A method for simultaneously separating and purifying two galloylated myricitrin from Myrica rubra leaves and its use

Abstract

The present invention discloses a method for simultaneously separating and purifying myricetin-3- O -(2''-galloyl)-α-L-rhamnoside and myricetin-3- O -(4''-galloyl)-α-L-rhamnoside, and its application in the preparation of α-glucosidase inhibitors. The method includes: alcohol extraction and concentration, solid-phase extraction column adsorption, and preparative liquid chromatography purification, to separately prepare high-purity myricetin-3- O -(2''-galloyl)-α-L-rhamnoside monomer and myricetin-3- O -(4''-galloyl)-α-L-rhamnoside monomer, and the purity of both can be as high as over 98%. Compared with the conventional methods for separating and purifying flavonols, this process has simple separation steps, is easy to operate, has a short time consumption, and causes little environmental pollution; through activity tests, it is found that the myricetin-3- O -(2''-galloyl)-α-L-rhamnoside monomer and myricetin-3- O -(4''-galloyl)-α-L-rhamnoside monomer can significantly inhibit the activity of α-glucosidase, can be used for the preparation of α-glucosidase inhibitors, and has great significance for the in-depth research and further development and utilization of bayberry leaves.

Description

Technical Field

[0001] The present invention relates to the field of separation and purification of natural products, and particularly to a method and use for simultaneously separating and purifying myricetin-3-O-(2”-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside from Myrica rubra leaves. Background Art

[0002] In recent decades, with the changes in people's lifestyles and living conditions, the incidence of diabetes and its complications has increased sharply and has now become a severe global health problem. Diabetes is a metabolic disease characterized by hyperglycemia. Long-term hyperglycemia can lead to metabolic disorders in patients, causing the occurrence of various metabolic syndromes such as obesity, hyperlipidemia, and hypertension. Currently, the high prevalence of diabetes has imposed a serious social and economic burden on countries, especially low- and middle-income countries. Therefore, it is urgent to take effective prevention and control measures to control the rapid development of metabolic syndromes such as diabetes.

[0003] α-Glucosidase inhibitors have been recommended as first-line drugs for the treatment of diabetes and are an effective method for treating diabetes and preventing related complications. They can improve insulin resistance and effectively prevent and treat metabolic syndromes such as diabetes and obesity. α-Glucosidase is a membrane-bound enzyme in the glycoside hydrolase GH31 family and is mainly present in the brush border cells of the small intestinal villus mucosa. After a person eats, α-glucosidase can hydrolyze carbohydrates in food into glucose, and the glucose is absorbed and enters the blood circulation, causing blood sugar to rise. Diabetic patients have impaired pancreatic islet function and blood sugar regulation ability and cannot effectively control the rapid increase in postprandial blood sugar levels. α-Glucosidase inhibitors can effectively control the rapid increase in postprandial blood sugar by delaying the decomposition and absorption of carbohydrates and the release of glucose, improve insulin resistance, and inhibit fat synthesis. Currently, the commonly used α-glucosidase inhibitors in clinical practice mainly include acarbose, voglibose, and miglitol, etc. However, the administration of these drugs usually causes adverse gastrointestinal reactions such as flatulence, passing gas, and diarrhea. Therefore, searching for new α-glucosidase inhibitors with strong inhibitory activity and lower toxicity and side effects is of great significance for the prevention and treatment of metabolic syndromes such as diabetes and obesity.

[0004] In recent years, it has been found that flavonoid compounds from various natural sources have strong inhibitory effects on α-glucosidase, which has attracted extensive attention from scholars at home and abroad. Flavonoids generally refer to a series of substances in which two benzene rings (A ring and B ring) are connected to each other through three carbon atoms (C ring), that is, the general name of a class of compounds with a C6-C3-C6 structure. According to the structural characteristics such as the degree of oxidation of the C ring and the connection position of the B ring, flavonoids can be further divided into flavonols, anthocyanins, flavones, flavanones, and flavanols, etc. In a comparative study on the α-glucosidase inhibitory activities of 27 dietary flavonoids, myricetin was screened out as the most active inhibitor (Jia Y., et al., Journal of Agricultural and Food Chemistry 67(37):10521-10533(2019)). Similarly, in another report on the α-glucosidase inhibitory activities of 15 flavonoids, myricetin also showed the strongest inhibitory effect, followed by fisetin and quercetin (He C., et al., Foods 8(9):355(2019)), and these substances all belong to flavonol compounds. This indicates that flavonol compounds such as myricetin may be α-glucosidase inhibitors with great development potential.

[0005] Myrica leaves are natural resources rich in flavonols such as myricetin. Myrica trees are evergreen throughout the year with lush branches and leaves. To avoid affecting the fruit setting rate due to apical dominance and promote the continuous, high-quality, and high-yield production of Myrica, farmers need to prune Myrica fruit trees in both spring and autumn, thus generating a large amount of waste Myrica leaves. These leaves are usually burned as firewood, increasing carbon emissions and causing environmental pollution. Ancient medical books recorded that Myrica leaves are bitter, slightly pungent, and warm in nature, and can be used to treat diseases such as diarrhea, jaundice hepatitis, lymph tuberculosis, and chronic pharyngitis. In recent years, a large number of studies have shown that the extracts of Myrica leaves have good activities such as scavenging free radicals, anti-inflammatory, and antibacterial. Therefore, the development and utilization of Myrica branches and leaves can not only solve the environmental pollution problems brought by waste treatment, but also turn waste into treasure, increase value and generate income, and bring benefits to human health. Myricetin specifically accumulates in Myrica leaves, and the content can be as high as more than 10 mg / g fresh weight. However, in the leaves, myricetin usually exists in the form of derivatives after being modified by glycosylation, galloylation, etc. However, due to the similar structures and small polarity differences of these flavonol derivatives, the separation of high-purity monomers is very difficult, and their structures cannot be accurately characterized, which greatly limits the exploration of the pharmacological activities of Myrica leaves and their further development and utilization.

