Compound extract of kudzuvine root and purple perilla as well as preparation method and application of compound extract

Abstract

The invention relates to the technical field of extraction and processing of radix puerariae and perilla frutescens, and particularly discloses a compound extract of radix puerariae and perilla frutescens as well as a preparation method and application of the compound extract. The preparation method comprises the following steps: slicing radix puerariae, cleaning with purple perilla, drying, adding water to adjust the water content, adding a leavening agent, and fermenting to a specific pH value to obtain a fermented product; and performing reflux extraction on the fermentation product by using a deep-eutectic solvent to obtain the deep-eutectic solvent extract. Mixing the extract with ethanol, and performing supercritical carbon dioxide extraction to obtain a supercritical carbon dioxide extract. Carrying out water extraction and alcohol precipitation treatment on the extract to obtain a primarily purified extract; and finally, separating by using macroporous resin to obtain the final compound extract of the radix puerariae and the perilla frutescens. According to the method, the extraction rate of flavonoid compounds and other bioactive substances in the compound extract is remarkably increased, so that the compound extract shows excellent effects in the aspects of reducing blood pressure, reducing blood sugar, reducing blood fat and the like, can be used as a daily diet supplement, and is widely applied to the fields of foods, medicines and health-care products.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction and processing of Pueraria lobata and Perilla frutescens, and particularly relates to a compound extract of Pueraria lobata and Perilla frutescens, a preparation method thereof, and an application thereof. Background Art

[0002] In recent years, with the continuous improvement of consumers' health awareness, functional foods, as a type of food category that can provide specific health benefits, have gradually become a market hotspot. Functional foods not only meet people's basic nutritional needs but also help prevent chronic diseases and regulate body functions by adding ingredients with specific physiological functions, such as plant extracts. For example, in daily baked foods such as biscuits and bread, consumers are increasingly inclined to choose functional products added with natural plant extracts. These natural products not only retain the delicious taste of traditional foods but also have high safety and endow additional health values, such as the effects of regulating blood lipid, blood sugar, and blood pressure.

[0003] Hypertension, hyperglycemia, and hyperlipidemia are common metabolic diseases in modern society, posing a serious threat to people's health. Pueraria lobata and Perilla frutescens are two traditional Chinese medicinal materials commonly used in traditional Chinese medicine in China, which have certain effects of lowering blood pressure, blood sugar, and blood lipid. At present, there has been some research on the extraction methods of Pueraria lobata and Perilla frutescens, but how to improve the extraction efficiency and the efficacy of the compound extract remains a problem to be solved.

[0004] The existing extraction technologies of Pueraria lobata and Perilla frutescens mainly include traditional solvent extraction method, ultrasonic extraction method, microwave extraction method, etc. These methods can extract the active ingredients in Pueraria lobata and Perilla frutescens to a certain extent, but have the following defects:

[0005] (1) The traditional extraction method does not completely extract the active ingredients, resulting in a low content of active ingredients in the extract, which affects its effects of lowering blood pressure, blood sugar, and blood lipid.

[0006] (2) Some extraction methods require high temperature and pressure, which may cause the destruction of the structure of active ingredients and reduce their activity.

[0007] (3) The organic solvents used in some extraction methods may cause environmental pollution and also have certain toxicity to the human body.

[0008] In view of this, a compound extract of Pueraria lobata and Perilla frutescens, a preparation method thereof, and an application thereof are proposed to solve the above problems. By optimizing the extraction process, the active ingredients in Pueraria lobata and Perilla frutescens are fully extracted, and a compound extract with high efficiency in lowering blood pressure, blood sugar, and blood lipid is prepared, providing new ideas and solutions for the development of industries such as food, medicine, and health products. Summary of the Invention

[0009] The present invention aims to solve the technical problems in the above-mentioned prior art that the extraction methods often fail to fully extract the active ingredients in Pueraria lobata and Perilla frutescens, resulting in low content of active ingredients in the extract, unstable purity and content, unstable product efficacy, and affecting its efficacy in lowering blood pressure, blood sugar and blood lipid. The traditional extraction and purification methods often have the problems of cumbersome steps and high production costs.

