CN111700902A - Hawthorn procyanidine-jujube polysaccharide composition and preparation method thereof - Google Patents
Hawthorn procyanidine-jujube polysaccharide composition and preparation method thereof Download PDFInfo
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- CN111700902A CN111700902A CN202010683895.9A CN202010683895A CN111700902A CN 111700902 A CN111700902 A CN 111700902A CN 202010683895 A CN202010683895 A CN 202010683895A CN 111700902 A CN111700902 A CN 111700902A
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- procyanidin
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- jujube polysaccharide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
- A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
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- Animal Behavior & Ethology (AREA)
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- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
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Abstract
The invention provides a hawthorn procyanidine-jujube polysaccharide composition and a preparation method thereof, belonging to the technical field of medicines and foods, wherein the hawthorn procyanidine-jujube polysaccharide composition comprises the following components in parts by weight: 21.82-22.02 parts of epicatechin, 13.02-13.22 parts of procyanidine B2, 1.30-1.50 parts of procyanidine B5, 3.46-3.66 parts of procyanidine C1 and 59.9-60.1 parts of jujube polysaccharide. The experimental results show that the composition can be used for synergistically and obviously improving the blood fat, blood sugar and inflammatory factor levels of rats with high fat diet, simultaneously improving the diversity of intestinal flora of the rats with high fat diet, improving the abundance of bacteroidetes and obesity-related bacteria (such as Akkermansia) and reducing the abundance of firmicutes.
Description
Technical Field
The invention belongs to the technical field of medicines and foods, and particularly relates to a hawthorn procyanidine-jujube polysaccharide composition and a preparation method thereof.
Background
Lipid metabolism disorder, insulin resistance and chronic low-grade systemic inflammation caused by long-term high-fat diet of residents in China are recognized as a great challenge seriously threatening the health of the residents in recent years. In addition to participating in important physiological processes such as food digestion, enteric nerve regulation and intraepithelial homeostasis in the body, the intestinal flora has recently been shown to be a medium connecting changes in food-borne metabolism and related chronic diseases in the body. An increasing number of studies have shown that an imbalance in the intestinal flora, in particular a decrease in its abundance and diversity, often leads to metabolic diseases (e.g. obesity, diabetes, etc.). Therefore, the intestinal flora is considered to be an important environmental factor for improving lipid metabolism disorder caused by high-fat diet. In terms of treatment, compared with chemotherapy treatment accompanied by side effects, the method for supplementing certain natural food components in diet can improve chronic disease conditions more safely and effectively and is more acceptable to people.
The hawthorn and the jujube are unique plant resources used as both medicine and food in China, and have high yield, but the related industrial chain is short, the key technology of high-valued processing is backward, and the added value of the product is low. Therefore, the deep excavation of the dual-purpose functions of medicine and food of hawthorn and jujube and the development of the nutritional functional food for preventing and improving chronic diseases are the problems which need to be solved urgently at the present stage and also accord with the policy guidance of the national compendium of healthy Chinese 2030.
Disclosure of Invention
In view of the above, the present invention provides a hawthorn procyanidin-jujube polysaccharide composition and a preparation method thereof, and the composition provided by the invention can synergistically and significantly improve the blood lipid, blood glucose and inflammatory factor levels of high-fat diet rats, and simultaneously can also improve the diversity of intestinal flora of the high-fat diet rats, increase the abundance of bacteroidetes and obesity-related bacteria (such as Akkermansia), and reduce the abundance of firmicutes.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a hawthorn procyanidine-jujube polysaccharide composition, which comprises the following components in parts by weight: 21.82-22.02 parts of epicatechin, 13.02-13.22 parts of procyanidine B2, 1.30-1.50 parts of procyanidine B5, 3.46-3.66 parts of procyanidine C1 and 59.9-60.1 parts of jujube polysaccharide.
Preferably, the composition comprises the following components in parts by weight: 21.92 parts of epicatechin, 13.12 parts of procyanidin B2, 1.40 parts of procyanidin B5, 3.56 parts of procyanidin C1 and 60 parts of jujube polysaccharide.