[0006] For example, Zhang et al. identified flavonol compounds in Myrica rubra leaves by LC-MS, but only inferred their possible structures based on the secondary fragment ion spectra, without performing precise structure analysis (Zhang, Y., PLoS One 11(12):e0167484(2016)); Chen Ping et al. ("Screening, isolation and purification of antibacterial components in Myrica rubra leaves", Food Science and Technology, 2011, Vol. 36, No. 2: 189-192) fractionated the ethanol extract of Myrica rubra leaves by multiple-step extraction with organic solvents, macroporous adsorption resin column chromatography, and gel column chromatography to obtain a crude flavonoid extract, but failed to purify a relatively pure flavonol monomer; Kim, H.H., et al. (Archives of Pharmacal Research, 36(12):1533-1540(2013)) separated and purified flavonols in Myrica rubra leaves. The crude extract of Myrica rubra leaves was subjected to repeated column chromatography and then reverse medium-pressure liquid chromatography to obtain flavonol monomers, but the extraction and purification processes were complex and the extraction efficiency was low, limiting the comprehensive utilization of Myrica rubra leaves. SUMMARY OF THE INVENTION

[0007] To solve the above technical problems, the present invention provides a method and use for simultaneously separating and purifying myricetin-3-O-(2”-O-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-O-galloyl)-α-L-rhamnoside from Myrica rubra leaves.

[0008] Based on this, the inventors used Myrica rubra leaves as raw materials to establish a separation and purification system combining ethanol extraction and concentration, solid-phase extraction column purification, and preparative liquid chromatography. For the first time, myricetin-3-O-(4”-O-galloyl)-α-L-rhamnoside (CAS No.: 85541-03-3) was isolated from Myrica rubra leaves, and it was found that this compound (IC 50 = 15.61 μM) has significantly better α-glucosidase inhibitory activity than the positive control drug acarbose (IC 50 = 369.15 μM) and myricetin (IC 50 = 1.77 μM). Secondly, myricetin-3-O-(2”-O-galloyl)-α-L-rhamnoside (CAS No.: 56939-52-7) was also isolated, and it was found that it can also significantly inhibit α-glucosidase activity (IC 50= 1.32 μM). These two compounds are isomers. Due to their similar structures and close polarities, they are difficult to separate and purify. In the latest reported literature (da Silva, G. L., et al., Natural Product Research 22: 1-5 (2021)), it was found that these two compounds are contained in Syzygium aromaticum leaves in the shape of a pear, but the separation and preparation of the two have not been achieved. Therefore, only the activities of the mixture containing these two substances were measured. The present invention has successfully established a purification system for rapidly and efficiently separating these two compounds, and can simultaneously prepare monomers of myricetin-3-O-(4”-galloyl)-α-L-rhamnoside and myricetin-3-O-(2”-galloyl)-α-L-rhamnoside with high purity (purity above 98%). Activity tests on the two purified flavonol monomers found that they have excellent α-glucosidase inhibitory activity, indicating that they have great potential to be developed into α-glucosidase inhibitors for the prevention and treatment of metabolic syndromes such as diabetes and obesity. This is of great significance for the further exploration of functional components in bayberry leaves, the exploration of pharmacological activities, and the improvement of the added value of the bayberry industry.

[0009] The present invention adopts the following technical solutions:

[0010] A method for simultaneously separating and purifying myricetin-3-O-(2”-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside from bayberry leaves, comprising:

[0011] (1) Alcohol extraction and concentration: Mix bayberry leaves with an alcohol solution, filter and collect the filtrate after ultrasonic extraction, remove the alcohol from the filtrate and concentrate it to obtain a crude extract of flavonols from bayberry leaves, and the volume percentage concentration of the alcohol solution is 50-100%;

[0012] (2) Adsorption on a solid-phase extraction column: Inject the crude extract of flavonols from bayberry leaves into a solid-phase extraction column, perform a first-gradient elution with a mobile phase, and post-treat the collected eluate to obtain a solid-phase extraction powder rich in myricetin-3-O-(2”-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside; preferably, the solid-phase extraction column is a C18 solid-phase extraction column;

[0013] The process of the first-gradient elution is as follows: First, perform a first elution with an alcohol solution with a volume percentage concentration lower than 40%, and then perform a second elution with an alcohol solution with a volume percentage concentration of 40%-60%, and collect the eluate after the second elution; the volume percentage concentration of the alcohol solution for the first elution is above 20%;

[0014] Preferably, perform the first elution with an alcohol solution with a volume percentage concentration of 30%;

[0015] Preferably, a 40% alcohol solution by volume is used for the second elution;

[0016] (3) Purification by preparative liquid chromatography: Using a solid-phase chromatography column, the solid-phase extraction powder obtained in step (2) is subjected to a second gradient elution with a mobile phase and then post-treated to obtain the target products: myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer; preferably, the solid-phase chromatography column used is a C18 solid-phase chromatography column;

[0017] The mobile phase: Phase A is selected from formic acid-aqueous solutions with a volume percentage concentration of 0.05% to 5% and trifluoroacetic acid-aqueous solutions with a volume percentage concentration of 0.05% to 5%, and Phase B is selected from acetonitrile-aqueous solutions with a volume percentage concentration of 40 to 60% and acid-acetonitrile-aqueous solutions with a volume percentage concentration of 40 to 60%, where the volume percentage concentration of the acid is 0.05% to 5%, and the acid is selected from formic acid and trifluoroacetic acid;

[0018] The process of the second gradient elution is as follows: the volume percentage concentration of Phase B rises from 20% to 60% within 0 to 10 minutes, from 60% to 90% within 10 to 30 minutes, from 90% to 100% within 30 to 35 minutes, and finally drops from 100% to 20% within 35 to 40 minutes, and the target products are collected separately;