[0010] To achieve the above object, the present invention provides the following technical solutions:

[0011] A preparation method of a compound extract of Pueraria lobata and Perilla frutescens, comprising the following steps:

[0012] S1. Wash and slice Pueraria lobata and Perilla frutescens respectively, dry them until the water content is lower than 10 wt%, put them into a fermentation tank, add water to adjust the water content to 45 wt%-55 wt% to obtain a fermentation substrate, add a fermentation agent, ferment at 40-45 °C until the pH is 4-4.5, take out, dehydrate and concentrate to obtain a fermented product;

[0013] Among them, the fermentation agent includes Lactobacillus bulgaricus, Candida lipolytica, Streptococcus thermophilus, Lactobacillus casei; the strain number of Lactobacillus bulgaricus is CI CC 25031; the strain number of Candida lipolytica is CI CC 32245; the strain number of Streptococcus thermophilus is CI CC 6038; the strain number of Lactobacillus casei is CI CC 20241;

[0014] S2. Reflux extract the fermented product with a deep eutectic solvent for 2-3 times, 1-2 h each time, filter, combine the extraction solutions, separate the deep eutectic solvent, and concentrate to obtain a deep eutectic solvent extract;

[0015] S3. Mix the deep eutectic solvent extract with a 90%-95% ethanol solution, stir evenly, put it into a supercritical carbon dioxide extraction instrument for supercritical extraction, recover ethanol, collect the extraction solution and concentrate to obtain a supercritical carbon dioxide extract;

[0016] S4. Perform water extraction and alcohol precipitation on the supercritical carbon dioxide extract to obtain a preliminarily purified extract;

[0017] S5. Separate the preliminarily purified extract by macroporous resin to obtain the compound extract of Pueraria lobata and Perilla frutescens.

[0018] Preferably, the dosage of Lactobacillus bulgaricus is 10 6 -10 7 CFU / g of fermentation substrate; the dosage of Candida lipolytica is 10 8 -10 9 CFU / g of fermentation substrate; the dosage of Streptococcus thermophilus is 10 7 -10 8CFU / g of fermentation substrate; the dosage of Lactobacillus casei is 10 5 -10 6 CFU / g of fermentation substrate.

[0019] Preferably, the mass ratio of kudzu root to perilla is (5 - 7):(3 - 5).

[0020] Preferably, the mass ratio of the fermented product to the deep eutectic solvent is 1:(30 - 40), and the water content of the deep eutectic solvent is 25wt% - 35wt%.

[0021] Preferably, the components in the deep eutectic solvent include betaine, glycerol, lactic acid, urea, citric acid, and water.

[0022] Preferably, the molar ratio of betaine, glycerol, lactic acid, urea, and citric acid is 1:(1 - 3):(0.5 - 2):(0.8 - 1.2):(0.5 - 2).

[0023] Preferably, the mass ratio of the deep eutectic solvent extract to the 90% - 95% ethanol solution is 1:(30 - 40), and the method for separating the deep eutectic solvent is bipolar electrodialysis membrane separation.

[0024] Preferably, the conditions for supercritical extraction are as follows: the dosage of carbon dioxide is 2 - 4 times that of the 90% - 95% ethanol solution, the pressure is 30 - 40 MPa, the temperature is 35 - 45 °C, and the time is 1 - 2 h.

[0025] Preferably, the specific steps of water extraction and alcohol precipitation are as follows:

[0026] Add the supercritical carbon dioxide extract to 8 - 12 times the amount of water, heat to 80 - 90 °C, extract for 1 - 2 hours, filter, and collect the extract;

[0027] Cool the extract to room temperature, add 95% - 98% ethanol until the ethanol concentration is 60% - 70%, stir evenly, let stand for 12 - 24 hours, then centrifuge, collect the precipitate, wash the precipitate successively with water, 50% ethanol solution, and 95% ethanol solution until neutral, evaporate the ethanol solution completely, and dry at 55 - 65 °C to obtain the preliminarily purified extract.

[0028] Preferably, the specific steps of macroporous resin separation are as follows: dissolve the preliminarily purified extract in water, pass it through the macroporous resin at a flow rate of 2.5 - 3.5 BV / h, elute successively with 3 - 5 BV of water and 2 - 4 BV of 45% - 55% ethanol solution, collect the eluate, concentrate and dry it under reduced pressure to obtain the kudzu root and perilla composite extract.

[0029] Preferably, the mass ratio of the preliminarily purified extract to water is 1:(10 - 30).