In the present invention, the preparation method of jujube polysaccharide preferably comprises the following steps:
(1) mixing jujube with water to obtain a mixture, mixing the mixture with cellulase to obtain an extract, performing water bath extraction on the extract at 50-60 ℃ for 5-8 hours to obtain an extract, and centrifuging the extract at 3500-4500 rpm for 8-12 min to obtain a supernatant;
(2) carrying out alcohol precipitation on the supernatant obtained in the step (1) to obtain a first precipitate;
(3) mixing the first precipitate obtained in the step (2) with a sevage reagent, oscillating for 10-20 min, and centrifuging at 3500-4500 rpm for 4-6 min to obtain an upper-layer water phase;
(4) carrying out alcohol precipitation on the upper-layer water phase obtained in the step (3) for 1-3 d, carrying out solid-liquid separation to obtain a solution, and centrifuging the solution at 3500-4500 rpm for 8-12 min to obtain a second precipitate;
(5) and (4) dialyzing the second precipitate obtained in the step (4) for 2-3 days, and then freeze-drying the solution in the dialysis bag to obtain the jujube polysaccharide.
Preferably, the volume ratio of the mass of the jujubes in the step (1) to the water is 1g: 10-15 ml, and the mass percentage of the cellulase in the extract is 0.2-0.4%.
Preferably, the alcohol precipitation conditions in step (2) include: the volume ratio of the alcohol solution used for alcohol precipitation to the supernatant is 3-5: 1, the mass percentage of the alcohol solution is 80-98%, and the temperature of alcohol precipitation is 3-5 ℃.
Preferably, the ratio of the mass of the first precipitate in the step (3) to the volume of the sevage reagent is 1g:1-3 ml.
Preferably, the alcohol precipitation conditions in step (4) include: the volume ratio of the alcohol solution used for alcohol precipitation to the upper-layer water phase is 2-4: 1, the mass percentage of the alcohol solution is 80-98%, and the temperature of alcohol precipitation is 3-5 ℃.
Preferably, the dialysis bag in the step (5) is a cellulose dialysis bag, and the cut-off molecular weight is 7000D.
The invention provides application of the hawthorn procyanidine-jujube polysaccharide composition in the technical scheme in preparing a medicine for improving intestinal flora disorder.
Preferably, the intestinal flora disorder is a high-fat diet-induced intestinal flora disorder.
Compared with the prior art, the invention has the following beneficial effects:
the animal test and the metagenome technical analysis result show that the prepared composition can synergistically and remarkably improve the blood fat, blood sugar and inflammatory factor levels of rats with high fat diet, achieve the purposes of reducing the blood fat and blood sugar of the rats and eliminating inflammation, simultaneously can improve the diversity of intestinal flora of the rats with high fat diet, improve the abundance of bacteroidetes and obesity-related bacteria (such as Akkermansia), reduce the abundance of firmicutes and achieve the purpose of improving the intestinal flora disorder caused by high fat diet.
In the invention, the composition is prepared by combining epicatechin, procyanidin B2, procyanidin B5, procyanidin C1 and jujube polysaccharide according to specific weight parts for the first time, and the active functions of the components are enriched and the purpose of improving intestinal flora disorder caused by high fat diet is achieved through the synergistic effect of the components according to the specific weight parts.
Drawings
FIG. 1 shows the improvement of the alpha-diversity of the intestinal flora of rats with high fat diet by the hawthorn procyanidine-jujube polysaccharide composition;
FIG. 2 shows the improvement of β -diversity of high-fat diet rat intestinal flora with the hawthorn procyanidin-jujube polysaccharide composition;
FIG. 3 shows the improvement of the composition (phylum level and genus level) and F/B value of the intestinal flora of rats with high fat diet by the hawthorn procyanidin-jujube polysaccharide composition.
