Hippophae rhamnoides peel polysaccharides, and preparation method therefor and use thereof

Abstract

Hippophae rhamnoides peel polysaccharides, and a preparation method therefor and a use thereof. The hippophae rhamnoides peel polysaccharides comprise the following monosaccharides: rhamnose, mannose, galactose, glucose, galacturonic acid, and arabinose. The preparation method comprises: degreasing, extracting, concentrating and performing alcohol precipitation to obtain water-soluble crude polysaccharides; and then using a non-polar macroporous adsorption resin to perform purification to obtain hippophae rhamnoides peel polysaccharides. The yield and content of the polysaccharides are high. By using the non-polar microporous adsorption resin to replace a relatively costly DEAE-cellulose and Sephadex gel, active polysaccharides in the Hippophae rhamnoides peel are enriched, the yield and purity of the polysaccharides are also greatly improved, and the polysaccharides have low production costs and are used for industrial batch production. The hippophae rhamnoides peel polysaccharides have significant effects of resisting ulcerative colitis, and alleviating constipation, indigestion, and intestinal flora disorder.

Description

Sea buckthorn peel polysaccharide and its preparation method and use Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine extraction, and particularly relates to seabuckthorn peel polysaccharide, a preparation method and application thereof. Background Art

[0002] Seabuckthorn, the dried, mature fruit of Hippophae rhamnoides Linn., a member of the Elaeagnaceae family, is a plant with both medicinal and edible properties. The seabuckthorn fruit consists of pulp (approximately 68%), seeds (approximately 23%), and peel (approximately 8%). Each component possesses highly medicinal value, containing over 100 bioactive compounds. Currently, research on the seabuckthorn fruit primarily focuses on the berry pulp and seeds. Numerous studies have reported that whole seabuckthorn fruit and seed meal are often used for the extraction of seabuckthorn seed oil, while seabuckthorn pulp is often used for the extraction of seabuckthorn flavonoids. Seabuckthorn oil has a rich chemical composition and unique properties, including various unsaturated fatty acids, carotenoids, and natural vitamin E, most of which are essential active ingredients for the human body. Seabuckthorn flavonoids have numerous physiological functions, such as anti-inflammatory, liver protection, obesity alleviation, and hypoglycemic effects. However, the seabuckthorn peel has not been effectively developed and utilized, primarily due to deficiencies in research related to component analysis, extraction and purification process optimization, and pharmacological activity evaluation.

[0003] The inventors' previous research found that the polysaccharide content in seabuckthorn peel is greater than the flavonoid content and the oil content, and the polysaccharide content is rich.

[0004] Polysaccharides are naturally occurring high-molecular-weight carbohydrates composed of a series of monosaccharides linked by glycosidic bonds. However, different Chinese herbal medicines have varying polysaccharide compositions, with variations in molecular weight, monosaccharide composition, glycosidic bonds, and biological functions. Therefore, the efficient extraction, separation, and purification of polysaccharides from plants, animals, and microorganisms is crucial for the development of therapeutic drugs and healthcare products.

[0005] Polysaccharide extraction methods often use water extraction and alcohol precipitation, after which the precipitate is collected to obtain crude polysaccharides. However, during the polysaccharide extraction process, pigments, proteins, and some small molecules are also dissolved along with the polysaccharide, necessitating a series of subsequent impurity removal and purification steps.

[0006] The polysaccharide impurity removal process mainly includes protein removal and pigment removal. The classic methods for polysaccharide protein removal mainly include the freeze-thaw method, the trichloroacetic acid (TCA) method, the Sevag method, etc. The freeze-thaw method is a physical protein removal method with less polysaccharide loss, but it is time-consuming and inefficient. The principle of the TCA method and the Sevag method is to use organic reagents to irreversibly denature the protein, so that it precipitates in the solution and the protein is removed by centrifugation. The TCA method and the Sevag method have high protein removal efficiency, but are prone to polysaccharide activity destruction and organic reagent residue problems. The classic methods for polysaccharide decolorization mainly include the activated carbon method and the H2O2 method. The principle of activated carbon decolorization is to use its good adsorption properties to adsorb impurities and pigments in the polysaccharide solution to achieve the decolorization effect of the polysaccharide solution, but it is easy to cause the polysaccharide and activated carbon to mix together, making it difficult to separate. The principle of H2O2 decolorization is to use oxidation to oxidize the pigment molecules in polysaccharides into colorless substances. The decolorization effect is good and the efficiency is high, but the concentration and time of hydrogen peroxide need to be controlled to avoid oxidation-induced destruction of polysaccharide molecules.

[0007] The polysaccharide purification process is usually carried out using techniques such as step-by-step precipitation, ion exchange chromatography, and gel chromatography. The step-by-step precipitation method utilizes the differences in polysaccharide structure, molecular weight, and polarity, as well as the differences in solubility in organic solvents, and continuously increases the concentration of organic reagents (methanol, ethanol, etc.) to precipitate polysaccharides from large to small molecular weight, but it is generally difficult to obtain uniform polysaccharides. Ion exchange chromatography uses chemically bonded ion exchangers or ion exchange resins as the stationary phase, and utilizes the differences in the ion exchange capacity or selectivity coefficient of the target polysaccharide to achieve separation. DEAE-cellulose columns are usually used to achieve good separation effects for neutral polysaccharides and acidic polysaccharides. This is because acidic polysaccharides contain anions that can be adsorbed by the chromatography column, and neutral polysaccharides flow out of the chromatography column first, thereby purifying the polysaccharides. Gel chromatography uses the exclusion effect of porous gels to separate molecules of different sizes, and can achieve efficient separation of high-molecular-weight substances. Cross-linked dextran gel Sephadex and agarose gel Sepharose are usually used to separate and purify polysaccharides. DEAE-cellulose columns and gel columns can produce good separation effects on polysaccharides. However, their disadvantages are that due to the small pores of the fillers, the chromatographic separation speed is slow and the fillers are expensive. They are only suitable for laboratory use and have certain limitations.

[0008] In recent years, macroporous resin adsorption technology has been applied in the extraction and purification of natural products, with particular application to flavonoids, alkaloids, and saponins. Macroporous resin adsorption selectively absorbs substances from a solution using physical adsorption, depending on their molecular weight and structure, thereby purifying the target substance. While macroporous resins can theoretically be used to purify polysaccharides, this can significantly shorten purification time and simplify the process. However, macroporous resins have weak adsorption and desorption capabilities for most polysaccharides, resulting in poor separation results. Consequently, limited research has been conducted on this method for polysaccharide purification. Consequently, selecting suitable resins based on the properties of polysaccharides is complex and difficult.

[0009] A search revealed that DEAE-cellulose and dextran gel columns are currently commonly used to purify and enrich crude seabuckthorn polysaccharides, yielding pharmacologically active seabuckthorn polysaccharides. Patent document CN115028753A discloses a uniform seabuckthorn polysaccharide with anti-tumor efficacy, its isolation and purification methods, and applications. The method involves extracting crude seabuckthorn dried fruit powder using flash extraction. The extract is then precipitated with anhydrous ethanol to remove impurities, deproteinized with Sevag reagent, and decolorized with H2O2 solution to obtain refined seabuckthorn polysaccharide. The polysaccharide is then purified using DEAE-52 fiber columns and Sephadex G-200 columns, yielding a 16.61% yield and a sugar content of 58.43 mg / g. Patent document CN110790848A discloses a method for preparing total seabuckthorn polysaccharides and their application. The method involves extracting defatted seabuckthorn fruit with distilled water, precipitating with anhydrous ethanol, and centrifuging the resulting polysaccharide precipitate at 40°C to produce a water-soluble crude polysaccharide. This crude polysaccharide is then subjected to the Sevag method for protein removal. The polysaccharide is then isolated and purified using ion exchange resin DEAE-52 and Sephadex G-150 to obtain total seabuckthorn polysaccharides with antioxidant and anti-fatigue properties. This method yields 10.61% total seabuckthorn polysaccharide. Furthermore, patent document CN103992299A discloses a method for simultaneously isolating and purifying multiple bioactive components from seabuckthorn seed meal. The seabuckthorn seed meal extract is eluted with AB-8 macroporous adsorption resin to collect seabuckthorn polysaccharides with a purity exceeding 80%.

[0010] The AB-8 macroporous resin used in patent document CN103992299A is a medium-polarity macroporous adsorption resin suitable for separating the polysaccharide components obtained after alcohol extraction of sea buckthorn seed meal. The monosaccharide composition and pharmacological activity of sea buckthorn seed meal polysaccharides are unknown.