[0030] Preferably, the macroporous resin is a styrene-based non-polar macroporous resin with an average pore diameter of 12 - 16 nm, a porosity of 45% - 50%, and a specific surface area of 450 - 500 m 2 / g. The macroporous resin is purchased from Tianjin Haoju Resin Technology Co., Ltd., and the model is ADS-8.

[0031] The present application also provides a compound extract of Pueraria lobata and Perilla frutescens, which is prepared by the above-mentioned preparation method of the compound extract of Pueraria lobata and Perilla frutescens.

[0032] The present application also provides an application of the above-mentioned compound extract of Pueraria lobata and Perilla frutescens in the preparation of functional products for regulating blood pressure, blood sugar, and blood lipid.

[0033] Before extracting Pueraria lobata and Perilla frutescens, fermenting them with specific fermenting agents first can improve the extraction efficiency of the active ingredients in Pueraria lobata and Perilla frutescens, thereby enhancing the efficacy of the compound extract. This is because these fermenting agents act synergistically. Lactobacillus bulgaricus and Streptococcus thermophilus produce lactic acid, reducing the pH value of the fermentation broth, inhibiting the growth of harmful microorganisms, protecting flavonoid compounds from oxidative damage, and promoting the dissolution and stability of flavonoid compounds. Candida lipolytica and Lactobacillus casei produce various enzymes, such as β-glucosidase, protease, cellulase, and amylase. These enzymes can decompose cell walls and macromolecular substances, releasing more active ingredients, such as flavonoid compounds and polysaccharides in Perilla frutescens and Pueraria lobata. In addition, these fermenting bacteria also have antioxidant effects, protecting the stability of flavonoid compounds, and producing organic acids and alcohol compounds, improving the flavor and aroma of the raw materials, and enhancing the sensory quality of the final product. Through these synergistic effects, the extraction rate and purity of the active ingredients can be significantly improved, thereby enhancing the efficacy of the compound extract.

[0034] Performing compound extraction on Pueraria lobata and Perilla frutescens, and applying the obtained compound extract to foods, drugs, and health products not only helps control high blood pressure, high blood sugar, and high blood lipid, but also has a positive impact on improving cardiovascular health and can be used as a daily dietary supplement. This is because Pueraria lobata and Perilla frutescens are rich in components such as flavonoid compounds (such as puerarin, daidzin, apigenin, luteolin, etc.), isoflavonoid compounds, polysaccharides, saponins, rosmarinic acid, perillaldehyde, etc., and these components act synergistically.

[0035] First, puerarin has a natural β-receptor antagonistic effect, which can block the excitation of adenylyl cyclase by adrenaline, thereby counteracting the pressor effect and achieving a blood pressure lowering effect. At the same time, puerarin and daidzein can also block α-receptors, causing the blood pressure to drop. The flavonoids in perilla indirectly affect blood pressure levels, maintain vascular health, and promote blood pressure stability by regulating the contraction and relaxation of vascular smooth muscle and exerting antioxidant and anti-inflammatory effects. The two work together in the blood pressure regulation system to enhance the blood pressure lowering effect. Secondly, puerarin can counteract the blood sugar increasing effect of adrenaline, enhance insulin sensitivity, and correct hyperinsulinemia, thereby reducing blood sugar. The flavonoids in perilla indirectly affect blood sugar levels by regulating insulin secretion and utilization. The two act together on the insulin signaling pathway, enhancing insulin sensitivity, reducing blood sugar levels, and reducing the risk of metabolic diseases such as diabetes.

[0036] In addition, puerarin can reduce the contents of total cholesterol and low-density lipoprotein, and reduce the deposition of fat on the arterial wall, while the active ingredients such as flavonoid compounds in perilla have the effect of reducing blood lipids. The two act together on the fat metabolism pathway, promoting the decomposition and utilization of fat, reducing blood lipid levels, and further reducing the risk of arteriosclerosis. Finally, the isoflavone compounds in pueraria help dilate the coronary arteries, improve cardiac and cerebral circulation, and prevent cardiovascular diseases. The active ingredients in perilla help reduce the inflammatory response and oxidative stress in the blood vessel wall, maintaining vascular health. The two act together on the cardiovascular system to improve cardiovascular health and reduce the risk of cardiovascular diseases through mechanisms such as dilating blood vessels, inhibiting oxidative stress and inflammatory responses. Therefore, the higher the content of active ingredients in the compound extract of pueraria and perilla, the more significant its effects of lowering blood pressure, blood sugar, and blood lipids.