[0019] Preferably, Phase A is selected from formic acid-aqueous solutions with a volume percentage concentration of 0.1% to 3%;

[0020] Preferably, in step (3), a preparative liquid column SunFire TM C18 OBM TM column (5μm, 19×250mm) is used. During the second gradient elution, the eluates at 27 to 28.5 minutes and 28.5 to 30 minutes are collected separately; more preferably, the column temperature is at room temperature and the flow rate is 3 - 6 mL / min;

[0021] Preferably, the solid-phase extraction powder prepared in step (2) is dissolved in methanol to a concentration of 100 - 200 mg / mL, injected into the preparative liquid for purification, and the single injection volume is 50 - 300 μL;

[0022] The alcohol solution refers to an aqueous solution of alcohol, and the alcohol is selected from methanol or ethanol;

[0023] Preferably, the volume percentage concentration of the alcohol solution is 80%;

[0024] Preferably, the time for ultrasonic extraction is 30 - 60 minutes;

[0025] Preferably, the mass-volume ratio of the myrica leaves to the methanol solution is 1:5 to 20; more preferably, the mass-volume ratio of the myrica leaves to the methanol solution is 1:10;

[0026] Preferably, the post-treatment refers to reduced pressure concentration and freeze-drying; more preferably, the conditions for the reduced pressure concentration are: vacuum rotary evaporation at 37 to 50 °C;

[0027] Preferably, the purity of the myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer and the myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer is above 98%; preferably, the purity is above 99%.

[0028] Preferably, in step (1), the filtrate is vacuum rotary evaporated at 37 to 50 °C to remove alcohol and concentrated to obtain a crude extract of myricetin from myrica leaves.

[0029] Preferably, in step (1), the process of collecting the filtrate is repeated 2 to 4 times, and the filtrates of 2 to 4 times are combined.

[0030] Preferably, in step (2), the gradient elution of the solid-phase extraction column is specifically as follows:

[0031] Inject the crude extract of myricetin from myrica leaves into the solid-phase extraction column, first rinse the solid-phase extraction column with deionized water, perform gradient elution with the mobile phase, and vacuum rotary evaporate the collected eluate to dryness at 37 to 45 °C to obtain the solid-phase extraction powder rich in the target product.

[0032] Among them, the function of rinsing with an alcohol solution below 40% in the first elution is to remove impurities such as sugar acids and other non-target flavonols such as myricitrin. If the concentration of the alcohol solution in the first elution is lower than 20%, the flavonols cannot be eluted.

[0033] Preferably, in step (2), the adsorption of the solid-phase extraction column is specifically as follows:

[0034] C18 After the solid-phase extraction column is activated, 4.5 BV of the myrica leaf extract is loaded onto each solid-phase extraction column; 4 BV of deionized water is used to wash away sugar acids; then 10 BV of a methanol solution with a volume percentage concentration of 30% is used for elution to remove some impurities and other non-target flavonols; then 4 BV of a methanol solution with a volume percentage concentration of 40% is used for elution, and the eluate of the 40% component is collected and vacuum rotary evaporated to dryness at 37 °C to 50 °C to obtain the solid-phase extraction powder rich in myricetin-3-O-(2”-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside.

[0035] Preferably, in step (3), phase B is selected from an acetonitrile - aqueous solution with a volume percentage concentration of 50%, an acid - acetonitrile - aqueous solution with a volume percentage concentration of 50%, the volume percentage concentration of the acid is 0.1% - 5%, and the acid is selected from formic acid and trifluoroacetic acid; preferably, the volume percentage concentration of the acid is 0.1% - 3%.

[0036] Preferably, in step (3), the preparative liquid chromatography purification is specifically as follows:

[0037] Use a preparative liquid chromatography column SunFire TM C18 OBM TM Column (5μm, 19×250mm), mobile phase: phase A: formic acid - aqueous solution with a volume percentage concentration of 0.1%, phase B: formic acid - acetonitrile - aqueous solution with a volume percentage concentration of 50% (wherein the volume percentage concentration of formic acid is 0.1%); the column temperature is at room temperature, and the flow rate is 3 - 6 mL / min;

[0038] Dissolve the solid - phase extraction powder prepared in step (2) with methanol to make its concentration reach 100 - 200 mg / mL, inject it into the preparative liquid chromatography for purification, and the single - injection volume is 50 - 300 μL;

[0039] Collect the eluates at 27 - 28.5 min and 28.5 - 30 min separately in tubes, combine the eluate rich in the pure target product, and after concentration under reduced pressure and freeze - drying, high - purity myricetin - 3 - O - (2'' - galloyl) - α - L - rhamnoside monomer and myricetin - 3 - O - (4'' - galloyl) - α - L - rhamnoside monomer can be obtained respectively.

[0040] The alcohol solution is methanol or ethanol. It is found that the concentration of the target product in the acetone extract is low and the target product in the sample cannot be fully extracted. Solvents such as ethyl acetate and petroleum ether cannot extract the target product (myricetin - 3 - O - (2'' - galloyl) - α - L - rhamnoside monomer and myricetin - 3 - O - (4'' - galloyl) - α - L - rhamnoside monomer), as shown in Example 16 and Figure 9 shown.

[0041] The volume percentage concentration of the alcohol solution is 50% - 100%. The aqueous solution of the target product is poor, and its lipophilicity is strong. Therefore, using an alcohol solution with a low volume concentration will reduce the extraction rate of the target product.

[0042] Preferably, the mass - to - volume ratio of the myrica leaves to the methanol solution is 1∶5 - 20. If the material - to - liquid ratio is too low, the extraction is insufficient; if the material - to - liquid ratio is too high, it will cause unnecessary waste of reagents.

