves lipid metabolism but also significantly increases high-density lipoprotein cholesterol, demonstrating excellent lipid metabolism regulation activity and providing a high-quality raw material basis for the development of related functional foods or drugs. The entire process is characterized by mild conditions, with ethanol and water as the main solvents, making it environmentally friendly. The macroporous adsorption resin used can be regenerated and reused multiple times, resulting in a long service life and effectively reducing material consumption and production costs. Furthermore, the method is simple in steps, has controllable parameters, is easy to scale up, has low dependence on equipment, and is suitable for large-scale industrial production. This invention effectively solves the problems of insufficient extraction and purification research and resource waste in sea buckthorn leaf research, providing an effective way to transform this abundant resource into high-value-added products and improve the overall economic benefits of the sea buckthorn industry. Attached Figure Description

[0016] Figure 1 Chromatogram of the total flavonoids test solution; Figure 2 The effects of different concentrations of flavonoid extracts on the lipid-lowering activity of HepG2 cells. Detailed Implementation

[0017] Example 1 The present invention discloses a method for extracting and purifying total flavonoids from sea buckthorn leaves, comprising the following steps: (1) Grind dried Chinese sea buckthorn leaves and weigh out 50g of sea buckthorn leaf powder; (2) The sea buckthorn leaf powder and 60% ethanol were extracted by ultrasonic extraction at 400W power for 25 minutes at a material-liquid ratio of 1:30. The mixture was then filtered. The filtrate was concentrated on a rotary evaporator to remove the ethanol. The concentration temperature was 40℃ and the vacuum degree was -0.1Mpa to obtain the crude extract. (3) Dilute the crude extract to 2 mg / ml and load it onto a macroporous resin XDA-6 chromatography column. The macroporous resin XDA-6 was activated before packing. The macroporous resin XDA-6 was wet-packed into a 4×50cm chromatography column with 60ml of packing material. The loading flow rate was 2 BV / h. After loading, the column was eluted with 3 BV of water and then immediately eluted with 3 BV of 10% ethanol. The eluent was collected and the ethanol was removed by rotary evaporator. After freeze-drying, the first purified product was obtained. The purity of the total flavonoids in the first purified product was 18.32%. (4) Dissolve the purified product to a concentration of 2 mg / ml and load it onto a macroporous resin D101 chromatography column. Macroporous resin D101 is activated before packing. Macroporous resin D101 is wet-packed into a 4×50cm chromatography column with 60ml of packing material. The loading flow rate is 1 BV / h. After loading, the column is eluted with 2 BV of water and then with 2 BV of 50% ethanol. The eluent is concentrated under reduced pressure at 40℃ and a vacuum of -0.1 MPa to remove the ethanol. The concentrate is then freeze-dried and pre-frozen at -80℃ for 12 hours at a vacuum of 10 Pa. The drying temperature is gradually and slowly increased to 0℃ and the drying time is maintained for 24 hours to obtain the purified total flavonoid extract of sea buckthorn leaves. The purity of the purified total flavonoid extract of sea buckthorn leaves is 35.55%.

[0018] Example 2 After grinding dried leaves of Chinese sea buckthorn, 50g of sea buckthorn leaf powder was weighed and ultrasonicated for 20min at 500W power with 50% ethanol at a material-to-liquid ratio of 1:20. The mixture was then filtered. The filtrate was concentrated on a rotary evaporator to remove ethanol at a concentration temperature of 45℃ and a vacuum degree of 0.08Mpa to obtain crude extract. The crude extract was diluted to 3 mg / ml and loaded onto a macroporous resin XDA-6 chromatography column. The XDA-6 column was a 4×50 cm column with 300 ml of activated macroporous resin wet-packed. The column was first washed with water to remove impurities. The sample was loaded at a flow rate of 2 BV / h, followed by elution with 3 BV of water and then with 20% ethanol. The eluent was removed from the ethanol using a rotary evaporator and freeze-dried to obtain the first-stage purified product. The total flavonoid purity was measured to be 19.76%. The purified product was dissolved to a concentration of 3 mg / ml and loaded onto a macroporous resin D101 chromatography column with 300 ml of packing material and a loading flow rate of 2 BV / h. After loading, the sample was eluted with 3 BV of water and then with 3 BV of 50% ethanol. The eluent was concentrated to remove the ethanol and then freeze-dried to obtain the purified total flavonoid extract of sea buckthorn leaves. The purity of the purified total flavonoid extract of sea buckthorn leaves was 35.7%.