[0011] While numerous studies have been conducted on seabuckthorn polysaccharides, the sources of these polysaccharides vary. Most are derived from seabuckthorn fruit, with very few from the pericarp. Polysaccharides from different sources vary in their monosaccharide composition, sugar residues, and molecular weight, leading to significant differences in their pharmacological activities. Furthermore, the DEAE-cellulose and dextran gel columns commonly used in the purification of seabuckthorn polysaccharides are expensive and slow to chromatographically separate. The yield and purity of the resulting seabuckthorn polysaccharides remain low, and functional evaluations of seabuckthorn polysaccharides are relatively limited. Therefore, further research is needed on seabuckthorn pericarp polysaccharides, their preparation methods, and their uses.

[0012] Summary of the Invention

[0013] To address the shortcomings of existing technologies, the present invention provides a sea buckthorn peel polysaccharide, its preparation method, and uses. By using a non-polar macroporous adsorption resin instead of a DEAE-cellulose column and a dextran gel column to treat crude polysaccharide obtained from sea buckthorn peel, the polysaccharide is enriched while simultaneously removing most impurities. This preparation method effectively improves the yield and purity of sea buckthorn polysaccharide, effectively preserves the polysaccharide's structure and biological activity, and eliminates residual organic solvents, making it suitable for large-scale production of sea buckthorn peel polysaccharide.

[0014] The inventors have discovered that the seabuckthorn peel polysaccharide of the present invention has a significant therapeutic effect on ulcerative colitis. A low-dose treatment group (50 mg / kg / day of seabuckthorn peel polysaccharide) was able to improve ulcerative colitis, while a high-dose treatment group (100 mg / kg / day of seabuckthorn peel polysaccharide) was able to significantly increase the body weight and colon length of mice with dextran sulfate (DSS)-induced ulcerative colitis (UC), reduce the disease activity index, significantly inhibit the increase in pro-inflammatory factors in the serum and colon tissue of DSS-induced UC mice, promote the secretion of anti-inflammatory factors, effectively inhibit the increase in intestinal permeability induced by DSS in UC mice, significantly reduce colon tissue damage in model mice, and restore the intestinal barrier function of colon tissue. Compared with the sea buckthorn fruit polysaccharide that has been reported to have a therapeutic effect on ulcerative colitis at a dose of 200 mg / kg / d [Wang Xinxu. Study on the regulatory mechanism of sea buckthorn polysaccharide on DSS-induced ulcerative colitis in mice [D]. Inner Mongolia Agricultural University, 2019.], the sea buckthorn peel polysaccharide provided by the present invention can achieve good therapeutic effects at a dose of 100 mg / kg / d, and its efficacy is better than the above-mentioned sea buckthorn fruit polysaccharide.

[0015] In addition, the inventors have found through research that the seabuckthorn peel polysaccharide of the present invention has a significant digestive function, can improve functional constipation and indigestion, and can also significantly improve intestinal flora disorders.

[0016] Therefore, the first object of the present invention is to provide a sea buckthorn peel polysaccharide.

[0017] The seabuckthorn peel polysaccharide provided by the invention comprises the following monosaccharides: rhamnose, mannose, galactose, glucose, galacturonic acid and arabinose, and the molar ratio thereof is 1:2.03-2.63:2.65-3.17:3.20-3.97:4.01-7.33:7.95-11.67.

[0018] Preferably, the molar ratio of rhamnose, mannose, galactose, glucose, galacturonic acid and arabinose is 1:2.10-2.63:2.65-3.17:3.20-3.97:4.10-6.35:9.60-11.30.

[0019] Preferably, the mass percentage of neutral sugar in the seabuckthorn peel polysaccharide is 82.73% to 85.31%.

[0020] Preferably, the molecular weight distribution range of the seabuckthorn peel polysaccharide is 2.5 kDa to 2.5×10 2 kDa.

[0021] Preferably, the mass percentage of uronic acid in the seabuckthorn peel polysaccharide is 16.90% to 23.78%.

[0022] Preferably, the sea buckthorn peel polysaccharide is yellow, an amorphous powder, has a fine texture, is soluble in water, and turns yellow when reacted with a phenol-sulfuric acid reagent.

[0023] The second object of the present invention is to provide a method for preparing seabuckthorn peel polysaccharide, which can obtain seabuckthorn peel polysaccharide in large quantities.

[0024] The present invention provides a method for preparing seabuckthorn peel polysaccharide, comprising the following steps:

[0025] (1) Defatting: adding dried seabuckthorn peel to an organic solvent for reflux, immersion or percolation extraction, discarding the extract, and obtaining defatted seabuckthorn peel residue;

[0026] (2) Extraction: drying the defatted seabuckthorn peel residue, soaking it in water, and then refluxing it for extraction, filtering it, and obtaining an extract;

[0027] (3) Concentrating: concentrating the extract and cooling it to obtain a concentrated solution;

[0028] (4) Alcohol precipitation: adding anhydrous ethanol to the concentrated solution or adding the concentrated solution to a high-concentration ethanol aqueous solution, allowing to stand, collecting the precipitate, and obtaining crude seabuckthorn peel polysaccharide;

[0029] (5) Purification: Dissolve the crude polysaccharide from seabuckthorn peel in water, add it to non-polar macroporous adsorption resin D101, D312, X-5 or HP-20, elute, concentrate and dry the eluate to obtain seabuckthorn peel polysaccharide.

[0030] Preferably, the method for preparing seabuckthorn peel polysaccharide comprises the following steps:

[0031] (1) Defatting: Dry the seabuckthorn peel, add an organic solvent with a solid-liquid ratio of 1:5-10 (kg / L) and perform reflux extraction. The extraction time is 2-3 hours, and the extraction is repeated 1-3 times. The extract is filtered and the extract is discarded to obtain the defatted seabuckthorn peel residue.

[0032] (2) Extraction: Dry the defatted seabuckthorn peel residue, add water (e.g., distilled water) with a solid-liquid ratio of 1:6-10 (kg / L) and soak the residue for several hours, then perform reflux extraction for 2-3 hours, perform extraction 2-3 times, filter, and combine the extracts;

[0033] (3) Concentration: Concentrating the extract to 1 / 50 to 1 / 4 of the original volume, cooling to room temperature, and obtaining a concentrated solution;

[0034] (4) Alcohol precipitation: adding anhydrous ethanol to the concentrated solution under stirring to make the volume concentration of ethanol 60-80%, or adding the concentrated solution to a high-concentration ethanol aqueous solution until the mass percentage of ethanol in the concentrated solution is 60-80%, standing at 4-25°C for 6-24 hours, discarding the supernatant, collecting the precipitate, and obtaining the crude seabuckthorn peel polysaccharide;

[0035] (5) Purification: Use non-polar macroporous adsorption resin D101, D312, X-5 or HP-20 to decolorize and deproteinize the crude polysaccharide from sea buckthorn peel; dissolve the crude polysaccharide from sea buckthorn peel in water, slowly add it to the non-polar macroporous adsorption resin, elute with water, collect the eluate, concentrate it to 1 / 80 to 1 / 2 of the original volume, and dry it to obtain sea buckthorn peel polysaccharide.

[0036] Preferably, in step (1), the organic solvent is ether, petroleum ether, ethyl acetate, a methanol aqueous solution with a mass percentage of 75% to 100%, or an ethanol aqueous solution with a mass percentage of 75% to 100%.

[0037] Preferably, in step (2), water (such as tap water) is added to the defatted seabuckthorn peel residue and the soaking time is 3 to 24 hours.

[0038] Preferably, in step (4), the mass percentage of the high-concentration ethanol aqueous solution is 80% to 100%.

[0039] Preferably, in step (5), the non-polar macroporous adsorption resin is D101 macroporous adsorption resin.

[0040] Preferably, in step (5), the mass concentration of the sea buckthorn peel crude polysaccharide loading solution is 0.005-0.03 kg / L, the loading amount is 0.01-0.15 kg / kg (polysaccharide / resin), the loading flow rate is 1-5 column volumes (BV) / h, and the sample is allowed to stand for 12-24 hours after loading.

[0041] Preferably, in step (5), the elution method is to use water (such as distilled water) for elution, the elution flow rate is 1 to 5 BV / h, and the elution volume is 1 to 10 BV.