[0043] Preferably, the ultrasonic extraction time is 30 - 60 min. If the ultrasonic time is too short, the extraction is insufficient; if the ultrasonic time is too long, the temperature of the extraction solution rises, affecting the extraction effect. It is repeated 2 - 4 times. If the extraction times are too few, the target components are not fully extracted; if the extraction times are too many, the solvent is wasted.

[0044] The present invention also provides the use of myricetin - 3 - O - (2'' - galloyl) - α - L - rhamnoside as an active ingredient in the preparation of an α - glucosidase inhibitor; preferably, the myricetin - 3 - O - (2'' - galloyl) - α - L - rhamnoside is isolated and purified according to any one of the methods described above.

[0045] The present invention also provides the use of myricetin - 3 - O - (4'' - galloyl) - α - L - rhamnoside as an active ingredient in the preparation of an α - glucosidase inhibitor; preferably, the myricetin - 3 - O - (4'' - galloyl) - α - L - rhamnoside is isolated and purified according to any one of the methods described above.

[0046] Preferably, the α - glucosidase inhibitor is a metabolic syndrome improver or a drug; preferably, the metabolic syndrome is at least one of the diseases such as type 2 diabetes, obesity, insulin resistance, and hyperinsulinemia; preferably, the improver is selected from functional foods, food additives, and supplements.

[0047] In addition to containing the above - mentioned myricetin - 3 - O - (2'' - galloyl) - α - L - rhamnoside and / or myricetin - 3 - O - (4'' - galloyl) - α - L - rhamnoside of the present invention, the drug of the present invention may also contain, for example, other active ingredients, pharmaceutically acceptable additives, etc. For the drug of the present invention, as specific dosage forms, for example, tablets, granules (including powders), capsules, liquid preparations (including syrups), etc. can be listed. Additives or substrates suitable for each dosage form can be appropriately used and manufactured according to the general methods described in pharmacopoeias, etc. In addition, as the administration route, there is no particular limitation. For example, oral administration and parenteral administration can be listed. As the above - mentioned parenteral administration, for example, intraoral administration, intratracheal administration, rectal administration, subcutaneous administration, intramuscular administration, and intravenous administration, etc. can be listed.

[0048] The metabolic syndrome improver of the present invention may also contain various additives, other supplements, etc. For example, it may contain other active ingredients, various vitamins such as vitamin C, amino acids, oligosaccharides, etc. The form of the improver of the present invention is not particularly limited. For example, tablets, granules (including powders), capsules, liquid preparations (including syrups), etc. can be listed.

[0049] The functional food of the present invention may also contain various additives, etc., for example, it may contain other active ingredients, etc. In addition, the form of the functional food of the present invention is not particularly limited, and examples thereof may include noodles, snacks, and functional beverages, etc.

[0050] The food additive of the present invention may also contain various additives, etc., for example, it may contain other active ingredients, etc. The form of the food additive of the present invention is not particularly limited, and examples thereof may include liquid, paste, powder, flake, and granular, etc. In addition, the food additive of the present invention also includes, for example, food additives for beverages.

[0051] Further activity tests found that the isolated and purified myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer has a significant inhibitory effect on α-glucosidase, and the IC 50 value is 1.32 μM, which is significantly better than the positive drug acarbose (IC 50 = 369.15 μM), and can be used as a new type of natural-source α-glucosidase inhibitor for controlling postprandial blood glucose and preventing and treating metabolic syndromes such as diabetes, obesity, and insulin resistance.

[0052] Further activity tests found that the isolated and purified myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer has a significant inhibitory effect on α-glucosidase, and the IC 50 value is 1.77 μM, which is significantly better than the positive drug acarbose (IC 50 = 369.15 μM), and can be used as a new type of natural-source α-glucosidase inhibitor for controlling postprandial blood glucose and preventing and treating metabolic syndromes such as diabetes, obesity, and insulin resistance.

[0053] Compared with the prior art, the present invention has the following advantages:

[0054] The present invention first isolated and purified the myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer and the myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer from myrtle leaves. Due to the similarity of these two flavonoid monomers, namely the myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer and the myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer, the prior art has been unable to solve the problem of isolation and purification. The present invention pioneered the purification of these two monomers, and the steps are simple and easy to operate, with short time consumption, little environmental pollution, and high purity of the purified monomers.

[0055] The raw materials of the present invention are easily obtainable and are isolated from bayberry leaves. The specific isolation steps are simple and easy to operate, time-consuming short, with little environmental pollution. The purified monomer has a high purity, and can also provide a reference for the isolation and purification of other plant active substances, which is of great significance for the research on the effective components of natural products.

[0056] The compounds myricetin-3-O-(2”-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside isolated by the present invention have various medicinal activities such as antioxidant, and can significantly inhibit the activity of α-glucosidase, and can be used to prepare α-glucosidase inhibitors, which is of great significance for the in-depth research and further development and utilization of bayberry leaves. Description of the Drawings

[0057] Figure 1 It is the high performance liquid chromatography (HPLC) diagram of the crude flavonol extract from bayberry leaves in Example 1. Among them, 2” represents the monomer of myricetin-3-O-(2”-galloyl)-α-L-rhamnoside, and 4” represents the monomer of myricetin-3-O-(4”-galloyl)-α-L-rhamnoside.

[0058] Figure 2 It is the high performance liquid chromatography (HPLC) diagram of the solid phase extraction powder rich in the target product in Example 1. Among them, 2” represents the monomer of myricetin-3-O-(2”-galloyl)-α-L-rhamnoside, and 4” represents the monomer of myricetin-3-O-(4”-galloyl)-α-L-rhamnoside.

[0059] Figure 3 It is the high performance liquid chromatography (HPLC) diagram of the finally isolated and purified myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer (a) and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer (b) in Example 1.

[0060] Figure 4 It is the high performance liquid chromatography (HPLC) diagram of the finally isolated and purified myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer (a) and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer (b) in Example 2.