[0019] Example 3 After grinding dried leaves of Chinese sea buckthorn, 50g of sea buckthorn leaf powder was weighed and ultrasonicated at 600W power for 30min with 70% ethanol at a material-to-liquid ratio of 1:40. The mixture was then filtered. The filtrate was concentrated on a rotary evaporator to remove ethanol at a concentration temperature of 42℃ and a vacuum degree of -0.1Mpa to obtain crude extract. The crude extract was diluted to 4 mg / ml and loaded onto a macroporous resin XDA-6 chromatography column. The XDA-6 column was pre-activated macroporous resin and wet-packed into a 4×50 cm column. The column was first washed with water to remove impurities, and then loaded at a flow rate of 2 BV / h. After loading, the column was eluted with 3 BV of water, followed by elution with 30% ethanol. The eluent was then removed from the ethanol using a rotary evaporator, and the product was freeze-dried to obtain the first-stage purified product. The total flavonoid purity was measured to be 21.13%. The purified product was dissolved to a concentration of 4 mg / ml and loaded onto a macroporous resin D101 chromatography column at a flow rate of 2 BV / h. After loading, the sample was eluted with 3 BV of water and then with 3 BV of 50% ethanol. The eluent was concentrated to remove the ethanol and then freeze-dried to obtain the purified total flavonoid extract of sea buckthorn leaves. The purity of the purified total flavonoid extract of sea buckthorn leaves was 36.7%.

[0020] Example 4 After grinding dried leaves of Chinese sea buckthorn, 50g of sea buckthorn leaf powder was weighed out and extracted with ultrasonically at 500W power for 25 minutes at a material-to-liquid ratio of 1:30 for 40% ethanol. The mixture was then filtered, and the filtrate was concentrated on a rotary evaporator to remove ethanol. The concentration temperature was 40℃ and the vacuum degree was -0.1Mpa to obtain crude extract. The crude extract was diluted to 3 mg / ml and loaded onto a macroporous resin XDA-6 chromatography column. The XDA-6 column was a 4×50 cm column with 300 ml of activated macroporous resin wet-packed. The column was first washed with water to remove impurities, and then loaded at a flow rate of 2 BV / h. After loading, the column was eluted with 3 BV of water, followed by elution with 40% ethanol. The ethanol was removed from the 40% eluent using a rotary evaporator, and the product was freeze-dried to obtain the first-stage purified product. The total flavonoid purity was measured to be 24.97%. The purified product was dissolved to a concentration of 3 mg / ml and loaded onto a macroporous resin D101 chromatography column with 300 ml of packing material and a loading flow rate of 2 BV / h. After loading, the sample was eluted with 3 BV of water and then with 3 BV of 50% ethanol. The eluent was concentrated to remove the ethanol and then freeze-dried to obtain the purified total flavonoid extract of sea buckthorn leaves. The purity of the purified total flavonoid extract of sea buckthorn leaves was 37.09%.

[0021] Example 5 After grinding dried leaves of Chinese sea buckthorn, 50g of sea buckthorn leaf powder was weighed out and extracted with ultrasonically at 600W power for 25 minutes at a material-to-liquid ratio of 1:30 for 60% ethanol. The mixture was then filtered, and the filtrate was concentrated on a rotary evaporator to remove ethanol. The concentration temperature was 40℃ and the vacuum degree was -0.1Mpa to obtain crude extract. The crude extract was diluted to 4 mg / ml and loaded onto a macroporous resin XDA-6 chromatography column. The XDA-6 column was pre-activated macroporous resin and wet-packed into a 4×50 cm column. The column was first washed with water to remove impurities, and the sample was loaded at a flow rate of 2 BV / h. After loading, the column was eluted with 3 BV of water, followed by elution with 50% ethanol. The eluent was then evaporated using a rotary evaporator to remove the ethanol. After freeze-drying, the purified product was obtained, and the total flavonoid purity was measured to be 14.88%. The purified product was dissolved to a concentration of 2 mg / ml and loaded onto a macroporous resin D101 chromatography column at a flow rate of 2 BV / h. After loading, the sample was eluted with 3 BV of water and then with 3 BV of 50% ethanol. The eluent was concentrated to remove the ethanol and then freeze-dried to obtain the purified total flavonoid extract of sea buckthorn leaves. The purity of the purified total flavonoid extract of sea buckthorn leaves was 35.3%.