Detailed Description
The invention provides a hawthorn procyanidine-jujube polysaccharide composition, which comprises the following components in parts by weight: 21.82-22.02 parts of epicatechin, 13.02-13.22 parts of procyanidine B2, 1.30-1.50 parts of procyanidine B5, 3.46-3.66 parts of procyanidine C1 and 59.9-60.1 parts of jujube polysaccharide; preferably comprising: 21.92 parts of epicatechin, 13.12 parts of procyanidin B2, 1.40 parts of procyanidin B5, 3.56 parts of procyanidin C1 and 60 parts of jujube polysaccharide. The sources of the epi-theophylline, the procyanidine B2, the procyanidine B5 and the procyanidine C1 are not particularly limited, and the epi-theophylline, the procyanidine B2, the procyanidine B5 and the procyanidine C1 can be obtained by adopting a conventional commercial or conventional preparation method. According to the composition, through the synergistic effect of specific amounts of components, the blood fat, blood sugar and inflammatory factor levels of rats with high-fat diet can be obviously improved, meanwhile, the diversity of intestinal flora of rats with high-fat diet can be improved, the abundance of bacteroides and obesity-related bacteria (such as Akkermansia) is improved, the abundance of firmicutes is reduced, and the purpose of improving intestinal flora disorder caused by high-fat diet is achieved.
In the present invention, the preparation method of jujube polysaccharide preferably comprises the following steps:
(1) mixing jujube with water to obtain a mixture, mixing the mixture with cellulase to obtain an extract, performing water bath extraction on the extract at 50-60 ℃ for 5-8 hours to obtain an extract, and centrifuging the extract at 3500-4500 rpm for 8-12 min to obtain a supernatant;
(2) carrying out alcohol precipitation on the supernatant obtained in the step (1) to obtain a first precipitate;
(3) mixing the first precipitate obtained in the step (2) with a sevage reagent, oscillating for 10-20 min, and centrifuging at 3500-4500 rpm for 4-6 min to obtain an upper-layer water phase;
(4) carrying out alcohol precipitation on the upper-layer water phase obtained in the step (3) for 1-3 d, carrying out solid-liquid separation to obtain a solution, and centrifuging the solution at 3500-4500 rpm for 8-12 min to obtain a second precipitate;
(5) and (4) dialyzing the second precipitate obtained in the step (4) for 2-3 days, and then freeze-drying the solution in the dialysis bag to obtain the jujube polysaccharide.
In the invention, the volume ratio of the mass of the jujube to the volume of the water is preferably 1g: 10-15 ml, more preferably 1g:10ml, the mass percentage content of the cellulase in the extract to be extracted is preferably 0.2-0.4%, more preferably 0.3%, and the extraction temperature of the extract to be extracted is preferably 50-60 ℃, more preferably 55 ℃. In the invention, the proper volume ratio of the jujube to the water can improve the extraction rate and the content of jujube polysaccharide, is more beneficial to the subsequent utilization of the jujube polysaccharide and can reduce the waste of water resources; the cellulase can decompose cellulose in the jujube into polysaccharide or monosaccharide, and the cellulase with a proper dosage can improve the extraction rate of jujube polysaccharide; the extraction efficiency can be improved by increasing the extraction temperature of the extract to be extracted, but the impurity content is higher, and the purity and the extraction rate of the jujube polysaccharide can be improved by proper extraction temperature.
In the present invention, the alcohol precipitation conditions in step (2) preferably include: the volume ratio of the alcohol solution used for alcohol precipitation to the supernatant is 3-5: 1, the mass percentage of the alcohol solution is 80-98%, the temperature of alcohol precipitation is 3-5 ℃, and the alcohol precipitation preferably comprises: the volume ratio of the alcohol solution used in alcohol precipitation to the supernatant is 4:1, the mass percentage of the alcohol solution is 95%, and the temperature of alcohol precipitation is 4 ℃.
In the invention, the ratio of the mass of the first precipitate to the volume of the sevage reagent is preferably 1g:1-3ml, more preferably 1g:1ml, and the shaking is preferably performed by shaking the table vigorously. In the invention, the sevage reagent can denature the protein in the first precipitate, remove the protein in the first precipitate and improve the purity of the final product jujube polysaccharide.