[0037] Using betaine, glycerol, lactic acid, urea, and citric acid as deep eutectic solvents to extract the active substances from perilla and kudzu root can improve the extraction selectivity and efficiency. This is because the active substances such as flavonoids in the two raw materials usually have certain polarity and hydrophobicity. In the deep eutectic solvent composed of betaine, glycerol, lactic acid, urea, and citric acid, betaine and urea stabilize the solvent structure by forming a strong hydrogen bond network, while glycerol and lactic acid optimize the extraction conditions by dissolving and adjusting the pH value. The multi-carboxyl groups of citric acid can interact with the active substances such as flavonoids, improving the extraction selectivity and efficiency. At the same time, the strong hydrogen bond interaction between urea and betaine binds the urea molecules, making it difficult for them to diffuse into the interior of lipase molecules, thus reducing the impact on the activity and extraction efficiency of the active substances. Using the deep eutectic solvent described in the present invention has higher solubility, better stability, and higher selectivity for active substances compared with traditional ethanol extraction. And through bipolar electrodialysis membrane separation technology, while removing the deep eutectic solvent, its active ingredients are recovered, greatly improving the extraction efficiency and product purity, and further enhancing its effects of lowering blood pressure, blood sugar, and blood lipid.

[0038] Selecting a specific macroporous resin to perform secondary separation and purification on the preliminarily purified extract can further improve the purity and content of the active ingredients in the composite extract. This may be because ADS-8, as a styrene-type non-polar adsorption resin, has certain ester groups on its surface, which is hydrophobic adsorption. Therefore, it can effectively adsorb the active ingredients such as flavonoids and isoflavonoids in kudzu root and perilla. The polar groups on the surface can also interact with the polar parts of molecules such as rosmarinic acid. At the same time, it will not adsorb sugars, inorganic acids, bases, salts, and small organic molecules with strong hydrophilicity, achieving the effect of selective separation, thus increasing the content of active substances in the composite extract. When eluting, by controlling the ethanol concentration, the active substances such as flavonoids can be eluted from the resin, avoiding the problems of structural damage of the active substances such as flavonoids caused by too high ethanol concentration and poor elution effect caused by too low ethanol concentration, ensuring the purity and yield of the active substances in the composite extract.

[0039] Compared with the prior art, the technical effects and advantages of the present invention are:

[0040] (1) The present invention provides a composite extract of kudzu root and perilla and its preparation method. Through the optimized combination of fermentation, deep eutectic solvent extraction, supercritical carbon dioxide extraction, water extraction and alcohol precipitation, and macroporous resin separation and purification, the extraction rate of flavonoids and other bioactive substances in the composite extract is significantly improved. The prepared composite extract not only helps to control hypertension, hyperglycemia and hyperlipidemia, but also has a positive impact on improving cardiovascular health. The raw materials are derived from plants, non-toxic and harmless, and can be used as a daily dietary supplement.

[0041] (2) Before extracting Pueraria lobata and Perilla frutescens, the present invention first ferments them with a specific fermenting agent, which can improve the extraction efficiency of the active ingredients in Pueraria lobata and Perilla frutescens, thereby enhancing the efficacy of the composite extract.

[0042] (3) The present invention uses betaine, glycerol, lactic acid, urea, and citric acid as deep eutectic solvents to extract the active substances in Perilla frutescens and Pueraria lobata, which can improve the extraction selectivity and efficiency.

[0043] (4) The present invention selects a specific macroporous resin to perform secondary separation and purification on the preliminarily purified extract, which can further improve the purity and content of the active ingredients in the composite extract. Specific Embodiments

[0044] The following will describe clearly and completely the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0045] Example 1

[0046] This example provides a composite extract of Pueraria lobata and Perilla frutescens, and the steps of its preparation method are as follows:

[0047] S1. Wash and slice Pueraria lobata and Perilla frutescens respectively, dry them to a water content of 10 wt%, put them into a fermentation tank, add water to adjust the water content of the total material to 50 wt% to obtain a fermentation substrate, add a fermenting agent, ferment at 44 °C until the pH reaches 4, take it out, dehydrate and concentrate to obtain a fermented product;