[0042] Preferably, in step (5), the drying method is spray drying, reduced pressure drying or freeze drying.

[0043] Preferably, in step (5), the decolorization rate is greater than 75%, the protein clearance rate is greater than 73%, and the polysaccharide retention rate is greater than 70%.

[0044] The third object of the present invention is to provide the use of the seabuckthorn peel polysaccharide in the preparation of medicines or functional health products for treating functional intestinal diseases.

[0045] Preferably, the intestinal functional disease includes one or more of ulcerative colitis, constipation, indigestion and intestinal flora disorder.

[0046] Preferably, the seabuckthorn peel polysaccharide exerts anti-inflammatory effects by promoting the secretion of anti-inflammatory factors.

[0047] In addition, the present invention provides a composition comprising the seabuckthorn peel polysaccharide or the seabuckthorn peel polysaccharide prepared by the preparation method, and pharmaceutically acceptable excipients.

[0048] The compositions, medicines, or functional health products of the present invention are prepared according to corresponding conventional pharmaceutical preparation methods. The compositions, medicines, or functional health products of the present invention use seabuckthorn peel polysaccharide as the sole active ingredient or as one of the active ingredients. Those skilled in the art can add appropriate auxiliary ingredients according to actual needs and prepare the corresponding dosage forms using conventional techniques, and the present invention is not particularly limited thereto.

[0049] The beneficial effects of the present invention are: providing a sea buckthorn peel polysaccharide, a preparation method thereof, and uses thereof. In the preparation method, a non-polar macroporous adsorption resin is used instead of a DEAE-cellulose column and a dextran gel column to enrich and purify the crude sea buckthorn peel polysaccharide, thereby obtaining a sea buckthorn peel polysaccharide with high application value. The yield of the polysaccharide preparation method of the present invention can reach more than 18.86%, and the neutral sugar content in the polysaccharide can reach more than 82.73%. Compared with the existing method, the purity and yield of the polysaccharide are greatly improved. The operation process of the preparation method is simple, the obtained polysaccharide has high purity, which can greatly reduce the cost of industrial production, avoid the residual organic solvent during the operation process, and conform to the concept of green development. DETAILED DESCRIPTION

[0050] Typical examples are listed below to further illustrate the present invention, but are not intended to limit the present invention in any form.

[0051] The following contents, unless otherwise specified, are percentages by mass.

[0052] Unless otherwise specified, the ethanol solutions mentioned below are aqueous solutions of ethanol.

[0053] Example 1

[0054] 600 kg of dried seabuckthorn peel was weighed and extracted with 4000 L of petroleum ether for 12 hours. After extraction, the petroleum ether extract was filtered, retaining the seabuckthorn peel residue. After defatting, the petroleum ether in the seabuckthorn peel residue was evaporated, and the residue was soaked in 4000 L of water for 6 hours. After that, the residue was refluxed and extracted three times, each for 2 hours, and filtered. The aqueous extracts were combined and the extract was transferred to a double-effect concentrator for vacuum concentration to a final concentration of 1200 L. After cooling to room temperature, 3 volumes of anhydrous ethanol were added to the concentrated extract. After standing at 15°C for 24 hours, the precipitate was collected. The precipitate was dissolved in water at a concentration of 0.01 kg / L, and the solution was added to D312 macroporous adsorption resin at a sample load of 0.05 kg / kg (polysaccharide / resin). The sample flow rate was controlled at 3 BV / h, and the mixture was allowed to stand for 12 hours after loading. Water was then used for elution, with the elution flow rate controlled at 3 BV / h and the elution volume at 6 BV. After completion, the eluate was collected and drawn into a double-effect concentrator for vacuum concentration, ultimately concentrating to 1800 L. After vacuum drying of the concentrate, sea buckthorn peel polysaccharide was obtained, with a neutral sugar content of 82.75%.

[0055] Example 2

[0056] 800 kg of dried seabuckthorn peel was weighed and degreased by adding 5500 L of 85% methanol-water solution. The extraction time was refluxed for 3 hours. After the extraction, the methanol extract was filtered out, and the seabuckthorn peel residue was retained. The seabuckthorn peel residue was treated once more according to the above-mentioned degreasing method. After the extraction, the methanol extract was filtered out. The methanol in the degreased seabuckthorn peel residue was evaporated, and the residue was soaked in 5500 L of water for 12 hours. After that, refluxed and extracted three times, each for 2 hours, and filtered. The aqueous extracts were combined, and the extracted liquid was transferred to a double-effect concentrator for vacuum concentration. The final concentration was 2000 L. After cooling to room temperature, 3 volumes of anhydrous ethanol were added to the concentrated extract. After standing at 4°C for 12 hours, the precipitate was collected. The precipitate was dissolved in water at a concentration of 0.015 kg / L, and the solution was added to HP-20 macroporous adsorption resin at a sample loading rate of 0.03 kg / kg (polysaccharide / resin). The sample loading flow rate was controlled at 4 BV / h, and the mixture was allowed to stand for 18 hours after loading. Water was then used for elution, with the elution flow rate controlled at 4 BV / h and the elution volume being 5 BV. After the elution was completed, the eluate was collected and the feed liquid was extracted into a double-effect concentrator for vacuum concentration, and finally concentrated to 2200 L. After freeze-drying, the concentrated solution obtained sea buckthorn peel polysaccharide with a neutral sugar content of 83.67%.

[0057] Example 3

[0058] Take 500kg dry seabuckthorn peel, add 3000L ethyl acetate and carry out diafiltration process, the processing time is 12h, after finishing, ethyl acetate extract is leached, obtain the seabuckthorn peel medicinal residue after degreasing.After degreasing, the ethyl acetate in the seabuckthorn peel medicinal residue is evaporated at 50 ℃, add 4000L water to soak the medicinal residue for 6h, reflux extraction 3 times afterwards, each 2h, filter.Merge water extract, extract feed liquid and enter double-effect concentrator and carry out concentrating under reduced pressure, finally be concentrated into 800L, after being cooled to room temperature, in 3200L 85% ethanolic solution, add concentrated extract, after leaving standstill 20h at 20 ℃, collect precipitation.This precipitation is dissolved in water according to 0.01kg / L concentration, solution is that 0.12kg / kg (polysaccharide / resin) adds in X-5 macroporous adsorption resin according to sample loading, and sample loading flow rate is controlled at 4BV / h, leaves standstill 12h after sample loading. Water was then used for elution, with the elution flow rate controlled at 3 BV / h and the elution volume at 6 BV. After completion, the eluate was collected and the feed liquid was drawn into a double-effect concentrator for vacuum concentration, ultimately concentrating to 1900 L. After spray drying, the concentrated liquid obtained sea buckthorn peel polysaccharide, with a neutral sugar content of 82.73%.

[0059] Example 4

[0060] 600 kg of dried seabuckthorn peel was weighed and refluxed with 4000 L of 80% ethanol. The medicinal material was defatted for 3 hours. After the extraction, the ethanol extract was filtered out, and the seabuckthorn peel residue was retained. The seabuckthorn peel residue was treated once more according to the above extraction method. After the extraction, the ethanol extract was filtered out. The ethanol in the defatted seabuckthorn peel residue was evaporated at 60°C, and the residue was soaked in 4000 L of water for 3 hours. After that, reflux extraction was performed three times, each for 2 hours, and filtered. The aqueous extracts were combined and the extract was transferred to a double-effect concentrator for vacuum concentration. The final concentration was 1000 L. After precooling at 10°C for 6 hours, the concentrated extract was added to 3000 L of 88% ethanol solution. After standing at 25°C for 22 hours, the precipitate was collected. The precipitate was dissolved in water at a concentration of 0.025 kg / L, and the solution was added to D101 macroporous adsorption resin at a sample loading rate of 0.15 kg / kg (polysaccharide / resin). The sample loading flow rate was controlled at 2 BV / h, and the mixture was allowed to stand for 12 hours after loading. Water was then used for elution, with the elution flow rate controlled at 3 BV / h and the elution volume being 5 BV. After the elution was completed, the eluate was collected and the feed liquid was extracted into a double-effect concentrator for vacuum concentration, and finally concentrated to 1500 L. After freeze-drying, the concentrated solution obtained sea buckthorn peel polysaccharide with a neutral sugar content of 85.31%.