[0022] Example 6 After grinding dried leaves of Chinese sea buckthorn, 50g of sea buckthorn leaf powder was weighed out and extracted with ultrasonically at a ratio of 1:30 for 40% ethanol for 25 minutes at 400W power. The filtrate was then filtered and concentrated on a rotary evaporator to remove ethanol. The concentration temperature was 43℃ ​​and the vacuum degree was 0.08Mpa to obtain crude extract. The crude extract was diluted to 2 mg / ml and loaded onto a macroporous resin XDA-6 chromatography column. The XDA-6 column was pre-activated macroporous resin and wet-packed into a 4×50 cm column. The column was first washed with water to remove impurities, and then loaded at a flow rate of 2 BV / h. After loading, the column was eluted with 3 BV of water, followed by elution with 60% ethanol. The eluent was then evaporated using a rotary evaporator to remove the ethanol. After freeze-drying, the purified product was obtained, and the total flavonoid purity was measured to be 13.05%. The purified product was dissolved to a concentration of 3 mg / ml and loaded onto a macroporous resin D101 chromatography column at a flow rate of 2 BV / h. After loading, the sample was eluted with 3 BV of water and then with 3 BV of 50% ethanol. The eluent was concentrated to remove the ethanol and then freeze-dried to obtain the purified total flavonoid extract of sea buckthorn leaves. The purity of the purified total flavonoid extract of sea buckthorn leaves was 35.05%.

[0023] Example 7 Determined by high performance liquid chromatography.

[0024] HPLC chromatographic conditions: Column GS-120-5-C18-AP (4.6 mm ID x 250 mm); Mobile phase: 0.4% phosphoric acid aqueous solution (A) - acetonitrile (B); Gradient elution program as follows: 0-15 min, 10-12% B; 15-38 min, 12-20% B; 38-48 min, 20-40% B; 48.58 min, 40-66% B; 58-62 min, 66-80% B; 62-64 min, 80-100% B; Detection wavelength: 260 nm; Flow rate: 0.8 mL / min; Column temperature: 30℃; Injection volume: 10 μL.

[0025] Preparation of reference solution: Take an appropriate amount of reference standard, accurately weigh it, and dissolve it in 80% methanol to obtain the solution.

[0026] Preparation of the test solution: Weigh 10 mg of this product (the purified total flavonoid extract of sea buckthorn leaves of this invention) accurately, place it in a 50 ml volumetric flask, and dissolve it in 80% methanol.

[0027] Accurately pipette 10 μl each of the reference solution and the test solution into the liquid chromatograph, measure and record the chromatograms. See Table 1.

[0028] UPLC separation was performed using an ACQUITY UPLC® BEH C18 1.7μm 2.1×50mm column. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile. Gradient elution conditions were as follows: 0–3 min (5% B), 3–4 min (5–9% B), 4–15 min (9–14% B), 15–21 min (14–60% B), 21–22 min (60–100% B), 22–23 min (100% B), 23–24 min (100–5% B), 24–28 min (5% B); injection volume: 2 µL; flow rate: 0.3 mL / min; column temperature: 36℃. Electrospray ionization (ESI) was used for data acquisition in negative ion detection mode. The QTOF-MS parameters in negative ion mode were as follows: capillary voltage 4000V, nebulizer voltage 25 psig, fragmentation voltage 400V, octodepletor RF 750V, dry gas temperature 225℃, dry gas flow rate 10 L / min, sheath gas temperature 350℃, sheath gas flow rate 12 L / min, and scan range 100-1500 m / z. Under MS / MS conditions, the collision energies were 10 eV, 20 eV, and 40 eV, and the mass range was 100~1500 m / z. By comparing with the standard, a total of 4 ellagitannin compounds (2, 3, 5, 6), 1 phenolic acid derivative (1), 14 flavonoid compounds (3, 4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19), 1 lignan compound (16), and 1 phenolic acid compound (7) were identified in its flavonoid components.

[0029] Table 1. Identification of total flavonoids

[0030]

[0031] Note: Duplicate numbers indicate peaks identified by liquid chromatography-mass spectrometry (LC-MS) analysis, which are mixtures.