[0061] Figure 5 It is the high performance liquid chromatography (HPLC) diagram of the finally isolated and purified myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer (a) and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer (b) in Example 3.

[0062] Figure 6a 、 Figure 6b and Figure 6cThe first-order ( Figure 6a ) and second-order ( Figure 6b ) ion fragmentation diagrams identified by LC-MS and the NMR identification diagram ( Figure 6c ) of the myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer obtained by separation and purification.

[0063] Figure 7a 、 Figure 7b and Figure 7c The first-order ( Figure 7a ) and second-order ( Figure 7b ) ion fragmentation diagrams identified by LC-MS and the NMR identification diagram ( Figure 7c ) of the myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer obtained by separation and purification.

[0064] Figure 8 The α-glucosidase inhibitory activity curves of the myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer and the myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer obtained by separation and purification are given, and the α-glucosidase inhibitory activity curve of acarbose is given for comparison.

[0065] Figure 9 The HPLC liquid phase detection diagrams of extracting bayberry leaves with solvents of different polarities. Detailed implementation manners

[0066] The present invention will be further described below in conjunction with specific embodiments. The following are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto:

[0067] Example 1

[0068] Weigh 20 g of bayberry leaves, add pure methanol solution according to the ratio of solid-liquid ratio 1:10 (w / v, g / mL), mix well, extract ultrasonically for 30 min, filter by suction after the ultrasonic extraction ends, repeat the extraction of the obtained filter residue under the above conditions once, combine the filtrates, and remove methanol by vacuum rotary evaporation at 40 °C to obtain a flavonol crude extract ( Figure 1 ).

[0069] First, activate the C18 solid-phase extraction column (waters 12 cc, 2 g) with 4 BV of methanol and 2 BV of water. Then, load the flavonol crude extract onto each solid-phase extraction column with 4.5 BV; wash away sugars and acids with 4 BV of deionized water; elute with 10 BV of methanol solution with a volume percentage concentration of 30% and 4 BV of methanol solution with a volume percentage concentration of 40% respectively, collect the eluate of the methanol solution with a volume percentage concentration of 40%, and evaporate to dryness under vacuum at 40 °C to obtain a solid-phase extraction powder rich in the target product ( Figure 2)。

[0070] Using a preparative liquid chromatography column SunFire TM C18 OBM TM Column (5μm, 19×250mm), mobile phase: Phase A: formic acid - aqueous solution with a volume percentage concentration of 0.1%, Phase B: 50% acetonitrile - aqueous solution (containing 0.1% formic acid); column temperature is 25°C, flow rate is 5 mL / min, gradient: 0 - 10 min, 20% - 60% B; 10 - 30 min, 60% - 90% B; 30 - 35 min, 90% - 100% B; 35 - 40 min, 100% - 20% B. Dissolve the solid phase extraction powder with methanol to make its concentration reach 100 mg / mL, inject it into the preparative liquid chromatography for separation, and the single injection volume is 100 μL. Detect under a Waters 2998 PAD detector, separately collect the eluates at 27 - 28.5 min and 28.5 - 30 min, concentrate under reduced pressure respectively, and vacuum freeze - dry to obtain myricetin - 3 - O - (2'' - galloyl) - α - L - rhamnoside monomer powder with a purity of 98.86%( Figure 3 a) in Figure 3 and myricetin - 3 - O - (4'' - galloyl) - α - L - rhamnoside monomer powder with a purity of 99%(

[0071] Example 2

[0072] Weigh 30 g of myrica leaves, add 80% methanol aqueous solution (volume ratio of methanol to water is 80:20) according to the ratio of solid - liquid ratio 1:10 (w / v, g / mL), mix well, extract ultrasonically for 30 min, filter by suction after the ultrasonic extraction ends, repeat the extraction of the obtained filter residue under the above conditions once, combine the filtrates, and remove methanol by vacuum rotary evaporation at 37°C to obtain a crude flavonol extract.

[0073] First, activate the C18 solid phase extraction column with 4 BV of methanol and 2 BV of water. Then, load the crude flavonol extract onto each solid phase extraction column with 4.5 BV; wash away sugars and acids with 4 BV of deionized water; elute with 10 BV of methanol solution with a volume percentage concentration of 30% and 4 BV of methanol solution with a volume percentage concentration of 40%, collect the eluate of the methanol solution with a volume percentage concentration of 40%, and evaporate to dryness by vacuum rotary evaporation at 37°C to obtain a solid phase extraction powder rich in the target product.

[0074] Using a preparative liquid chromatography column SunFire TM C18 OBM TMColumn (5μm, 19×250mm), mobile phase: Phase A: formic acid - aqueous solution with a volume percentage concentration of 0.1%, Phase B: 50% acetonitrile - aqueous solution (containing 0.1% formic acid); column temperature is 25°C, flow rate is 5 mL / min, gradient: 0 - 10 min, 20% - 60% B; 10 - 30 min, 60% - 90% B; 30 - 35 min, 90% - 100% B; 35 - 40 min, 100% - 20% B. Dissolve the solid phase extraction powder with methanol to make its concentration reach 150 mg / mL, inject it into the preparative liquid phase for separation, and the single injection volume is 150 μL. Detect under Waters 2998 PAD detector, separately collect the eluates at 27 - 28.5 min and 28.5 - 30 min, concentrate under reduced pressure respectively, and freeze-dry in vacuum to obtain myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer powder with a purity of 98.19%( Figure 4 a) in Figure 4 and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer powder with a purity of 98.32%(

[0075] Example 3

[0076] Weigh 60 g of myrica leaves, add 80% methanol aqueous solution (volume ratio of methanol to water is 80:20) according to the ratio of solid-liquid ratio 1∶10 (w / v, g / mL), mix well, extract ultrasonically for 30 min, filter by suction after ultrasonic extraction, repeat the extraction of the obtained filter residue under the above conditions once, combine the filtrates, and remove methanol by vacuum rotary evaporation at 37°C to obtain a crude flavonol extract.