[0061] Example 5

[0062] 900 kg of dried seabuckthorn peel was weighed and refluxed with 5500 L of 75% ethanol. The medicinal material was defatted for 3 hours. After extraction, the ethanol extract was filtered out, and the seabuckthorn peel residue was retained. The seabuckthorn peel residue was treated once more according to the above extraction method. After extraction, the ethanol extract was filtered out. The ethanol in the defatted seabuckthorn peel residue was evaporated at 50°C, and the residue was soaked in 5500 L of water for 5 hours. After that, reflux extraction was performed twice, each for 3 hours, and filtered. The aqueous extracts were combined and the extract was transferred to a double-effect concentrator for vacuum concentration. The final concentration was 950 L. After cooling to room temperature, 2 volumes of anhydrous ethanol were added to the concentrated extract. After standing at 4°C for 12 hours, the precipitate was collected. The precipitate was dissolved in water at a concentration of 0.015 kg / L, and the solution was added to D101 macroporous adsorption resin at a sample loading rate of 0.05 kg / kg (polysaccharide / resin). The sample loading flow rate was controlled at 4 BV / h, and the mixture was allowed to stand for 12 hours after loading. Water was then used for elution, with the elution flow rate controlled at 3 BV / h and the elution volume being 6 BV. After the elution was completed, the eluate was collected and the feed liquid was extracted into a double-effect concentrator for vacuum concentration, and finally concentrated to 3000 L. After spray drying, the concentrated solution was obtained to obtain sea buckthorn peel polysaccharide, with a neutral sugar content of 84.58%.

[0063] Example 6

[0064] Take 600kg dry seabuckthorn peel, add 3000L 95% ethanol solution and carry out reflux extraction, extraction time is 2h, after extraction finishes, ethanol extract is leached, retain seabuckthorn peel medicinal residues, reprocess 1 seabuckthorn peel medicinal residues according to the above-mentioned extraction method, after extraction finishes, ethanol extract is leached. After defatting, the ethanol in the seabuckthorn peel medicinal residues is evaporated, add 3600L water to soak the medicinal residues for 10h, reflux extraction 2 times, each 3h, filter afterwards. Combine the aqueous extracts, extract the feed liquid and enter the double-effect concentrator for concentrating under reduced pressure, finally be concentrated into 1700L, after being cooled to room temperature, add concentrated extract in 5500L 80% ethanol solution, after leaving standstill for 24h at 4 ℃, collect precipitation. The precipitate was dissolved in water at a concentration of 0.03 kg / L, and the solution was added to D312 macroporous adsorption resin at a sample loading rate of 0.12 kg / kg (polysaccharide / resin). The sample loading flow rate was controlled at 4 BV / h, and the mixture was allowed to stand for 24 hours after loading. Water was then used for elution, with the elution flow rate controlled at 2 BV / h and the elution volume being 4 BV. After the elution was completed, the eluate was collected and the feed liquid was extracted into a double-effect concentrator for vacuum concentration, and finally concentrated to 1500 L. After freeze-drying, the concentrated solution obtained sea buckthorn peel polysaccharide with a neutral sugar content of 83.95%.

[0065] Comparative Example 1

[0066] A sea buckthorn seed meal polysaccharide [a method for simultaneously separating and purifying multiple bioactive components in sea buckthorn seed meal, patent application publication number CN 103992299 A], the preparation method of which comprises the following steps:

[0067] 0.5 kg of sea buckthorn seed meal obtained after supercritical CO2 extraction of sea buckthorn seed oil was extracted with 5 L of 75% ethanol solution, 25 g of cellulase and 10 g of pectinase were added, and the mixture was extracted three times under 500 W ultrasonic wave, with the pH controlled at 3.0 and the temperature controlled at 40°C, and each extraction time was 2 hours; the three extracts were combined, concentrated and recovered with a rotary evaporator to recover the ethanol, and the obtained aqueous solution was filtered through a 200-mesh filter cloth to obtain a filtrate; the filtrate was adsorbed on an AB-8 macroporous resin, eluted with water until colorless, and the eluate was collected to obtain a crude polysaccharide, which was freeze-dried, pre-frozen at -50°C for 2 hours, and then vacuum-dried for 24 hours to obtain a powdered sea buckthorn seed meal polysaccharide. The neutral sugar content of the polysaccharide was determined to be 83.1%.

[0068] Comparative Example 2

[0069] A seabuckthorn fruit polysaccharide [Wang Xinxu. Study on the regulatory mechanism of seabuckthorn polysaccharide on DSS-induced ulcerative colitis in mice [D]. Inner Mongolia Agricultural University, 2019.], the preparation method of which comprises the following steps:

[0070] 100g of dried seabuckthorn fruit was ground in a mortar, passed through a 30-mesh sieve, and defatted by adding 800mL of petroleum ether. The filtrate was recovered, ultrasonically cracked with distilled water, and subjected to hot water extraction. The aqueous extract was concentrated under reduced pressure at 80°C, and Sevag reagent was added to remove protein. Anhydrous ethanol was added to a beaker containing the deproteinized sample solution to a concentration of 80%, and the solution was precipitated with alcohol in a 4°C refrigerator. After 48 hours, a precipitate was precipitated to obtain a crude polysaccharide. The polysaccharide solution was dissolved in distilled water to form a 5% polysaccharide solution, and concentrated ammonia water was adjusted to a pH of approximately 8.0. H2O2 was added dropwise below 50°C until it turned light yellow. The solution was kept warm for 2 hours, loaded into a dialysis membrane (molecular weight cut-off of 8000-14000), dialyzed against running water for 72 hours, then against distilled water for 48 hours, and freeze-dried to obtain a seabuckthorn fruit polysaccharide having a neutral sugar content of 60.01%.

[0071] The following test examples further illustrate the physicochemical properties of the seabuckthorn peel polysaccharide of the present invention, the purification effect evaluation of the method for preparing the polysaccharide using non-polar macroporous adsorption resins (D101, D312, X-5, HP-20), and the therapeutic effect of the polysaccharide on intestinal functional diseases.

[0072] Test Example 1: Determination of neutral sugar, protein and uronic acid content in seabuckthorn peel polysaccharide

[0073] Neutral sugar content determination: The neutral sugar content was determined using the phenol-sulfuric acid method. Standard glucose solutions of 0.00, 0.02, 0.04, 0.06, 0.08, 0.10, and 0.12 mg / mL were prepared to generate a standard curve. The polysaccharides obtained in Examples 1 to 6 were accurately weighed and prepared into a 0.1 mg / mL solution. 2 mL of the sample solution was accurately pipetted into a test tube, and 2 mL of 6% phenol and 6 mL of concentrated H2SO4 were added in sequence. The mixture was mixed and allowed to stand for 10 minutes, then placed in a 100°C water bath for 20 minutes. After cooling to room temperature, the absorbance was measured at 490 nm. The neutral sugar contents of the polysaccharides were calculated using the standard curve to be 82.75%, 83.67%, 82.73%, 85.31%, 84.58%, and 83.95%, respectively.

[0074] Protein content determination: The protein content was determined by the Coomassie Brilliant Blue method. 0.00, 0.02, 0.04, 0.06, 0.08, 0.10, and 0.12 mg / mL bovine serum albumin standard solutions were prepared to create a standard curve. Accurately weigh the polysaccharides obtained in Examples 1 to 6 and prepare a 0.2 mg / mL solution. Accurately pipette 3 mL of the sample solution into test tubes, add 3 mL of Coomassie Brilliant Blue reagent, mix well, and measure the absorbance at 595 nm. The protein contents calculated using the standard curve were 2.54%, 2.24%, 2.33%, 2.11%, 2.17%, and 2.19%, respectively.

[0075] Determination of uronic acid content: The uronic acid content was determined using the sulfuric acid-carbazole method. Galacturonic acid standard solutions at concentrations of 0.00, 0.02, 0.04, 0.06, 0.08, 0.10, and 0.12 mg / mL were prepared to generate a standard curve. The polysaccharides obtained in Examples 1-6 were accurately weighed and prepared into a 0.1 mg / mL solution. Accurately pipette 2 mL of sample solution into a test tube, add 6 mL of concentrated H₂SO₄ to each solution, shake well, and cool to room temperature. Then, add 1 mL of 0.15% carbazole, shake well, and let stand for 30 minutes. Measure the absorbance at 530 nm, and calculate the uronic acid contents using the standard curve to be 21.72%, 22.50%, 23.78%, 16.90%, 16.95%, and 17.53%, respectively.