[0048] Among them, the fermenting agent includes Lactobacillus bulgaricus, Candida lipolytica, Streptococcus thermophilus, and Lactobacillus casei; the fermenting agents are all commercially available. The strain number of Lactobacillus bulgaricus is CICC 25031; the strain number of Candida lipolytica is CICC 32245; the strain number of Streptococcus thermophilus is CICC 6038; the strain number of Lactobacillus casei is CICC 20241;

[0049] S2. Reflux extract the fermented product with a deep eutectic solvent 3 times, each time for 1.5 h, filter, combine the extraction solutions, separate the deep eutectic solvent, and concentrate to obtain a deep eutectic solvent extract;

[0050] S3. Mix the deep eutectic solvent extract and 90% ethanol solution, stir evenly, put it into a supercritical carbon dioxide extraction instrument for supercritical extraction, recover ethanol, collect the extraction solution and concentrate to obtain a supercritical carbon dioxide extract;

[0051] S4. Subject the supercritical carbon dioxide extract to water extraction and ethanol precipitation to obtain a preliminarily purified extract;

[0052] S5. Separate the preliminarily purified extract using macroporous resin to obtain the compound extract of Pueraria lobata and Perilla frutescens.

[0053] The mass ratio of Pueraria lobata to Perilla frutescens is 3:2.

[0054] The fermenting agents are Lactobacillus bulgaricus, Candida lipolytica, Streptococcus thermophilus, and Lactobacillus casei.

[0055] The dosage of Lactobacillus bulgaricus is 10 6 CFU / g of the fermentation substrate; the dosage of Candida lipolytica is 10 8 CFU / g of the fermentation substrate; the dosage of Streptococcus thermophilus is 10 7 CFU / g of the fermentation substrate; the dosage of Lactobacillus casei is 10 5 CFU / g of the fermentation substrate.

[0056] The mass ratio of the fermented product to the deep eutectic solvent is 1:35.

[0057] The components in the deep eutectic solvent are betaine, glycerol, lactic acid, urea, citric acid, and water.

[0058] The water content of the deep eutectic solvent is 30%.

[0059] The molar ratio of betaine, glycerol, lactic acid, urea, and citric acid is 1:2:1:1:1.

[0060] The mass ratio of the deep eutectic solvent extract to the 90% ethanol solution is 1:35.

[0061] The method for separating the deep eutectic solvent is bipolar electrodialysis membrane separation, and the specific implementation method refers to Example 1 described in the patent authorization announcement number "CN115531490B".

[0062] The conditions for supercritical extraction are: the dosage of carbon dioxide is 3 times that of the 90% ethanol solution, the pressure is 35 MPa, the temperature is 40 °C, and the time is 1.5 h.

[0063] The specific steps of water extraction and ethanol precipitation are as follows:

[0064] Add the supercritical carbon dioxide extract to 10 times the amount of water, heat to 85 °C, extract for 1.5 hours, filter, and collect the extract;

[0065] Cool the extract to room temperature, add 95% ethanol until the ethanol concentration is 65%, stir evenly, let stand for 20 hours, then centrifuge, collect the precipitate, wash the precipitate successively with water, 50% ethanol solution, and 95% ethanol solution until neutral, evaporate the ethanol solution completely, and dry at 60 °C to obtain a preliminarily purified extract.

[0066] The specific steps for macroporous resin separation are as follows: Dissolve the preliminarily purified extract in water, pass it through the macroporous resin at a flow rate of 3 BV / h, elute successively with 4 BV of water and 3 BV of 50% ethanol solution, collect the eluate, and concentrate and dry it under reduced pressure to obtain the composite extract of Pueraria lobata and Perilla frutescens.

[0067] The mass ratio of the preliminarily purified extract to water is 1:20.

[0068] The macroporous resin is a styrene-type non-polar macroporous resin, with an average pore diameter of 12 - 16 nm, a porosity of 45% - 50%, and a specific surface area of 450 - 500 m 2 / g, purchased from Tianjin Haoju Resin Technology Co., Ltd., ADS-8.

[0069] Example 2

[0070] Based on Example 1, the difference between this example and Example 1 is that the dosage of Lactobacillus bulgaricus is 10 7 CFU / g of fermentation substrate; the dosage of Candida lipolytica is 10 9 CFU / g of fermentation substrate; the dosage of Streptococcus thermophilus is 10 8 CFU / g of fermentation substrate; the dosage of Lactobacillus casei is 10 6 CFU / g of fermentation substrate.