In the present invention, the alcohol precipitation conditions in step (4) preferably include: the volume ratio of the alcohol solution used for alcohol precipitation to the upper-layer water phase is 2-4: 1, the mass percentage of the alcohol solution is 80-98%, the temperature of alcohol precipitation is 3-5 ℃, and the alcohol precipitation preferably comprises the following components: the volume ratio of the alcohol solution used in alcohol precipitation to the upper aqueous phase is 3:1, the mass percentage of the alcohol solution is 95%, the temperature of the alcohol precipitation is 4 ℃,
in the present invention, the dialysis bag is preferably a cellulose dialysis bag, and the cut-off molecular weight is preferably 7000D. In the present invention, the dialysis can remove the salt in the second precipitate, and improve the purity of the jujube polysaccharide.
The invention also provides application of the hawthorn procyanidine-jujube polysaccharide composition in the technical scheme in preparing a medicine for improving intestinal flora disorder. In the present invention, the intestinal flora disturbance is preferably a high-fat diet-induced intestinal flora disturbance. In the invention, the intestinal flora comprises one or more than two of verrucomicrobia, firmicutes, actinomycetes, bacteroidetes and proteobacteria. In the present invention, the intestinal flora further includes one or more than two of genera consisting of akkermansia, lactobacillus, bifidobacterium, bacteroides, blautia, lachnospira, oscillatoria, peptostreptococcus and escherichia.
In the present invention, the preparation method of epicatechin, procyanidin B2, procyanidin B5, and procyanidin C1 preferably comprises the steps of:
(1) mixing the hawthorn and an ethanol solution, and extracting for 150min at the temperature of 60-90 ℃ to obtain an ethanol extracting solution;
(2) cooling the ethanol extract obtained in the step (1) to room temperature, and extracting the ethanol extract with ethyl acetate to obtain an upper layer extract;
(3) purifying the upper layer extract liquid obtained in the step (2) by adopting macroporous resin AB-8, and collecting a hawthorn procyanidine resolving liquid;
(4) performing rotary evaporation concentration and freeze-drying on the hawthorn procyanidin analytic solution obtained in the step (3) to obtain hawthorn procyanidin;
(5) dissolving the hawthorn procyanidin obtained in the step (4) in 0.5-2 ml of methanol, filtering to obtain a sample solution, injecting the sample solution into a preparative high performance liquid chromatograph, and separating to obtain epicatechin, procyanidin B2, procyanidin B5 and procyanidin C1.
In the invention, the ratio of the mass of the hawthorn to the volume of the ethanol solution is preferably 1g: 15-30 mL, more preferably 1g:25mL, and the mass percentage of the ethanol solution is 50-80%, more preferably 70%. In the invention, the extraction rate and the content of procyanidine of hawthorn can be improved by the proper proportion of hawthorn and ethanol solution and the concentration of the ethanol solution.
In the present invention, the conditions for extracting the ethanol extract with ethyl acetate preferably include: the extraction time is 20-30 min, and the ethyl acetate: extracting 3-5 times with ethanol extract in a volume ratio of 1-2: 1; more preferably, it comprises: extraction time 25min, ethyl acetate: extracting with ethanol at a volume ratio of 1.5:1 for 4 times.
In the present invention, the conditions for purifying the upper layer extract by the macroporous resin AB-8 preferably include: the analysis solution is an ethanol solution with the mass percent of 40-60%, the pH value of the analysis solution is 4-6, and the diameter-height ratio is 1: 30-50; more preferably, it comprises: the analysis solution is an ethanol solution with the mass percent of 50%, the pH value of the analysis solution is 5, and the diameter-height ratio is 1: 40.