[0076] Test Example 2: Monosaccharide composition analysis of seabuckthorn peel polysaccharides

[0077] PMP derivatization: Prepare 10 mg / mL Man, Rha, GlcA, GalA, Glc, Gal, Ara, and Fuc standard solutions, pipette 400 μL of each into eight 50 mL round-bottom flasks, add 200 μL of 0.3 mol / L NaOH solution and 400 μL of 0.5 mol / L PMP methanol solution, mix well, blow with nitrogen for 5 min, place in a 70°C water bath for 2 h, cool to room temperature, adjust the pH to 7 with 0.3 mol / L HCl, then add 2 mL of H2O and 4 mL of CHCl3, extract, retain the aqueous phase, and extract three times with CHCl3. Collect the aqueous derivatized samples, filter through a 0.45 μm water filter, and use for HPLC analysis.

[0078] Complete acid hydrolysis of seabuckthorn peel polysaccharide: Accurately weigh 10 mg of each polysaccharide obtained in Examples 1-6 to prepare a 0.2 mg / mL solution. Add 1 mL of 2 mol / L trifluoroacetic acid solution, aerate with nitrogen for 5 minutes, seal, and incubate in a 100°C water bath for 8 hours until the polysaccharide is completely acid-hydrolyzed. Cool the acid-hydrolyzed sample to room temperature, centrifuge (4000 rpm, 5 minutes), and collect the supernatant. Add 5 mL of methanol to the supernatant, spin dry, and repeat 2-3 times to remove excess trifluoroacetic acid. Add 2 mL of distilled water, and adjust the pH to 7 with 2 mol / L NaOH solution to obtain the complete acid hydrolyzate of seabuckthorn peel polysaccharide.

[0079] PMP derivatization of sea buckthorn peel polysaccharide: 400 μL of sea buckthorn peel polysaccharide fully hydrolyzed sample was subjected to PMP derivatization according to the standard.

[0080] HPLC conditions: A DIONEX UltiMate chromatography system was used. Column: Accliam™ 120C18 (4.6 mm id × 250 mm, 5 μm, Thermo Fisher); flow rate: 1.0 mL / min; injection volume: 10 μL; column temperature: 30°C; detection wavelength: 250 nm; mobile phase: 100 mmol / L aqueous ammonium acetate (pH 5.0): tetrahydrofuran: acetonitrile = 81:2:17.

[0081] The monosaccharide compositions and molar ratios of the seabuckthorn peel polysaccharides obtained in Examples 1 to 6 were measured based on the retention time and peak area of ​​the monosaccharides:

[0082] Example 1, rhamnose: mannose: galactose: glucose: galacturonic acid: arabinose = 1: 2.13: 2.71: 3.30: 4.22: 9.65.

[0083] Example 2, rhamnose: mannose: galactose: glucose: galacturonic acid: arabinose = 1: 2.42: 2.69: 3.67: 5.25: 9.88.

[0084] Example 3, rhamnose: mannose: galactose: glucose: galacturonic acid: arabinose = 1: 2.35: 2.74: 3.81: 6.25: 10.13.

[0085] Example 4, rhamnose: mannose: galactose: glucose: galacturonic acid: arabinose = 1: 2.55: 3.05: 3.86: 6.31: 11.24.

[0086] Example 5, rhamnose: mannose: galactose: glucose: galacturonic acid: arabinose = 1: 2.62: 3.14: 3.96: 4.82: 10.56.

[0087] Example 6, rhamnose: mannose: galactose: glucose: galacturonic acid: arabinose = 1: 2.59: 3.07: 3.93: 4.17: 9.81.

[0088] Test Example 3: Determination of molecular weight distribution range of seabuckthorn peel polysaccharide

[0089] Chromatographic column: TSK-Gel G3000; mobile phase: 0.1 mol / L sodium sulfate; column temperature: 35 ° C; flow rate: 0.5 mL / min; detector: differential detector. The standard solution was prepared using various dextran series standards (180, 2700, 5250, 9750, 13050, 36800, 64650 Da), and a standard curve was drawn. The polysaccharides obtained in Examples 1 to 6 were weighed and dissolved in 400 μL of mobile phase so that the concentration of all samples was 5 mg / mL. According to the chromatographic peak position of the dextran standard curve and the polysaccharide sample spectrum, the molecular weight distribution range of the polysaccharide samples was calculated to be: 4.063 kDa to 2.289 × 10 2 kDa, 2.912 kDa~1.594×10 2 kDa, 3.637 kDa~1.757×10 2 kDa, 3.589 kDa~2.500×10 2 kDa, 4.832 kDa~1.872×10 2 kDa, 2.500 kDa~1.337×10 2 kDa.

[0090] Experimental Example 4: Evaluation of the method for preparing polysaccharides using non-polar macroporous adsorption resin, including the determination of three indicators: decolorization rate, protein clearance rate, and polysaccharide retention rate

[0091] Decolorization rate determination: The polysaccharides obtained before and after treatment with the non-polar macroporous adsorption resin in Examples 1 to 6 were prepared into 1 mg / mL solutions, centrifuged at 3000 r / min, filtered, and the absorbance was measured at 450 nm. According to Formula 1, the decolorization rates achieved by the processes of Examples 1 to 6 were 77.34%, 76.23%, 79.21%, 75.96%, 75.73%, and 76.51%, respectively. Decolorization rate (100%) = [(A 脱色前 -A 脱色后 ) / A 脱色前 ]×100% (1)

[0092] Protein clearance rate determination: The protein content was determined by the Coomassie Brilliant Blue method. 0.00, 0.02, 0.04, 0.06, 0.08, 0.10, and 0.12 mg / mL bovine serum albumin standard solutions were prepared to create a standard curve. Accurately weigh the polysaccharides obtained before and after treatment with the non-polar macroporous adsorption resin in Examples 1 to 6 and prepare a 0.2 mg / mL solution. Accurately draw 3 mL of the sample solution and place it in a test tube, add 3 mL of Coomassie Brilliant Blue reagent, mix well, and measure the absorbance at 595 nm. The protein content of the polysaccharide was calculated using the standard curve. According to Formula 2, the protein clearance rates achieved by the processes of Examples 1 to 6 were 75.84%, 73.74%, 74.65%, 73.19%, 73.23%, and 74.39%, respectively.

[0093] Determination of Polysaccharide Retention Rate: The polysaccharide content was determined using the phenol-sulfuric acid method. Standard glucose solutions at concentrations of 0.00, 0.02, 0.04, 0.06, 0.08, 0.10, and 0.12 mg / mL were prepared to generate a standard curve. The polysaccharides obtained before and after treatment with the non-polar macroporous adsorption resin in Examples 1-6 were accurately weighed and prepared into a 0.1 mg / mL solution. 2 mL of the sample solution was accurately pipetted into a test tube, followed by the addition of 2 mL of 6% phenol and 6 mL of concentrated H₂SO₄. The mixture was mixed and allowed to stand for 10 minutes, then placed in a 100°C water bath for 20 minutes. After cooling to room temperature, the absorbance was measured at 490 nm. The polysaccharide content was calculated using the standard curve. According to Equation 3, the polysaccharide retention rates after treatment in Examples 1-6 were 70.94%, 74.75%, 71.94%, 78.94%, 76.53%, and 75.83%, respectively.

[0094] Test Example 5: Evaluation of the anti-ulcerative colitis activity of seabuckthorn peel polysaccharide in vivo

[0095] Experimental animals: Kunming mice, male, 6-8 weeks old.

[0096] Experimental groups: Mice were randomly divided into 7 groups: a normal group (Con), a model group (DSS), a sea buckthorn peel polysaccharide treatment group obtained in Example 1 (50 mg / kg / d, 100 mg / kg / d), a sea buckthorn seed meal polysaccharide treatment group obtained in Comparative Example 1 (100 mg / kg / d), a sea buckthorn fruit polysaccharide treatment group obtained in Comparative Example 2 (100 mg / kg / d), and a 100 mg / kg / d sulfasalazine (SAZ) positive control group.