[0077] First activate the C18 solid phase extraction column with 4 BV of methanol and 2 BV of water. Then load the crude flavonol extract onto each solid phase extraction column with 4.5 BV; wash away sugars and acids with 4 BV of deionized water; elute with 10 BV of methanol solution with a volume percentage concentration of 30% and 4 BV of methanol solution with a volume percentage concentration of 40%, collect the eluate of the methanol solution with a volume percentage concentration of 40%, and evaporate to dryness by vacuum rotary evaporation at 37°C to obtain a solid phase extraction powder rich in the target product.

[0078] Use the preparative liquid phase column SunFire TM C18 OBM TMColumn (5 μm, 19 × 250 mm), mobile phase: Phase A: pure water containing 0.1% formic acid system, Phase B: 50% acetonitrile containing 0.1% formic acid system; column temperature was 25 °C, flow rate was 5 mL / min, gradient: 0 - 10 min, 20% - 60% B; 10 - 30 min, 60% - 90% B; 30 - 35 min, 90% - 100% B; 35 - 40 min, 100% - 20% B. The powder rich in the target product was dissolved in methanol to make its concentration reach 150 mg / mL, and then injected into the preparative liquid phase for separation. The single injection volume was 200 μL. Detection was carried out under a Waters 2998 PAD detector, and the eluates at 27 - 28.5 min and 28.5 - 30 min were collected and fractionally collected, concentrated under reduced pressure respectively, and freeze-dried in vacuum to obtain myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer powder with a purity of 98.30%( Figure 5 a) in Figure 5 , and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer powder with a purity of 98.13%(

[0079] According to the separation method of Example 1, some parameters in the process were changed, and the following Examples 4 - 13 were carried out (Note: the concentrations in the table are all volume percentages, the 2” monomer refers to myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer; the 4” monomer refers to myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer).

[0080]

[0081]

[0082] Example 14

[0083] The structures of the two compound monomers separated in the above three examples were analyzed by high-resolution LC-MS and NMR techniques. The primary and secondary ion fragmentation diagrams identified by high-resolution LC-MS and the NMR identification diagrams are shown in Appendices Figure 6a 、 Figure 6b and Figure 6c , Figure 7a 、 Figure 7b and Figure 7c . Thus, it was determined that the two purified monomers were isomers, namely myricetin-3-O-(2”-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside.

[0084] Myricetin-3-O-(2”-galloyl)-α-L-rhamnoside. 1313C NMR (151 MHz, DMSO-d6) δ 177.44 (C-4), 164.94 (COO), 164.19 (C-7), 161.25 (C-5), 157.49 (C-2), 156.39 (C-9), 145.78 (C-3' / 5'), 145.44 (C-3"' / 5"'), 138.53 (C-4"'), 136.55 (C-4'), 133.32 (C-3), 119.38 (C-1'), 119.24 (C-1"'), 108.84 (C-2' / 6'), 107.93 (C-2"' / 6"'), 103.97 (C-10), 98.70 (C-6), 98.33 (C-1"), 93.54 (C-8), 71.75 (C-4"), 71.68 (C-2"), 70.65 (C-5"), 68.55 (C-3"), 17.56 (C-6").

[0085] Myricetin-3-O-(4”-galloyl)-α-L-rhamnoside. 13 13C NMR (151 MHz, DMSO-d6) δ 177.78 (C-4), 165.70 (COO), 164.30 (C-7), 161.30 (C-5), 157.63 (C-2), 156.47 (C-9), 145.81 (C-3' / 5'), 145.38 (C-3"' / 5"'), 138.27 (C-4"'), 136.50 (C-4'), 134.85 (C-3), 120.00 (C-1"'), 119.61 (C-1'), 108.99 (C-2' / 6'), 107.93 (C-2"' / 6"'), 104.03 (C-10), 102.71 (C-1"), 98.70 (C-6), 93.57 (C-8), 73.90 (C-4"), 70.87 (C-2"), 68.61 (C-3"), 67.78 (C-5"), 17.46 (C-6").

[0086] Example 15

[0087] α-Glucosidase can hydrolyze 4-nitrophenyl-β-D-glucopyranoside (PNPG) to generate p-nitrophenol (pNP). Under alkaline conditions, pNP has a maximum absorption value at 405 nm. By measuring the concentration of pNP in the reaction solution with a microplate reader, the inhibitory activity of the sample against α-glucosidase can be detected.

[0088] The α-glucosidase was diluted to 0.2 U / mL with 0.1 mol / L buffer (pH = 6.8). During the determination, 112 μL of phosphate buffer (0.1 mol / L, pH = 6.8) and 20 μL of α-glucosidase (0.2 U / mL) were added to a 96-well plate. Subsequently, 8 μL of inhibitor, namely myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer or myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer or acarbose at gradient concentrations, was added. It was left standing at 37 °C for 15 min, then 20 μL of PNPG with a concentration of 2.5 mmol / L was added. After reacting at 37 °C for 15 min, 80 μL of Na 2 CO 3 solution (2.5 mmol / L) was added, and the absorbance (OD test ) at 405 nm was measured with an enzyme-linked immunosorbent assay reader. The one without enzyme was used as the blank control (OD blank ), the one without sample but with enzyme was controlOD test , the one without sample and without enzyme was controlOD blank , and acarbose was used as the positive control. The calculation formula for the enzyme activity inhibition rate is:

[0089]

[0090] According to the enzyme activity inhibition rate of the test monomers at gradient concentrations on α-glucosidase, the IC 50 value was calculated using SPSS. The results showed that both myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer had significant inhibitory effects on α-glucosidase, and the IC 50 values were 1.32 μM and 1.77 μM respectively, which were significantly better than the positive drug acarbose (IC 50 = 369.15 μM). This indicates that myricetin-3-O-(2”-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside are good α-glucosidase inhibitors, can be used for the development of drugs for the treatment of diabetes, and can be applied to improve insulin resistance and prevent and treat metabolic syndromes such as diabetes and obesity.