In the present invention, the chromatographic conditions for preparing a high performance liquid chromatograph preferably include: the chromatographic column is SunAireTMC18(4.6mm × mm, 5m), wherein the mobile phase A is 0.4% glacial acetic acid water solution, the mobile phase B is acetonitrile, the detection wavelength is 280nm, the sample introduction amount is 100mL, the flow rate is 16mL/min, gradient elution is carried out according to the steps of 0-10 min, 5% → 15% B, 10-15 min, 15% → 35% B, 15-20 min, 35% → 50% B, 20-30 min, 50% → 80% B, 30-65 min, 80% → 30% B, 65-75 min, 30% B, 75-80 min, 30% → 10% B, 80-85 min and 10% B, 12.675min procyanidin B2 and 14.611min surface catechin collection, 20.472min surface procyanidin C1 and 38.032min procyanidin collection B5.. in the method, the high performance liquid phase conditions are set differently, the finally separated components are different, and procyanidin can be accurately separated from procyanidin extract, such as catechin, hawthorn fruit catechin, 2B, surface catechin, 4625 and 4625B and surface procyanidin through the chromatographic conditions set by the chromatographic conditions of the method.
In the invention, the procyanidine B2, the epicatechin, the procyanidine C1 and the procyanidine B5 are taken as components and combined with the jujube polysaccharide to prepare the hawthorn proanthocyanidin-jujube polysaccharide composition according to a specific dosage.
The preparation method of the hawthorn procyanidine-jujube polysaccharide composition comprises the following steps: mixing procyanidin B2, epicatechin, procyanidin C1, procyanidin B5 and fructus Jujubae polysaccharide at a certain proportion, and mixing well to obtain fructus crataegi procyanidin-fructus Jujubae polysaccharide composition.
The application method of the hawthorn procyanidine-jujube polysaccharide composition comprises the following steps: the composition is dissolved in distilled water according to the gavage dosage of rats, and the obtained solution is subjected to gavage.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Preparing the components of the hawthorn procyanidine:
(1) removing seeds of fructus crataegi, drying, making into powder, mixing with 70% ethanol at a mass volume ratio of 1g to 25ml, extracting at 80 deg.C for 120min to obtain ethanol extractive solution
(2) Cooling the ethanol extract to room temperature, and extracting the ethanol extract by using ethyl acetate: extracting with ethanol at a volume ratio of 1.5:1 for 25min for 4 times, and collecting the upper layer extractive solution;
(3) purifying the upper layer extractive solution with macroporous resin AB-8, collecting fructus crataegi procyanidin eluate, wherein the eluate is 50% ethanol solution with pH of 5 and diameter-height ratio of 1:40 (cm);
(4) performing rotary evaporation, concentration and freeze-drying on the hawthorn procyanidin resolving liquid to obtain a hawthorn procyanidin extract with the purity of 81.20%;
(5) qualitative and quantitative detection of each component of the hawthorn proanthocyanidin extract by a high performance liquid chromatograph: dissolving 100mg fructus crataegi procyanidin extract in 1.0mL methanol, filtering with 0.22 μm filter to obtain sample solution, injecting into analytical high performance liquid chromatograph under the chromatographic conditions: the chromatographic column is SunAireTMC18(4.6mm × 250mm, 5m), wherein the mobile phase A is 0.4% glacial acetic acid water solution, the mobile phase B is acetonitrile, the sample size is 10 mu L, the flow rate is 1mL/min, the detection wavelength is 280nm, the column temperature is 25 ℃, and the contents are calculated according to the following formula of 0-10 min, 5% → 15% B, 10-15 min, 15% → 35% B, 15-20 min, 35% → 50% B, 20-30 min and 50%And (3) carrying out gradient elution on the components of the hawthorn procyanidin → 80% B, 30-65 min, 80% → 30% B, 65-75 min, 30% B, 75-80 min, 30% → 10% B, 80-85 min and 10% B, and measuring the components and the content of the hawthorn procyanidin. The results are shown in Table 1.