[0097] Experimental methods: UC mice were induced with DSS. After one week of adaptive feeding, 56 Kunming mice aged 6-8 weeks were randomly divided into a normal group of 8 and a modeling group of 48. Starting from the first day, the normal group was given normal drinking water and continued until the 14th day; the modeling group was given 2.5% DSS to induce the UC model until the 7th day. After the modeling group was successfully established, they were randomly divided into a model group, a sea buckthorn peel polysaccharide treatment group obtained in Example 1 (50 mg / kg / d, 100 mg / kg / d), a sea buckthorn seed meal polysaccharide treatment group obtained in Comparative Example 1 (100 mg / kg / d), a sea buckthorn fruit polysaccharide treatment group obtained in Comparative Example 2 (100 mg / kg / d), and a 100 mg / kg / d sulfasalazine positive control group, with 8 mice in each group. Starting from the 8th day, the normal group and the model group were gavaged with 0.2 mL of normal saline every day, which lasted until the 14th day; the 50 mg / kg / d and 100 mg / kg / d sea buckthorn peel polysaccharide treatment groups were gavaged with 0.2 mL of sea buckthorn peel polysaccharide solution every day according to the body weight of the mice, and the sea buckthorn seed meal polysaccharide treatment group obtained in Comparative Example 1 (100 mg / kg / d) and the sea buckthorn fruit polysaccharide treatment group obtained in Comparative Example 2 (100 mg / kg / d) were operated in the same way, which lasted until the 14th day; the positive control group was gavaged with 0.2 mL of SAZ once a day according to the body weight of the mice, which lasted until the 14th day.

[0098] After administration on day 14, mice were fasted for 18 hours but not water. On day 15, blood was collected from the eye sockets. The blood was allowed to stand at room temperature for 30 minutes, centrifuged at 4000 rpm at 4°C for 10 minutes, and serum was collected and stored at -80°C. Mice were sacrificed by cervical dislocation, dissected, and colon tissue was cut, measured, and photographed. The terminal 0.5-0.8 cm of the colon was circularly excised, fixed with 4% paraformaldehyde for 24-48 hours, dehydrated, embedded in paraffin, and sectioned for subsequent immunohistochemistry (IHC) analysis of colon tissue lesions and intestinal barrier function. The upper colon of the mouse was cut open, the contents removed, and rinsed with saline. After absorbing the liquid, the intestinal mucosal tissue was scraped with a glass slide and stored at -80°C for subsequent related experiments.

[0099] Observation of various physical signs of mice and disease activity index (DAI) scoring: During the experiment, the mice in each group were weighed and recorded every other day, the fecal characteristics were recorded, and the occult blood was detected using a fecal test kit. This continued until the 14th day. The DAI score of the mice was calculated according to Table 1.

[0100] Table 1 DAI points principle

[0101] IHC was used to detect the expression of intestinal barrier function proteins in UC mice. After wax was completely melted, sections were immersed in xylene for 20 minutes. Dewaxing was then performed in anhydrous ethanol, 95% ethanol, 85% ethanol, 75% ethanol, and distilled water. Antigen retrieval was performed using citric acid antigen retrieval buffer (pH 6.0) according to standard procedures. 0.3% Triton was added to the tissue for permeabilization, and the slides were washed with PBS. Sections were placed in a humidified chamber and inactivated with a drop of PBS. Endogenous catalase was inactivated and the sections were blocked with goat serum. ZO-1 or OCLN antibody dilution was added and incubated at room temperature for 2 hours. After washing with PBS, secondary antibody dilution was added and incubated at room temperature for 1 hour. After washing with PBS, DAB solution was added to the sections for color development. Cell nuclei were counterstained with hematoxylin. After washing, sections were dehydrated and transparentized in 75% ethanol, 85% ethanol, 95% ethanol, anhydrous ethanol, and xylene, and mounted with neutral resin. Three mice were randomly selected from each group, and three fields of view (n=9) were selected for observation and photography under a 200x microscope. The images were analyzed using Image-Pro Plus software.

[0102] ELISA assay for inflammatory factors in mouse serum and colon tissue: The levels of TNF-α and IL-10 in the serum and colon tissue of each group of mice were determined according to the procedures of the mouse ELISA assay kit. 20 mg of colonic mucosa was weighed and added with normal saline at a ratio of 1:3. The mixture was homogenized until no lumps remained. The mixture was centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatant was then used to assay the levels of TNF-α and IL-10 in the colon tissue of the mice using a mouse ELISA assay kit. Serum was removed from a -80°C freezer, thawed at room temperature, and then the levels of TNF-α and IL-10 in the serum were determined according to the procedures of the mouse ELISA assay kit.

[0103] Statistical analysis: Data were expressed as mean ± standard error (mean ± SE), and the data were subjected to homogeneity of variance test and analysis of variance (One-way ANOVA) or t-test (Student's t-test).

[0104] Experimental results:

[0105] Effects of seabuckthorn peel polysaccharides on body weight and DAI in UC mice:

[0106] During the experiment, the weight of mice in the normal group continued to increase. In contrast, from day 1 of DSS-induced modeling to day 7, the model mice lost approximately 10%-20% of their weight; from day 1 of modeling to day 14, the model group lost approximately 30% of their weight. In the treatment group, the weight of mice with DSS-induced UC began to increase steadily from day 8 to day 14. Compared with the model group, the weight of mice in the 50mg / kg / day and 100mg / kg / day seabuckthorn peel polysaccharide treatment groups and the positive control group increased significantly (see Table 2). This suggests that seabuckthorn peel polysaccharide can inhibit weight loss in UC mice.

[0107] From day 1 of modeling, the DAI of all DSS-induced mice groups continued to rise. Compared to the DSS group, the DAI of the treated groups steadily decreased after administration of seabuckthorn peel polysaccharide, seabuckthorn seed meal polysaccharide, seabuckthorn fruit polysaccharide, or SAZ on day 8. Continued administration continued until day 14, when the DAI scores of all groups were significantly lower compared to the DSS group. Compared to the groups treated with the same dose of seabuckthorn seed meal polysaccharide or seabuckthorn fruit polysaccharide, the 100 mg / kg / d seabuckthorn peel polysaccharide treatment group showed superior effects (see Table 2). These results indicate that administration of seabuckthorn peel polysaccharide significantly alleviated the severity of DSS-induced UC in mice.

[0108] Table 2 Effects of seabuckthorn peel polysaccharides on body weight and DAI scores of UC mice (n=8)

[0109] Note: Compared with the model group, * indicates significant difference, *P<0.05, **P<0.01.

[0110] Effects of seabuckthorn peel polysaccharides on colon length in UC mice:

[0111] Compared with the normal group, the model group showed a 28.79% shortened colon length. Colon shortening was significantly improved in the Hippophae rhamnoides peel polysaccharide-treated group or the positive control group, with superior effects compared to the Hippophae rhamnoides seed meal polysaccharide-treated and Hippophae rhamnoides fruit polysaccharide-treated groups. Compared with the model group, treatment with 100 mg / kg / d Hippophae rhamnoides peel polysaccharide and 100 mg / kg / d SAZ increased colon length in UC mice by 24.68% and 12.02%, respectively. The therapeutic effect of 100 mg / kg / d Hippophae rhamnoides peel polysaccharide was more pronounced than that of SAZ (see Table 3). These results indicate that Hippophae rhamnoides peel polysaccharide treatment reduced colon damage in UC mice, demonstrating its ability to inhibit DSS-induced colon damage.

[0112] Table 3 Effects of seabuckthorn peel polysaccharides on colon length in UC mice (n=8)

[0113] Note: Compared with the model group, * indicates significant difference, *P<0.05, **P<0.01.

[0114] Effects of seabuckthorn peel polysaccharides on intestinal barrier function in UC mice:

[0115] The relative expression levels of ZO-1 and OCLN proteins were counted. Compared with the normal group, the tight junction structures ZO-1 and OCLN proteins between the colonic mucosal epithelial cells of the model group mice were reduced or even absent, and the integrity of the tight junction structure was destroyed, resulting in severe damage to the colon barrier function. However, compared with the model group, the expression of ZO-1 and OCLN proteins was significantly increased after treatment with seabuckthorn peel polysaccharide, and the therapeutic effect was better than that of the seabuckthorn seed meal polysaccharide treatment group and the seabuckthorn fruit polysaccharide treatment group. The protective effect of 100 mg / kg / d seabuckthorn peel polysaccharide on the intestinal barrier function of the colon tissue of UC mice was equivalent to that of the positive control group. Seabuckthorn peel polysaccharide treatment reduced the intestinal permeability and intestinal function impairment of UC mice, suggesting that seabuckthorn peel polysaccharide has a significant therapeutic effect on DSS-induced UC, as shown in Table 4.

[0116] Table 4 Statistics of relative expression of ZO-1 and OCLN in mouse colon tissue (n=9)

[0117] Note: Compared with the model group, * indicates significant difference, *P<0.05, **P<0.01.