[0071] Example 3

[0072] Based on Example 1, the difference between this example and Example 1 is that the molar ratio of betaine, glycerol, lactic acid, urea, and citric acid is 1:1:2:1:2.

[0073] Comparative Example 1

[0074] The difference between this comparative example and Example 1 is that the starter culture is Lactobacillus bulgaricus, Candida lipolytica, and Streptococcus thermophilus.

[0075] The dosage of Lactobacillus bulgaricus is 10 6 CFU / g of fermentation substrate; the dosage of Candida lipolytica is 10 8 CFU / g of fermentation substrate; the dosage of Streptococcus thermophilus is 10 7 CFU / g of fermentation substrate.

[0076] Comparative Example 2

[0077] The difference between this comparative example and Example 1 is that the dosage of Lactobacillus bulgaricus is 10 5 CFU / g of fermentation substrate; the dosage of Candida lipolytica is 10 6 CFU / g of fermentation substrate; the dosage of Streptococcus thermophilus is 10 6CFU / g of fermentation substrate; the dosage of Lactobacillus casei is 10 4 CFU / g of fermentation substrate.

[0078] Comparative Example 3

[0079] The difference between this comparative example and Example 1 is: S2. The fermented product was refluxed and extracted 3 times with 70% ethanol aqueous solution for 1.5 h each time, filtered, the extraction solutions were combined, and concentrated to obtain an extract.

[0080] Comparative Example 4

[0081] The difference between this comparative example and Example 1 is: The components in the deep eutectic solvent are glycerol, lactic acid, urea, citric acid, and water.

[0082] The molar ratio of glycerol, lactic acid, urea, and citric acid is 2:1:1:1.

[0083] Comparative Example 5

[0084] The difference between this comparative example and Example 1 is: The macroporous resin is a polar macroporous resin, with an average pore diameter of 1.3 - 1.4 nm and a specific surface area of 480 - 520 m 2 / g.

[0085] The macroporous resin was purchased from Bengbu Sanyi Technology Co., Ltd., and the model is A - 8.

[0086] Comparative Example 6

[0087] The difference between this comparative example and Example 1 is: The specific steps of macroporous resin separation are: The preliminarily purified extract was dissolved in water, passed through the macroporous resin at a flow rate of 3 BV / h, eluted successively with 4 BV of water and 3 BV of 80% ethanol solution, the eluate was collected, concentrated under reduced pressure and dried to obtain the compound extract of Pueraria lobata and Perilla frutescens.

[0088] Comparative Example 7

[0089] The difference between this comparative example and Example 1 is: For the compound extract of Pueraria lobata and Perilla frutescens, its preparation method, the steps are:

[0090] S1. After washing and slicing Pueraria lobata and Perilla frutescens respectively, they were dried until the water content was lower than 10 wt%, put into a fermentation tank, the water content was adjusted to 50 wt% with water to obtain a fermentation substrate, a fermenting agent was added, and fermentation was carried out at 44 °C until the pH reached 4, then taken out, dehydrated and concentrated to obtain a fermented product;

[0091] S2. The fermented product was refluxed and extracted 3 times with the deep eutectic solvent for 1.5 h each time, filtered, the extraction solutions were combined, after separating the deep eutectic solvent, it was concentrated to obtain a deep eutectic solvent extract;

[0092] S3. The deep eutectic solvent extract was subjected to water extraction and alcohol precipitation treatment to obtain a preliminarily purified extract;

[0093] S4. Subject the preliminarily purified extract to macroporous resin separation to obtain the compound extract of Pueraria lobata and Perilla frutescens.

[0094] Performance test

[0095] Refer to the method in Patent CN114306544B and use mouse experiments to test the blood glucose-lowering performance of the tea bags. After feeding the mice with high-fat, high-sugar, and high-salt diets for 10 weeks, ensure that the fasting blood glucose content in all mice increases by more than 4 times. Administer the drug by gavage to the mice, with the daily oral gavage dose being 1 g / kg·Bw. Test the changes in fasting blood glucose and cholesterol levels in the mice after 30 days.

[0096] Blood glucose decrease rate = (blood glucose before the test - blood glucose after the test) / blood glucose before the test × 100%;

[0097] Cholesterol decrease rate = (cholesterol before the test - cholesterol after the test) / cholesterol before the test × 100%.