(6) Preparing each component of the hawthorn procyanidin extract by a preparative high performance liquid chromatograph: dissolving the obtained 100mg fructus crataegi procyanidin extract in 1.0mL methanol, filtering with 0.45 μm filter to obtain sample solution, and injecting into preparative high performance liquid chromatograph under the chromatography conditions including: the chromatographic column is SunAireTMC18(4.6mm × 250mm, 5m), wherein the mobile phase A is 0.4% glacial acetic acid water solution, the mobile phase B is acetonitrile, the detection wavelength is 280nm, the sample injection amount is 100 mu L, the flow rate is 16mL/min, and gradient elution is carried out according to the following steps of 0-10 min, 5% → 15% B, 10-15 min, 15% → 35% B, 15-20 min, 35% → 50% B, 20-30 min, 50% → 80% B, 30-65 min, 80% → 30% B, 65-75 min, 30% B, 75-80 min, 30% → 10% B, 80-85 min and 10% B, 12.675min collecting procyanidin B2, 14.611min collecting epicatechin, 20.472min collecting procyanidin C1 and 38.032min procyanidin B5, so as to obtain catechin table 2, procyanidin B5 and procyanidin C1.
TABLE 1 Crataegus oxyacantha procyanidin extract major components and contents
Example 2
Preparing jujube polysaccharide:
(1) removing cores of fructus Jujubae, drying, making into powder, mixing fructus Jujubae powder with distilled water at a mass volume ratio of 1g:10mL, adding 0.3% cellulase (m/v), extracting at 55 deg.C in water bath for 5 hr, centrifuging the water-bath extractive solution at 4000r/min for 10min to obtain supernatant;
(2) the supernatant was reprecipitated with 4 volumes of 95% ethanol solution at 4 ℃ overnight to give a first precipitate.
(3) Removing protein in the first precipitate by using a sevage reagent, mixing the first precipitate and the sevage reagent at a mass-to-volume ratio of 1:1, violently oscillating for 15min by using a shaking table, centrifuging for 5min at 4000r/min, and repeating the process for many times until denatured protein disappears to obtain an upper-layer water phase.
(4) Slowly sucking out the upper water phase by using a liquid transfer gun, adding a 95% ethanol solution with the volume being 3 times that of the upper water phase, standing for 2d at 4 ℃, taking out the ethanol extraction solution, and centrifuging for 10min at 4000r/min to obtain a second precipitate.
(5) Dissolving the second precipitate in water with appropriate volume, pouring the solution into a cellulose dialysis bag with cut-off molecular weight of 7000D, sealing, dialyzing at low temperature for 2D, removing small molecular substances, and freeze drying the solution in the dialysis bag to obtain fructus Jujubae polysaccharide. The content of jujube polysaccharide is 72.5% by using a phenol-sulfuric acid method.
Example 3
The hawthorn procyanidin-jujube polysaccharide composition is prepared from the components (epicatechin, procyanidin B2, procyanidin B5 and procyanidin C1) of the hawthorn procyanidin prepared in example 1 and the jujube polysaccharide prepared in example 2, and the technical scheme is as follows:
the hawthorn procyanidine-jujube polysaccharide composition comprises the following components in parts by weight: 21.92 parts of epicatechin, 13.12 parts of procyanidin B2, 1.40 parts of procyanidin B5, 3.56 parts of procyanidin C1 and 60 parts of jujube polysaccharide.
And (2) fully and uniformly mixing the procyanidin B2, the epicatechin, the procyanidin C1, the procyanidin B5 and the jujube polysaccharide according to the weight parts to obtain the hawthorn procyanidin-jujube polysaccharide composition.
Example 4
Hawthorn procyanidine-jujube polysaccharide composition for improving conditions of high-fat diet-induced rat blood fat, blood sugar and inflammatory factors
Male Wistar rats, 60 rats aged 5 weeks, were acclimated to a new environment for one week, and then randomly divided into 5 groups of 11 rats each, namely, a negative control group (CON), a high fat model group (HFD), the group of crataegus procyanidin extracts prepared in example 1, the group of jujube polysaccharides prepared in example 2, and the group of crataegus procyanidin-jujube polysaccharides composition prepared in example 3 (HPC). The negative control rats were given a standard diet and the remaining rats were given a high fat diet for 8 weeks. Meanwhile, the hawthorn procyanidine extract group, the jujube polysaccharide group and the hawthorn procyanidine-jujube polysaccharide composition group are respectively added with 200mg/kg of hawthorn procyanidine extract, jujube polysaccharide and hawthorn procyanidine-jujube polysaccharide composition for 8 weeks, and equal amounts of distilled water are respectively added to the negative control group and the high-fat model group.