[0118] Effects of seabuckthorn peel polysaccharide on TNF-α and IL-10 in serum and colon tissue of UC mice:

[0119] Compared with the normal group, the level of TNF-α in the colon tissue of the model group mice was significantly increased, indicating that the inflammatory response in the colon tissue of UC mice was severe. However, the TNF-α level in the colon tissue of the mice in the sea buckthorn peel polysaccharide treatment group, sea buckthorn seed meal polysaccharide treatment group, and sea buckthorn fruit polysaccharide treatment group was significantly lower than that in the DSS group, and the effect of 100 mg / kg / d sea buckthorn peel polysaccharide treatment was better than that of the same dose sea buckthorn seed meal polysaccharide treatment group, sea buckthorn fruit polysaccharide treatment group, and SAZ treatment group. Similarly, compared with the normal group, DSS-induced TNF-α in the serum of UC mice was significantly increased; sea buckthorn peel polysaccharide treatment can effectively reduce the TNF-α level in the serum of UC mice, and the effect of 100 mg / kg / d sea buckthorn peel polysaccharide treatment group is comparable to that of SAZ, and the effect is better than that of the same dose sea buckthorn seed meal polysaccharide treatment group and sea buckthorn fruit polysaccharide treatment group.

[0120] In mouse serum samples, IL-10 levels were significantly reduced in the model group compared to the normal group. Compared to the model group, administration of 50 mg / kg / day and 100 mg / kg / day of sea buckthorn peel polysaccharide promoted IL-10 secretion, significantly increasing its concentration. Furthermore, the effect of 100 mg / kg / day of sea buckthorn peel polysaccharide on IL-10 secretion was superior to that of the sea buckthorn seed meal polysaccharide and sea buckthorn fruit polysaccharide treatment groups, and comparable to that of the positive drug SAZ. Similar to the results of serum samples, DSS-induced IL-10 levels in colonic tissue of model mice were significantly reduced, but administration of 100 mg / kg / day of sea buckthorn peel polysaccharide or SAZ significantly increased levels of this anti-inflammatory factor, with a greater effect than that of the sea buckthorn seed meal polysaccharide and sea buckthorn fruit polysaccharide treatment groups. These results suggest that sea buckthorn peel polysaccharide can exert its anti-inflammatory effects by promoting the secretion of anti-inflammatory factors, inhibiting the inflammatory response in DSS-induced UC mice, thereby achieving therapeutic effects. See Table 5 for details.

[0121] Table 5 Effects of seabuckthorn peel polysaccharide on TNF-α and IL-10 in serum and colon tissue of UC mice (n=5)

[0122] Note: Compared with the model group, * indicates significant difference, *P<0.05, **P<0.01.

[0123] Test Example 6: Evaluation of the activity of seabuckthorn peel polysaccharide in improving functional constipation and functional dyspepsia

[0124] Experimental animals: SD rats, male, 120-150 g.

[0125] Experiment 1: Effects of seabuckthorn peel polysaccharides on defecation function in rats

[0126] Experimental Methods: A constipation model was established by oral gavage with loperamide hydrochloride. Thirty male SD rats were selected and acclimated for one week. All rats were randomly divided into a normal group, a model group, three dose groups of seabuckthorn peel polysaccharide (50 mg / kg / day for the high dose group, 25 mg / kg / day for the medium dose group, and 12.5 mg / kg / day for the low dose group), and a positive control group receiving 0.6 g / kg / day of Ma Ren Wan. Six groups, each with five rats, were used for the intervention period. From days 1 to 7, rats in the three dose groups of seabuckthorn peel polysaccharide were gavaged with 1 mL of Ma Ren Wan (0.6 g / kg / day) according to their body weight. The normal and model groups were gavaged with an equal volume of normal saline. Following the intervention period, rats were fasted for 16 hours. For modeling, the normal group was gavaged with 1 mL of normal saline, while the model, positive control, and seabuckthorn peel polysaccharide-treated groups were gavaged with 1 mL of 3 mg / kg loperamide hydrochloride. 0.5 hours later, the normal group and the model group were gavaged with Chinese ink (10 mL / kg), while the positive control group and each dose group were gavaged with Chinese ink (10 mL / kg) containing the corresponding amount of the test substance. The time of first black stool, the number and weight of black stool particles within 6 hours were recorded.

[0127] Experiment 2: Effects of seabuckthorn peel polysaccharides on small intestinal motility in rats

[0128] Experimental Methods: A constipation model was established by oral gavage with loperamide hydrochloride. Thirty male Sprague-Dawley rats were selected and acclimated for one week. All rats were randomly divided into a normal group, a model group, three dose groups of seabuckthorn peel polysaccharide (50 mg / kg / day for the high dose group, 25 mg / kg / day for the medium dose group, and 12.5 mg / kg / day for the low dose group), and a positive control group receiving 0.6 g / kg / day of Ma Ren Wan. Six groups, each with five rats, were used for the intervention period from days 1 to 7. Each of the three dose groups of seabuckthorn peel polysaccharide was administered orally (1 mL) based on rat body weight. The positive control group was administered orally (1 mL) of Ma Ren Wan (0.6 g / kg / day) based on rat body weight. The normal and model groups were each administered orally with an equal volume of normal saline. Following the intervention period, rats were fasted for 16 hours but not water. Modeling was initiated by oral gavage with 1 mL of normal saline in the normal group and 1 mL of 3 mg / kg loperamide hydrochloride in the model, positive control, and seabuckthorn peel polysaccharide treatment groups. 0.5 hours later, the normal group and the model group were gavaged with Chinese ink (10 mL / kg), while the positive control group and each dose group were gavaged with Chinese ink (10 mL / kg) containing the corresponding amount of the test substance. After 25 minutes of precise timing, the animals were immediately killed by cervical dislocation. The abdominal cavity was opened and the mesentery was separated. The intestinal loop from the pylorus to the ileocecal region was cut and the small intestine was gently pulled into a straight line. The intestinal length was measured as the "total small intestine length" and the distance from the pylorus to the front of the ink propagation was measured as the "ink propagation length."

[0129] The formula for calculating the small intestinal propulsion rate is as follows: small intestinal propulsion rate = ink propulsion length (cm) / total small intestine length (cm) × 100%.

[0130] Experiment 3: Effects of seabuckthorn peel polysaccharides on the digestive function of rats

[0131] Experimental Methods: Twenty male Sprague-Dawley rats were selected and acclimated for one week. All rats were randomly divided into four groups, each containing five rats, and three doses of seabuckthorn peel polysaccharide (50 mg / kg / day, 25 mg / kg / day, and 12.5 mg / kg / day). The intervention period lasted from days 1 to 30. All animals in each group were fasted for 24 hours before the end of the experiment. Gastric fluid was collected using isoflurane anesthesia and pyloric ligation, and the volume per unit time was measured. A 100 mm long glass capillary tube was filled with fresh egg white and placed in hot steam for 2 minutes to allow the protein to coagulate within the tube. The tube was then stored at 4°C and cut into 20 mm long tubes before use. One mL of gastric fluid was placed in a 50 mL Erlenmeyer flask, 15 mL of 0.05 mol / L hydrochloric acid solution was added, shaken, and placed into two freshly prepared tubes. Plug the bottle and incubate in a 37°C incubator for 24 hours. Remove the protein tubes and use a vernier caliper to measure the length (mm) of the transparent portion at each end of each protein tube. Calculate the average of the values ​​at the four ends. Calculate the pepsin activity and pepsin excretion. Pepsin activity unit (μ / mL) = average length of the transparent portion of the four ends of the protein tube. 2 ×16 Pepsin output (μ / h) = Pepsin activity × gastric fluid volume per hour

[0132] Statistical analysis: Data were expressed as mean ± standard error (mean ± SE), and the data were subjected to homogeneity of variance test and analysis of variance (One-way ANOVA) or t-test (Student's t-test).

[0133] Experimental Results: Compared with the model group, the number of fecal particles, fecal weight, fecal water content, and small intestinal propulsion rate in the rats treated with seabuckthorn peel polysaccharide increased significantly over 6 hours, and the time to first defecation decreased significantly (see Tables 6 and 7). After seabuckthorn peel polysaccharide treatment, the rats' hourly gastric fluid output, pepsin activity, and pepsin excretion increased significantly (see Table 8). This suggests that seabuckthorn peel polysaccharide promotes digestion and improves functional constipation.