[0091] Example 16

[0092] Screening of extraction solvents

[0093] 0.1 g of freeze-dried myrica leaves powder was weighed and dissolved in 1 mL of each of the 5 selected organic solvents. After ultrasonic extraction for 30 min, it was centrifuged at 10000 rpm for 10 min to take the supernatant. It was extracted again according to the above steps, and the supernatants of the two extractions were combined for HPLC detection and analysis.

[0094] The relative content of the specified compound in the solution can be determined by the peak area detected by HPLC. As Figure 9 shown, it was found that the concentration of the target product in the acetone extract was low and the target product in the sample could not be fully extracted. The target product was not detected in the ethyl acetate and petroleum ether extracts. The target products refer to: myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer. Therefore, methanol and ethanol are more suitable for the extraction of the two target products from myrtle leaves.

[0095] Comparative Example 1

[0096] Weigh 20 g of myrtle leaves, add pure methanol solution in a ratio of solid-to-liquid of 1:10 (w / v, g / mL), mix well, extract ultrasonically for 30 min, filter by suction after the ultrasonic extraction is completed, repeat the extraction of the obtained filter residue under the above conditions once, combine the filtrates, and remove methanol by vacuum rotary evaporation at 40 °C to obtain a crude flavonol extract.

[0097] First, activate C18 solid-phase extraction column (waters 12 cc, 2 g) with 4 BV of methanol and 2 BV of water. Then load 4.5 BV of the crude flavonol extract onto each solid-phase extraction column; wash with 4 BV of deionized water to remove sugars and acids; elute with 10 BV of methanol solution with a volume percentage concentration of 10% and 4 BV of methanol solution with a volume percentage concentration of 40% respectively, collect the eluate of the methanol solution with a volume percentage concentration of 40%, and evaporate to dryness under vacuum at 40 °C to obtain a solid-phase extraction powder rich in the target product.

[0098] Use the preparative liquid column SunFire TM C18 OBM TMColumn (5 μm, 19×250 mm), mobile phase: Phase A: formic acid - aqueous solution with a volume percentage concentration of 0.1%, Phase B: 50% acetonitrile - aqueous solution (containing 0.1% formic acid); column temperature is 25 °C, flow rate is 5 mL / min, gradient: 0 - 10 min, 20% - 60% B; 10 - 30 min, 60% - 90% B; 30 - 35 min, 90% - 100% B; 35 - 40 min, 100% - 20% B. Dissolve the solid phase extraction powder with methanol to make its concentration reach 100 mg / mL, inject it into the preparative liquid phase for separation, and the single injection volume is 100 μL. Detect under a Waters 2998 PAD detector, separately collect the eluates at 27 - 28.5 min and 28.5 - 30 min, concentrate under reduced pressure respectively, and vacuum freeze-dry to obtain myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer powder with a purity of 80.18% and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer powder with a purity of 75.25% in sequence.

[0099] Comparative Example 2

[0100] Weigh 20 g of myrica leaves, add pure methanol solution according to the ratio of solid-to-liquid ratio of 1:10 (w / v, g / mL), mix well, extract ultrasonically for 30 min, filter by suction after the ultrasonic extraction is over, repeat the extraction of the obtained filter residue under the above conditions once, combine the filtrates, and remove methanol by vacuum rotary evaporation at 40 °C to obtain a crude flavonol extract.

[0101] First, activate C18 with 4 BV of methanol and 2 BV of water Solid phase extraction column (waters 12 cc, 2 g), then load the crude flavonol extract onto each solid phase extraction column with 4.5 BV; wash with 4 BV of deionized water to remove sugar acids; elute with 10 BV of methanol solution with a volume percentage concentration of 20% and 4 BV of methanol solution with a volume percentage concentration of 30% respectively, collect the eluate of the methanol solution with a volume percentage concentration of 30%, and vacuum rotary evaporate to dryness at 40 °C, and it is found that the obtained solid phase extraction powder does not contain the target product.

[0102] Comparative Example 3

[0103] Weigh 20 g of myrica leaves, add petroleum ether solution according to the ratio of solid-to-liquid ratio of 1:10 (w / v, g / mL), mix well, extract ultrasonically for 30 min, filter by suction after the ultrasonic extraction is over, repeat the extraction of the obtained filter residue under the above conditions once, combine the filtrates, and it is found that the obtained crude extract does not contain the target product.

Claims

1. A method for simultaneously separating and purifying myricetin-3-O-(2”-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside from Myrica rubra leaves, characterized in that, it includes: (1) Alcohol extraction and concentration: Mix Myrica rubra leaves with an alcohol solution, filter and collect the filtrate after ultrasonic extraction, remove the alcohol from the filtrate and concentrate it to obtain a crude extract of flavonols from Myrica rubra leaves, and the volume percentage concentration of the alcohol solution is 50-100%; (2) Adsorption on a solid-phase extraction column: Inject the crude extract of flavonols from Myrica rubra leaves into a solid-phase extraction column, perform a first gradient elution with a mobile phase, and post-treat the collected eluate to obtain a solid-phase extraction powder rich in myricetin-3-O-(2”-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside; The process of the first gradient elution is as follows: First, perform the first elution with an alcohol solution with a volume percentage concentration lower than 40%, and then perform the second elution with an alcohol solution with a volume percentage concentration of 40%-60%, and collect the eluate after the second elution; the volume percentage concentration of the alcohol solution for the first elution is above 20%; (3) Purification by preparative liquid chromatography: Use a solid-phase chromatographic column, perform a second gradient elution on the solid-phase extraction powder obtained in step (2) with a mobile phase and then perform post-treatment to obtain the target products: myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer respectively; The mobile phase: Phase A is selected from formic acid-aqueous solution with a volume percentage concentration of 0.05%-5% and trifluoroacetic acid-aqueous solution with a volume percentage concentration of 0.05%-5%, and Phase B is selected from acetonitrile-aqueous solution with a volume percentage concentration of 40-60% and acid-acetonitrile-aqueous solution with a volume percentage concentration of 40-60%, and the volume percentage concentration of the acid is 0.05%-5%, and the acid is selected from formic acid and trifluoroacetic acid; The process of the second gradient elution is as follows: The volume percentage concentration of Phase B rises from 20% to 60% within 0-10 min, rises from 60% to 90% within 10-30 min, rises from 90% to 100% within 30-35 min, and finally drops from 100% to 20% within 35-40 min, and collect the target products respectively; The alcohol solution refers to an aqueous solution of alcohol, and the alcohol is selected from methanol or ethanol.

