CN113354748A - Dendrobium officinale leaf polysaccharide and preparation and application thereof - Google Patents
Dendrobium officinale leaf polysaccharide and preparation and application thereof Download PDFInfo
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- CN113354748A CN113354748A CN202110523365.2A CN202110523365A CN113354748A CN 113354748 A CN113354748 A CN 113354748A CN 202110523365 A CN202110523365 A CN 202110523365A CN 113354748 A CN113354748 A CN 113354748A
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- 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
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/898—Orchidaceae (Orchid family)
- A61K36/8984—Dendrobium
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses dendrobium officinale leaf polysaccharide and preparation and application thereof. The preparation method comprises the following steps in sequence: (1) cleaning Dendrobium officinale leaves, drying and crushing; (2) soaking the treated material in 60-95 vol% ethanol water solution for extraction to remove impurities, wherein the extraction times are 2-6 times, and the concentration of the ethanol water solution used in the next extraction is higher than that of the ethanol water solution used in the previous extraction, so as to obtain a soaked sample; (3) extracting the soaked sample with water to obtain a water extract; (4) carrying out enzymolysis by protease; (5) decoloring with active carbon; (6) concentrating the supernatant, adding ethanol water solution, standing overnight, and centrifuging to obtain precipitate; (7) dissolving the precipitate in distilled water, and freeze drying to obtain Dendrobium officinale leaf polysaccharide. The dendrobe leaf polysaccharide prepared by the invention has the effects of improving immunodeficiency and recovering normal health, and can be used for preparing food or health-care products with the effect of improving immunodeficiency.
Description
(I) technical field
The invention relates to the technical field of natural polymers, in particular to dendrobium officinale leaf polysaccharide, preparation thereof and application thereof in preparing foods or health-care products with the effect of improving immunodeficiency.
(II) technical background
Dendrobium officinale (Dendrobium officinale Kimura et Migo) is a perennial epiphytic herb of Orchidaceae, is sweet in taste and slightly cold in nature, has the effects of promoting the production of body fluid, nourishing stomach, nourishing yin, clearing heat, moistening lung, tonifying kidney and the like, and is a traditional and rare Chinese traditional medicinal material. In modern food and drug industries, dendrobium is applied to various diseases by virtue of good treatment efficacy. However, in the traditional cognition, only the stem of dendrobium has application value, and other parts such as flowers and leaves are usually directly discarded or simply treated, so that great resource waste is caused. Researches in recent years find that some active ingredients in the dendrobium leaves can have treatment effects on some diseases, so that the application value of the dendrobium leaves is effectively discovered, and the waste of production resources in the industry is reduced.
The polysaccharide is the main active ingredient in the dendrobium officinale, and has the characteristics of high content, wide efficacy and the like. Researches show that the dendrobe polysaccharide has a treatment effect on various diseases and has extremely low toxic and side effects. However, the current research mainly focuses on the efficacy of dendrobium stem polysaccharide, and the research on the activity and application of dendrobium leaf polysaccharide is very little. In addition, a large amount of harmful organic solvent is often added in the extraction process of the polysaccharide, so that the risk of the product is increased.
Research shows that polysaccharide cannot be directly absorbed and utilized by human body due to its specific macromolecular structure, and can produce relevant influence on the body only after being fermented by intestinal microorganisms. In the existing research, the possible physiological activity of the dendrobium officinale leaf polysaccharide is determined only through in vitro experiments or in vitro simulated fermentation experiments, and the function of the dendrobium officinale leaf polysaccharide in human body immune regulation cannot be exactly explained.
Patent CN 107286269A discloses two preparation methods and applications of Dendrobium officinale leaf polysaccharide with immunoregulation function. The invention utilizes petroleum ether to extract and remove lipid, a water extraction and alcohol precipitation method to obtain crude polysaccharide, and an ion exchange column is used for separating and obtaining two polysaccharide components, wherein the polysaccharide has the effect of regulating immune macrophages, but the actual effect cannot be confirmed only through in vitro verification, and the production process is complex, the yield is low, and the application cost is higher.
Patent CN 111825779A discloses a method for extracting dendrobium officinale polysaccharide. The dendrobium polysaccharide is obtained by soaking dendrobium powder in absolute ethyl alcohol, and performing water extraction and alcohol precipitation after ultrasonic treatment. The invention does not introduce harmful organic solvent in the extraction process, improves the product safety, but needs long-time ultrasound in the extraction process, has larger energy consumption, has insufficient impurity removal operation, and can not definitely remove a large amount of flavonoid pigment in the product.