After the experiment was finished, the rats were fasted for 12h, anesthetized, and then soaked in 75% alcohol for 1min for sterilization. Collecting blood from rats of negative control group, high lipid model group, fructus crataegi proanthocyanidin extract group, fructus Jujubae polysaccharide group, and fructus crataegi procyanidin-fructus Jujubae polysaccharide composition group, centrifuging at 3000g for 10min, and collecting serum. Detecting Total Cholesterol (TC), Triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in serum by adopting a biochemical standard kit of Nanjing Biotechnology engineering Limited company, wherein the detection results are shown in a table 2; the ELISA kit of Shanghai-derived leaf bioengineering company Limited is adopted to detect the tumor necrosis factor alpha (TNF-alpha), the interleukin-1 beta (IL-1 beta), the interleukin-4 (IL-4) and the interleukin-10 (IL-10) in the serum, and the detection results are shown in Table 3.
TABLE 2 improvement of blood lipid of high-fat diet rat by fructus crataegi procyanidine-fructus Jujubae polysaccharide composition
Note: p < 0.05, P < 0.01 compared to negative controls; compared with the model control group, # P < 0.05, # P < 0.01.
TABLE 3 amelioration of high fat diet of inflammatory factors in rats with hawthorn procyanidins-jujube polysaccharide composition
Note: p < 0.05, P < 0.01 compared to negative controls; compared with the model control group, # P < 0.05, # P < 0.01.
After treating each group of rats for 8 weeks, after fasting the rats in each group for 12 hours, tail-cutting method is adopted to collect fasting blood samples of the rats in a negative control group, a high fat model group, a hawthorn proanthocyanidin extract group, a jujube polysaccharide group and a hawthorn proanthocyanidin-jujube polysaccharide composition group, 2.5g/kg bw of glucose solution (40%) is orally taken to all the rats after blood collection, then blood glucose is recorded at 0, 0.5, 1 and 2 hours respectively, and the area under the glucose tolerance curve is calculated. Rats were tested for fasting glucose (fasting 12h) and oral glucose tolerance using a blood glucose meter (Super glucocard II, Shiga, japan). The results are shown in Table 4.
TABLE 4 improvement of blood sugar in high-fat diet rat by fructus crataegi procyanidine-fructus Jujubae polysaccharide composition
Note: p < 0.05, P < 0.01 compared to negative controls; compared with the model control group, # P < 0.05, # P < 0.01.
The results of the experimental data shown in tables 2-4 show that the hawthorn procyanidin-jujube polysaccharide composition can obviously improve the blood fat, blood sugar and inflammatory factor levels of rats with high-fat diet, and the hawthorn procyanidin-jujube polysaccharide composition has better effect of improving the blood fat and blood sugar of rats with high-fat diet than a single hawthorn procyanidin extract or jujube polysaccharide, so that the hawthorn procyanidin and jujube polysaccharide have better synergistic effect after being combined, and obvious technical effects are obtained.
Example 5
Improvement of high-fat diet induced rat intestinal flora by hawthorn procyanidine-jujube polysaccharide composition
Male Wistar rats, 60, 5 weeks old, were acclimated to the new environment for one week and then randomly divided into 3 groups of 11 rats each, negative control group (CON), high fat model group (HFD), and hawthorn procyanidin-jujube polysaccharide composition group (HPC) prepared in example 3. The negative control rats were given a standard diet and the remaining rats were given a high fat diet for 8 weeks. Meanwhile, the hawthorn procyanidine-jujube polysaccharide composition is respectively given 200mg/kg for 8 weeks, and distilled water with the same amount is given to both the negative control group and the high-fat model group. After rat model creation, rats of the negative control group, the high fat model group and the hawthorn procyanidine-jujube polysaccharide composition group prepared in example 3 were sacrificed, and the cecal contents of the rats were taken out in a sterile environment, and the change of the flora therein was analyzed by metagenome. The results are shown in FIGS. 1-3.