[0032] Example 8 Experimental Objective: To test the lipid-lowering activity of purified sea buckthorn leaf flavonoids. Step 1: Accurately weigh the purified sea buckthorn leaf flavonoid powder, dissolve it in DMSO to prepare solutions of 50 μg / ml, 100 μg / ml, and 150 μg / ml, and store at -4℃ for later use; Step 2: HepG2 cell culture: Mix HepG2 cells with cell culture medium (DMEM medium containing 10% FBS and 1% antibiotics) thoroughly, and then add the mixture to a culture dish. Incubate the culture dish in a cell culture incubator at 37°C and 5% CO2. Change the medium every 48 hours. When the cell growth density reaches 80%-90%, passage the cells or proceed with subsequent experiments. Step 3: Establish a cellular lipid deposition model: HepG2 cells were divided into groups of 5 × 10⁻⁶ cells. 6 After inoculating into a six-well plate and incubating for 24 hours, discard the old solution in the plate, wash twice with PBS, add 0.5 mM FFA (OA:PA=2:1) ​​working solution and incubate for 24 hours, discard the old solution in the well, wash twice with PBS, and continue to culture with different concentrations of flavonoid purified product; The experiment was divided into 6 groups: normal group, model group (0.5mM FFA), three graded drug groups (0.5mM FFA + 50μg / ml flavonoid purified product, 0.5mM FFA + 100μg / ml flavonoid purified product, 0.5mM FFA + 150μg / ml flavonoid purified product), and positive control group (0.5mM FFA + 20μg / ml simvastatin). Step 4: Determination of TG, TC, LDL-C, and HDL-C: After 24 hours of intervention with different concentrations of drugs, the old solution in the plate was discarded, the plate was washed twice with PBS, and after digestion with trypsin, it was centrifuged at 1000 r / min for 10 min at 4°C. The contents of TG, TC, HDL-C, and LDL-C in the homogenate were determined according to the instructions of the triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) kits from Nanjing Jiancheng Biotechnology Co., Ltd. Each experiment was repeated three times, and the results are shown in the table below. Figure 2 .

[0033] Figure 2 The effects of different concentrations of flavonoid extracts on the lipid-lowering activity of HepG2 cells were investigated. The results showed that, # compared with the normal group, p < 0.01; * compared with the model group, p < 0.01 (n = 3). Depend on Figure 2 It can be seen that, compared with the blank control group, after treatment with 0.5mMFFA, the levels of TC, TG, and LDL-C in cells were significantly increased, while the level of HDL-C was significantly decreased. After treatment with different concentrations of total flavonoid extract, the levels of TC, TG, and LDL-C were all reduced to a certain extent, while the level of HDL-C was increased. 150μg / ml of total flavonoid extract can significantly reduce the levels of TC and TG in cells and increase the level of HDL-C.

[0034] Comparative Example The operation process is the same as in Example 1. The macroporous resins used for purification are macroporous resin XDA-6 and macroporous resin AB-8. The purification is performed once using macroporous resin XDA-6 and then again using macroporous resin AB-8. After collecting the eluent to remove ethanol, the purified total flavonoid extract of sea buckthorn leaves is obtained by freeze-drying. The purity of the purified total flavonoid extract of sea buckthorn leaves is 31.5%.

[0035] In summary, this invention employs ultrasound-assisted extraction. After screening seven macroporous resins, the extract is purified using macroporous resin XDA-6 and then further purified using macroporous resin D101 to obtain a total flavonoid extract with a purity of 35.0-37.09%. Furthermore, this extract (150 μg / ml) can significantly reduce TC and TG levels and increase HDL-C levels in HepG2 cells, indicating that this total flavonoid extract has the potential to regulate lipid metabolism and promote weight loss.

[0036] This invention not only enables the recycling and utilization of sea buckthorn leaves, but also enriches and purifies the total flavonoids in the leaves to obtain a high-purity total flavonoid extract. Furthermore, cellular level assays show that this extract has a positive regulatory effect on lipid metabolism in vivo, providing a foundation for the development and utilization of lipid-lowering products based on sea buckthorn leaf flavonoids.