[0098] Refer to the method in the paper "Hypotensive Effect of Pueraria lobata Fermentation Broth on Spontaneously Hypertensive Rats" published by Su Lei et al. Select spontaneously hypertensive rats and administer the drug by gavage to the mice, with the daily oral gavage dose being 1 g / kg·Bw. Test the diastolic and systolic blood pressures of the mice after 30 days.

[0099] Diastolic blood pressure decrease rate = (diastolic blood pressure before the test - diastolic blood pressure after the test) / diastolic blood pressure before the test × 100%;

[0100] Systolic blood pressure decrease rate = (systolic blood pressure before the test - systolic blood pressure after the test) / systolic blood pressure before the test × 100%.

[0101] The results are shown in Table 1.

[0102] Table 1 Measurement results

[0103]

[0104] According to statistics, for the Pueraria lobata and Perilla frutescens compound extract prepared in Examples 1-3 of the present invention, the decline rates of fasting blood glucose, cholesterol, diastolic blood pressure, and systolic blood pressure are all relatively high, indicating excellent effects in reducing blood sugar, blood lipid, and blood pressure. In Comparative Example 1, Lactobacillus casei was not added; in Comparative Example 2, the dosage of each fermenting agent was relatively low; in Comparative Example 3, deep eutectic solvent extraction was not used; in Comparative Example 4, betaine was not added; in Comparative Example 5, the specified macroporous resin was not used; in Comparative Example 6, the ethanol solution concentration was too high during elution; in Comparative Example 7, supercritical extraction treatment was not carried out. The decline rates of fasting blood glucose, cholesterol, diastolic blood pressure, and systolic blood pressure of the prepared Pueraria lobata and Perilla frutescens compound extract are all relatively poor. Therefore, the Pueraria lobata and Perilla frutescens compound extract prepared by the method of the present application helps to control hypertension, hyperglycemia, and hyperlipidemia, and has a positive impact on improving cardiovascular health. The raw materials are derived from plants, non-toxic and harmless, and can be used as a daily dietary supplement.

[0105] Through the optimized combination of fermentation, deep eutectic solvent extraction, supercritical carbon dioxide extraction, water extraction and alcohol precipitation, and macroporous resin separation and purification, the present invention significantly improves the extraction rate of flavonoids and other bioactive substances in the compound extract. This means that more active ingredients can be obtained from Pueraria lobata and Perilla frutescens, thereby enhancing the efficacy of the product. Fermentation treatment can improve the extraction efficiency of active ingredients in Pueraria lobata and Perilla frutescens, and then improve the efficacy of the compound extract. The function of the fermenting agent not only protects the active ingredients, but also may produce new beneficial ingredients, making the compound extract show better effects in controlling hypertension, hyperglycemia, and hyperlipidemia.

[0106] Using a deep eutectic solvent composed of betaine, glycerol, lactic acid, urea, and citric acid improves the selectivity and efficiency of extraction. This solvent can better dissolve and stabilize the target components, while reducing the extraction of unnecessary components and improving the purity of the final product.

[0107] The use of deep eutectic solvents reduces the dependence on traditional organic solvents and reduces the impact on the environment. At the same time, the extremely low water content makes the separation of deep eutectic solvents more efficient, reducing energy consumption and costs.

[0108] Through secondary separation and purification with a specific macroporous resin, the purity and content of active ingredients in the compound extract are further improved. This selective adsorption not only removes impurities, but also ensures that the structure of the active ingredients is not damaged, thereby improving the yield and stability of the product.

[0109] The compound extract is rich in various bioactive ingredients, such as puerarin, flavonoids, etc., which have a positive effect on improving cardiovascular health, helping to dilate blood vessels, reduce blood pressure, and regulate blood sugar and blood lipids. The raw materials are derived from plants, non-toxic and harmless, suitable for use as a daily dietary supplement, increasing consumers' confidence and acceptance.

[0110] The compound extract of Pueraria lobata and Perilla frutescens provided by the present invention is not only applicable to the food field, but also can be applied to the fields of medicine, health products, etc., and has broad application prospects.

[0111] In summary, the present invention has remarkable technical effects and advantages in aspects such as improving extraction efficiency, enhancing product efficacy, environmentally friendly production, and increasing product purity and yield.