2. The method according to claim 1, characterized in that, in step (1), the filtrate is evaporated to remove alcohol and concentrated under vacuum at 37-50 °C to obtain a crude extract of flavonols from Myrica rubra leaves.

3. The method according to claim 1, characterized in that, in step (1), the process of collecting the filtrate is repeated 2-4 times, and the filtrates of 2-4 times are combined.

4. The method according to claim 1, characterized in that, in step (1), the volume percentage concentration of the alcohol solution is 80%.

5. The method according to claim 1, characterized in that, In step (1), the time for ultrasonic extraction is 30 to 60 min.

6. According to the method described in claim 1, wherein, in step (1), the mass-volume ratio of the myrica leaves to the alcohol solution is 1:5 to 20.

7. According to the method described in claim 1, wherein, in step (2), the solid-phase extraction column is a C18 solid-phase extraction column.

8. According to the method described in claim 1, wherein, in step (2), the first elution is carried out with an alcohol solution having a volume percentage concentration of 30%.

9. According to the method described in claim 1, wherein, in step (2), the second elution is carried out with an alcohol solution having a volume percentage concentration of 40%.

10. According to the method described in claim 1, wherein, in step (2), gradient elution of the solid-phase extraction column is specifically as follows: Inject the crude extract of myricetin from myrica leaves into the solid-phase extraction column, first rinse the solid-phase extraction column with deionized water, carry out gradient elution with the mobile phase, and vacuum rotary evaporate the collected eluate at 37 to 45 °C to obtain the solid-phase extraction powder rich in the target product.

11. According to the method described in claim 1, wherein, in step (2), the adsorption of the solid-phase extraction column is specifically as follows: C18 Sep- After the solid-phase extraction column was activated, 4.5 BV of the myrica leaf extract was loaded onto each solid-phase extraction column; deionized water (4 BV) was used to wash away sugars and acids; then, a methanol solution with a volume percentage concentration of 30% (10 BV) was used for elution to remove some impurities and other non-target flavonols; then, a methanol solution with a volume percentage concentration of 40% (4 BV) was used for elution, and the eluate of the 40% fraction was collected and rotary evaporated to dryness under vacuum at 37 °C to 50 °C, obtaining a solid-phase extraction powder rich in myricetin-3-O-(2”-galloyl)-α-L-rhamnoside and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside.

12. According to the method described in claim 1, wherein, in step (3), the solid-phase chromatography column used is a C18 solid-phase chromatography column.

13. According to the method described in claim 1, wherein, in step (3), phase A is selected from formic acid-aqueous solutions having a volume percentage concentration of 0.1% to 3%.

14. According to the method described in claim 1, wherein, In step (3), a preparative liquid chromatography column SunFire TM C18 OBM TM column is used. During the second gradient elution, the eluents in the time ranges of 27 - 28.5 min and 28.5 - 30 min are collected separately; the column temperature is at room temperature, and the flow rate is 3 - 6 mL / min.

15. According to the method described in claim 1, wherein, in step (3), dissolve the solid-phase extraction powder prepared in step (2) with methanol to make its concentration reach 100 - 200 mg / mL, inject it into the preparative liquid phase for purification, and the single injection volume is 50 - 300 μL; 16. According to the method described in claim 1, wherein, in step (3), phase B is selected from acetonitrile-aqueous solutions having a volume percentage concentration of 50% and acid-acetonitrile-aqueous solutions having a volume percentage concentration of 50%, the volume percentage concentration of the acid is 0.1% to 5%, and the acid is selected from formic acid and trifluoroacetic acid.

17. According to the method described in claim 1, wherein, in step (3), the purification by preparative liquid chromatography is specifically as follows: Using the preparative liquid chromatography column SunFire TM C18 OBM TM column, mobile phase: Phase A: formic acid - aqueous solution with a volume percentage concentration of 0.1%, Phase B: formic acid - acetonitrile - aqueous solution with a volume percentage concentration of 50%, where the volume percentage concentration of formic acid is 0.1%; column temperature is room temperature, flow rate is 3 - 6 mL / min; Dissolve the solid-phase extraction powder prepared in step (2) with methanol to make its concentration reach 100 - 200 mg / mL, inject it into the preparative liquid phase for purification, and the single injection volume is 50 - 300 μL; Collect the eluates at 27 to 28.5 min and 28.5 to 30 min separately in tubes, combine the eluate rich in the pure target product, and carry out vacuum concentration and freeze drying to respectively obtain high-purity myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer and myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer.

18. According to the method described in claim 1, wherein, The post-treatment refers to decompression concentration and freeze-drying; the conditions for decompression concentration are: vacuum rotary evaporation at 37-50 °C; 19. According to the method described in claim 1, it is characterized in that the purity of the myricetin-3-O-(2”-galloyl)-α-L-rhamnoside monomer and the myricetin-3-O-(4”-galloyl)-α-L-rhamnoside monomer is above 98%.

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