Claims

1. A method for extracting and purifying total flavonoids from sea buckthorn leaves, characterized in that, Includes the following steps: (1) Pulverize the dried leaves of sea buckthorn to obtain sea buckthorn leaf powder; (2) Add the powder obtained in step (1) to an ethanol aqueous solution with a volume fraction of 50%-70% at a material-to-liquid ratio of 1:(20-40), extract it by ultrasonic extraction at a power of 400-600W for 20-30 minutes, filter it, concentrate the filtrate to remove ethanol, and obtain crude extract. (3) Dilute the crude extract to 2-5 mg / ml and load it onto the macroporous resin chromatography column of the purified crude extract. The specific surface area of ​​the macroporous resin of the purified crude extract is 450-600 m² / g and the average pore size is 10-15 nm. First, wash with water to remove impurities, and then elute with a gradient of 10%-60% ethanol solution at a flow rate of 0.5-2 BV / h and an elution volume of 2-4 BV. (4) Collect the eluent from step (3), concentrate the eluent to remove ethanol, freeze dry to obtain a first-purified product; (5) Dissolve the first-purified product to a concentration of 2-5 mg / ml and load it onto a macroporous resin chromatography column for purifying the first-purified product. The specific surface area of ​​the macroporous resin for purifying the first-purified product is ≥700 m² / g and the average pore size is 8.5-11 nm. The loading flow rate is 1-3 BV / h. After loading, elute with 2-4 BV of water and then elute with 2-4 BV of 10%-60% ethanol. Concentrate the eluent to remove the ethanol and freeze-dry to obtain the purified total flavonoid extract of sea buckthorn leaves. The purity of the purified total flavonoid extract of sea buckthorn leaves is 35.05-37.09%.

2. The method as described in claim 1, characterized in that: The sea buckthorn mentioned in step (1) is Chinese sea buckthorn, which is a seed source from Zhangxian County, Gansu Province; the moisture content of the dried sea buckthorn leaves is 5%-8%, and the dried sea buckthorn leaves are crushed and passed through a 40-60 mesh sieve.

3. The method as described in claim 1, characterized in that: The filtrate in step (2) is concentrated to remove ethanol by using a rotary evaporator to concentrate the filtrate to remove ethanol. The concentration temperature is 40-45℃ and the vacuum degree is -0.1-0.08 MPa. The eluent in step (4) is concentrated to remove ethanol by using a rotary evaporator to concentrate the eluent to remove ethanol. The concentration temperature is 35-40℃, the vacuum degree is -0.1-0.08 MPa, the freezing temperature is -70--90℃, and the dryness is 100 Pa. The eluent in step (5) is concentrated to remove ethanol by using a rotary evaporator to concentrate the eluent to remove ethanol. The concentration temperature is 35-40℃, the vacuum degree is -0.1-0.08 MPa, the freezing temperature is -70--90℃, and the dryness is 100 Pa.

4. The method as described in claim 1, characterized in that: The macroporous resin used in step (3) to purify the crude extract is XDA-6 macroporous resin. In step (3), the XDA-6 macroporous resin is activated before being loaded into the chromatography column. The activation step is to soak it in anhydrous ethanol solution for 24h to 48h for activation, and to continuously rinse it with distilled water until the eluent is clear. Finally, the activated adsorbent that has been rinsed clean is placed in an oven to dry for later use.

5. The method as described in claim 1, characterized in that: In step (3), the diameter-to-height ratio of the chromatography column is 1:(10-20), the chromatography column is 2×30cm-4×50cm, and the packing volume is 60-300ml.

6. The method as described in claim 1, characterized in that: The macroporous resin used to purify the first-stage purified product in step (5) is D101 macroporous resin. Before loading the D101 macroporous resin into the chromatography column in step (5), it is activated by soaking it in anhydrous ethanol solution for 24-48 hours, rinsing it continuously with distilled water until the eluent is clear, and finally drying the cleaned and activated adsorbent in an oven for later use.

7. The method as described in claim 1, characterized in that: In step (5), the diameter-to-height ratio of the chromatography column is 1:(10-20), the chromatography column is 2×30cm-4×50cm, and the packing volume is 60ml-300ml.

8. The use of the total flavonoid extract obtained by the method according to any one of claims 1-8 in the preparation of lipid-lowering drugs, characterized in that: The lipid-lowering activity includes reducing intracellular levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL-C), and increasing levels of high-density lipoprotein (HDL-C).

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