[0112] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a composite extract of kudzu root and perilla, characterized in that: The following steps are involved: S1, slicing the kudzu root and washing and drying it together with the perilla until the water content is less than 10wt%, putting it into a fermentation tank, adding water to adjust the water content to 45wt%-55wt% to obtain a fermentation substrate, adding a fermentation agent, fermenting at 40-45°C until the pH is 4-4.5, taking it out, dehydrating and concentrating it to obtain a fermentation product; S2, extracting the fermented product with a low eutectic solvent by reflux for 2-3 times, each time for 1-2 hours, filtering, combining the extracts, separating the low eutectic solvent, and concentrating to obtain a low eutectic solvent extract; S3, mixing the low eutectic solvent extract and 90%-95% ethanol solution, stirring evenly, and placing in a supercritical carbon dioxide extractor for supercritical extraction, recovering ethanol, collecting and concentrating the extract to obtain a supercritical carbon dioxide extract; S4, subjecting the supercritical carbon dioxide extract to water extraction and alcohol precipitation to obtain a preliminarily purified extract; S5. Separating the preliminarily purified extract using a macroporous resin to obtain a composite extract of kudzu root and perilla.

2. The method for preparing a composite extract of kudzu root and perilla according to claim 1, characterized in that: The fermentation agents include Lactobacillus bulgaricus, Candida lipolytica, Streptococcus thermophilus, and Lactobacillus casei; the strain number of Lactobacillus bulgaricus is CICC 25031; the strain number of Candida lipolytica is CICC 32245; the strain number of Streptococcus thermophilus is CICC6038; and the strain number of Lactobacillus casei is CICC 20241.

3. The method for preparing a composite extract of kudzu root and perilla according to claim 1, characterized in that: The dosage of Lactobacillus bulgaricus is 10 6 -10 7 CFU / g fermentation substrate; the dosage of Candida lipolytica is 10 8 -10 9 CFU / g fermentation substrate; the dosage of thermophilic Streptococcus is 10 7 -10 8 CFU / g fermentation substrate; the dosage of Lactobacillus casei is 10 5 -10 6 CFU / g fermentation substrate; And / or, the conditions of supercritical extraction are: the amount of carbon dioxide is 2-4 times that of 90%-95% ethanol solution, the pressure is 30-40 MPa, the temperature is 35-45° C., and the time is 1-2 h.

4. The method for preparing a composite extract of kudzu root and perilla according to claim 1, characterized in that: The ingredients in the deep eutectic solvent include betaine, glycerol, lactic acid, urea, citric acid and water.

5. The method for preparing a composite extract of kudzu root and perilla according to claim 4, characterized in that: The molar ratio of betaine, glycerol, lactic acid, urea and citric acid is 1:(1-3):(0.5-2):(0.8-1.2):(0.5-2).

6. The method for preparing a composite extract of kudzu root and perilla according to claim 1, characterized in that: The specific steps of water extraction and alcohol precipitation are: Add the supercritical carbon dioxide extract to 8-12 times the amount of water, heat to 80-90°C, extract for 1-2 hours, filter, and collect the extract; The extract was cooled to room temperature, 95%-98% ethanol was added to make the ethanol concentration 60%-70%, stirred evenly and allowed to stand for 12-24 hours, then centrifuged and the precipitate was collected. The precipitate was washed with water, 50% ethanol solution and 95% ethanol solution in sequence until neutral, the ethanol solution was evaporated, and dried at 55-65°C to obtain a preliminarily purified extract.

7. The method for preparing a composite extract of kudzu root and perilla according to claim 1, characterized in that: The specific steps of macroporous resin separation are: dissolving the preliminarily purified extract in water, passing it through the macroporous resin at a flow rate of 2.5-3.5BV / h, eluting with 3-5BV water and 2-4BV 45%-55% ethanol solution in sequence, collecting the eluate, concentrating and drying under reduced pressure to obtain the composite extract of Pueraria lobata and Perilla frutescens.

8. The method for preparing a composite extract of kudzu root and perilla according to claim 1, characterized in that: The macroporous resin is a styrene-type non-polar macroporous resin with an average pore size of 12-16nm, a porosity of 45%-50%, and a specific surface area of ​​450-500m 2 / g.

9. A composite extract of kudzu root and perilla, characterized in that: The kudzu root and perilla composite extract is prepared by the preparation method of the kudzu root and perilla composite extract according to any one of claims 1 to 8.

10. Use of the composite extract of Pueraria root and Perilla frutescens according to claim 9 in preparing functional products for regulating blood pressure, blood sugar and blood lipids.

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