[0134] Table 6 Effects of seabuckthorn peel polysaccharide on defecation function in constipated rats (n=5)

[0135] Note: Compared with the model group, * indicates significant difference, *P<0.05, **P<0.01.

[0136] Table 7 Effects of seabuckthorn peel polysaccharide on small intestinal motility in constipated rats (n=5)

[0137] Note: Compared with the model group, * indicates significant difference, *P<0.05, **P<0.01.

[0138] Table 8 Effects of seabuckthorn peel polysaccharide on gastric juice volume and pepsin in rats (n=5)

[0139] Note: Compared with the normal group, * indicates significant difference, *P<0.05, **P<0.01.

[0140] Test Example 7: Evaluation of the activity of seabuckthorn peel polysaccharide in improving intestinal flora

[0141] Experimental animals: BALB / C mice, male, 18-22 g.

[0142] Experimental method: 40 mice that were adaptively fed were randomly divided into a normal group of 8 mice and a modeling group of 32 mice. On the 1st to 10th day, the modeling group was gavaged with 375 mg / kg / d of the antibiotic cefixime, and the normal group was gavaged with an equal volume of normal saline. After 10 days of modeling, the modeling group was successfully divided into a model group and a sea buckthorn peel polysaccharide treatment group (high-dose group 100 mg / kg, medium-dose group 50 mg / kg, low-dose group 25 mg / kg). On the 11th to 20th day, the sea buckthorn peel polysaccharide treatment group was treated, and the normal control group was gavaged with an equal volume of normal saline. During the experiment, mouse feces were collected. 24 hours after the last administration, the contents of the ileum and cecum were removed under sterile conditions and gradiently diluted to 10 -9 , were inoculated on selective culture medium to detect intestinal flora. After blood was collected from mice, they were anesthetized and killed, and tissues were obtained and stored at -80℃.

[0143] Statistical analysis: Data were expressed as mean ± standard error (mean ± SE), and the data were subjected to homogeneity of variance test and analysis of variance (One-way ANOVA) or t-test (Student's t-test).

[0144] Experimental results: The 50mg / kg / d and 100mg / kg / d sea buckthorn peel polysaccharide treatment groups can gradually restore the body weight of mice with intestinal flora imbalance, see Table 9; each sea buckthorn peel polysaccharide treatment group can restore the intestinal bacteria count of mice with intestinal flora imbalance to normal levels, which is significantly different from the model group, see Table 10.

[0145] Table 9 Effects of seabuckthorn peel polysaccharides on body weight of mice with intestinal flora imbalance model (n=8)

[0146] Note: Compared with the model group, * indicates significant difference, *P<0.05, **P<0.01.

[0147] Table 10 Effects of seabuckthorn peel polysaccharides on the number of normal flora in intestinal flora imbalance model mice (n=8)

[0148] Note: Compared with the model group, * indicates significant difference, *P<0.05, **P<0.01.

[0149] Although the specific embodiments of the present invention have been described in detail, they should not be construed as limiting the scope of protection of this patent. Within the scope described by the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A seabuckthorn peel polysaccharide, characterized in that: The monosaccharide composition includes the following monosaccharides: rhamnose, mannose, galactose, glucose, galacturonic acid and arabinose, and the molar ratio thereof is 1: 2.03-2.63: 2.65-3.17: 3.20-3.97: 4.01-7.33: 7.95-11.

67.

2. The seabuckthorn peel polysaccharide according to claim 1, characterized in that The mass percentage of neutral sugar in the seabuckthorn peel polysaccharide is 82.73% to 85.31%; the molecular weight distribution range of the seabuckthorn peel polysaccharide is 2.5 kDa to 2.5×10 2 kDa.

3. The seabuckthorn peel polysaccharide according to claim 1, characterized in that The mass percentage of uronic acid in the seabuckthorn peel polysaccharide is 16.90% to 23.78%.

4. The seabuckthorn peel polysaccharide according to claim 1, characterized in that The molar ratio of rhamnose, mannose, galactose, glucose, galacturonic acid and arabinose is 1:2.10-2.63:2.65-3.17:3.20-3.97:4.10-6.35:9.60-11.

30.

5. A method for preparing seabuckthorn peel polysaccharide, comprising the following steps: (1) defatting: adding dried seabuckthorn peel to an organic solvent for reflux, immersion or percolation extraction, discarding the extract, and obtaining defatted seabuckthorn peel residue; (2) Extraction: drying the defatted seabuckthorn peel residue, soaking it in water, then refluxing it for extraction, filtering it, and obtaining an extract; (3) Concentrating: concentrating the extract and cooling it to obtain a concentrated solution; (4) alcohol precipitation: adding anhydrous ethanol to the concentrated solution or adding the concentrated solution to a high concentration ethanol aqueous solution, allowing to stand, collecting the precipitate, and obtaining crude seabuckthorn peel polysaccharide; (5) Purification: Dissolve the crude seabuckthorn peel polysaccharide in water, add it to non-polar macroporous adsorption resin D101, D312, X-5 or HP-20, elute, concentrate and dry the eluate to obtain seabuckthorn peel polysaccharide.

6. The method for preparing seabuckthorn peel polysaccharide according to claim 5, comprising the following steps: (1) Defatting: drying the seabuckthorn peel, adding an organic solvent with a solid-liquid ratio of 1:5 to 10 kg / L to perform reflux extraction, the extraction time is 2 to 3 hours, the extraction is performed 1 to 3 times, filtering, discarding the extract, and obtaining the defatted seabuckthorn peel residue; (2) Extraction: drying the defatted seabuckthorn peel residue, adding water with a solid-liquid ratio of 1:6 to 10 kg / L to soak the residue for several hours, and then performing reflux extraction for 2 to 3 hours, performing extraction 2 to 3 times, filtering, and combining the extracts; (3) Concentrating: concentrating the extract to 1 / 50 to 1 / 4 of the original volume, cooling to room temperature, and obtaining a concentrated solution; (4) Alcohol precipitation: under stirring, adding anhydrous ethanol to the concentrated solution to make the volume concentration of ethanol 60-80%, or adding the concentrated solution to a high-concentration ethanol aqueous solution until the mass percentage of ethanol in the concentrated solution is 60-80%, standing at 4-25° C. for 6-24 hours, discarding the supernatant, collecting the precipitate, and obtaining the crude seabuckthorn peel polysaccharide; (5) Purification: Use non-polar macroporous adsorption resin D101, D312, X-5 or HP-20 to decolorize and deproteinize the crude seabuckthorn peel polysaccharide; dissolve the crude seabuckthorn peel polysaccharide in water, slowly add it to the non-polar macroporous adsorption resin, elute with water, collect the eluate, concentrate it to 1 / 80 to 1 / 2 of the original volume, and dry it to obtain seabuckthorn peel polysaccharide.

7. The method for preparing seabuckthorn peel polysaccharide according to claim 6, characterized in that: In step (1), the organic solvent is ether, petroleum ether, ethyl acetate, a methanol aqueous solution with a mass percentage of 75% to 100%, or an ethanol aqueous solution with a mass percentage of 75% to 100%; In step (2), water is added to the defatted seabuckthorn peel residue and the soaking time is 3 to 24 hours; In step (4), the mass percentage of the high concentration ethanol aqueous solution is 80% to 100%; In step (5), the drying method is spray drying, reduced pressure drying or freeze drying; In step (5), the non-polar macroporous adsorption resin is preferably D101 macroporous adsorption resin; In step (5), the mass concentration of the crude polysaccharide loading solution of seabuckthorn peel is 0.005-0.03 kg / L, the loading amount of polysaccharide / resin is 0.01-0.15 kg / kg, the loading flow rate is 1-5 column volumes BV / h, and the loading is allowed to stand for 12-24 hours after completion; In step (5), the elution method is to use water elution, the elution flow rate is 1 to 5 BV / h, and the elution volume is 1 to 10 BV.

8. Use of the seabuckthorn peel polysaccharide according to claim 1 or the seabuckthorn peel polysaccharide prepared by the preparation method according to claim 5 in the preparation of medicines or functional health products for treating intestinal functional diseases.

9. The use according to claim 8, characterized in that The intestinal functional diseases include one or more of ulcerative colitis, constipation, indigestion and intestinal flora disorder.

10. A composition comprising the seabuckthorn peel polysaccharide according to claim 1 or the seabuckthorn peel polysaccharide prepared by the preparation method according to claim 5, and pharmaceutically acceptable excipients.

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