The results of experimental analysis shown in fig. 1-3 show that the hawthorn procyanidin-jujube polysaccharide composition can improve the diversity of intestinal flora of high-fat diet rats, so that the abundance of bacteroidetes is improved, the abundance of firmicutes is reduced, and the abundance of bacteria related to obesity is also improved, such as Akkermansia.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The hawthorn procyanidin-jujube polysaccharide composition is characterized by comprising the following components in parts by weight: 21.82-22.02 parts of epicatechin, 13.02-13.22 parts of procyanidine B2, 1.30-1.50 parts of procyanidine B5, 3.46-3.66 parts of procyanidine C1 and 59.9-60.1 parts of jujube polysaccharide.
2. The hawthorn procyanidin-jujube polysaccharide composition of claim 1, comprising the following components in parts by weight: 21.92 parts of epicatechin, 13.12 parts of procyanidin B2, 1.40 parts of procyanidin B5, 3.56 parts of procyanidin C1 and 60 parts of jujube polysaccharide.
3. The hawthorn procyanidin-jujube polysaccharide composition of claim 1, wherein the preparation method of jujube polysaccharide comprises the following steps:
(1) mixing jujube with water to obtain a mixture, mixing the mixture with cellulase to obtain an extract, performing water bath extraction on the extract at 50-60 ℃ for 5-8 hours to obtain an extract, and centrifuging the extract at 3500-4500 rpm for 8-12 min to obtain a supernatant;
(2) carrying out alcohol precipitation on the supernatant obtained in the step (1) to obtain a first precipitate;
(3) mixing the first precipitate obtained in the step (2) with a sevage reagent, oscillating for 10-20 min, and centrifuging at 3500-4500 rpm for 4-6 min to obtain an upper-layer water phase;
(4) carrying out alcohol precipitation on the upper-layer water phase obtained in the step (3) for 1-3 d, carrying out solid-liquid separation to obtain a solution, and centrifuging the solution at 3500-4500 rpm for 8-12 min to obtain a second precipitate;
(5) and (4) dialyzing the second precipitate obtained in the step (4) for 2-3 days, and then freeze-drying the solution in the dialysis bag to obtain the jujube polysaccharide.
4. The hawthorn procyanidin-jujube polysaccharide composition as claimed in claim 3, wherein the mass-to-water volume ratio of the jujube in step (1) is 1g: 10-15 ml, and the mass percentage content of cellulase in the extract is 0.2-0.4%.
5. The hawthorn procyanidin-jujube polysaccharide composition of claim 3, wherein the alcohol precipitation conditions of step (2) comprise: the volume ratio of the alcohol solution used for alcohol precipitation to the supernatant is 3-5: 1, the mass percentage of the alcohol solution is 80-98%, and the temperature of alcohol precipitation is 3-5 ℃.
6. The hawthorn procyanidin-jujube polysaccharide composition of claim 3, wherein the ratio of the mass of the first precipitate in step (3) to the volume of sevage reagent is 1g:1-3 ml.
7. The hawthorn procyanidin-jujube polysaccharide composition of claim 3, wherein the alcohol precipitation conditions of step (4) comprise: the volume ratio of the alcohol solution used for alcohol precipitation to the upper-layer water phase is 2-4: 1, the mass percentage of the alcohol solution is 80-98%, and the temperature of alcohol precipitation is 3-5 ℃.
8. The hawthorn procyanidin-jujube polysaccharide composition of claim 3, wherein the dialysis bag of step (5) is a cellulose dialysis bag with a cut-off molecular weight of 7000D.
9. Use of the hawthorn procyanidin-jujube polysaccharide composition of claim 1 or 2 in the preparation of a medicament for ameliorating a disturbance in intestinal flora.
10. Use according to claim 4, wherein the intestinal flora disorder is a high-fat meal resulting in an intestinal flora disorder.
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