Patent CN 107974475 a discloses a process for extracting polysaccharide from dendrobium officinale leaves by an enzyme degradation method. The invention obtains crude polysaccharide from dendrobium leaves in a mode of multiple enzyme degradation and obtains pure polysaccharide in a dialysis mode.
Patent CN 108464392A discloses an application of Dendrobium officinale polysaccharide in preparation of microecological preparation. The invention uses petroleum ether for degreasing, ethanol for removing monosaccharide, and ethanol precipitation is carried out after multiple times of water decoction to obtain polysaccharide.
Therefore, at present, the preparation method of the dendrobium officinale leaf polysaccharide and the active function of the dendrobium officinale leaf polysaccharide in organisms need to be further studied.
Disclosure of the invention
The invention aims to solve the first technical problem of providing a preparation method of dendrobium officinale leaf polysaccharide with an effect of improving immunodeficiency.
The second technical problem to be solved by the invention is to provide dendrobium officinale leaf polysaccharide with an effect of improving immunodeficiency.
The third technical problem to be solved by the invention is to provide the application of the dendrobium officinale leaf polysaccharide in preparing foods or health-care products with the effect of improving immunodeficiency.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect, the invention provides a preparation method of dendrobium officinale leaf polysaccharide, which is carried out according to the following steps:
(1) cleaning Dendrobium officinale leaves, drying and crushing;
(2) soaking the material treated in the step (1) in an ethanol water solution with the volume concentration of 60-95% for extraction to remove impurities, wherein the extraction times are 2-6 times, and the concentration of the ethanol water solution used in the next extraction is higher than that of the ethanol water solution used in the previous extraction, so as to obtain a soaked sample;
(3) removing ethanol in the soaked sample obtained in the step (2) completely, and adding water for extraction to obtain a water extract;
(4) adding protease into the water extract obtained in the step (3) for enzymolysis reaction, inactivating, and centrifuging to obtain supernatant;
(5) adding activated carbon into the supernatant obtained in the step (4) for decoloring, and centrifuging to obtain a supernatant;
(6) concentrating the supernatant obtained in the step (5), adding 80-95 vol% ethanol water, standing overnight, and centrifuging to obtain precipitate;
(7) and (4) dissolving the precipitate obtained in the step (6) in distilled water, and freeze-drying to obtain the dendrobium officinale leaf polysaccharide.
Preferably, in the step (1), the drying temperature is 50-90 ℃; the particle size of the crushed particles is 20-60 meshes.
Preferably, in step (2), the concentration of the aqueous ethanol solution used in the subsequent extraction is at least 5%, more preferably at least 10% higher than the concentration of the aqueous ethanol solution used in the previous extraction.
Preferably, in step (3), the ratio of the mass of the added water to the mass of the soaked sample after the ethanol is removed is 1:10-1:60, the extraction temperature is 60-95 ℃, the extraction time is 1-6h, the extraction times are 2-5 times, and finally the water extract is combined.
Preferably, in the step (4), the protease is papain, the enzymolysis temperature is 30-50 ℃, the enzymolysis time is 2-5h, and the inactivation mode is to keep the temperature at 80-100 ℃ for 15-60 min.
Preferably, in the step (5), activated carbon is added to make the mass concentration of the activated carbon be 0.1-0.2%.
In a second aspect, the invention provides the dendrobium officinale leaf polysaccharide prepared according to the preparation method.
In a third aspect, the invention provides application of the dendrobium officinale leaf polysaccharide in preparation of foods or health-care products with the efficacy of improving immunodeficiency.
The dendrobium officinale leaf polysaccharide prepared by the invention can be used as a raw material to be applied to the fields of health-care food and the like.
Compared with the prior art, the invention has the following beneficial effects:
(1) compared with the existing preparation method of the dendrobium officinale leaf polysaccharide, the preparation method of the dendrobium officinale leaf polysaccharide adopted by the invention is simple in process, only ethanol precipitated polysaccharide and activated carbon are used for decoloring, and the most active flavonoid active substances are retained to the greatest extent while pigment removal is ensured; only protease is used for degrading protein impurities in the polysaccharide, and a large amount of separation and purification steps are not needed. In addition, in the preparation process, toxic organic reagents are not used, few foreign substances are introduced, and the foreign substances are all components allowed to be added in food, so that the safety of the product is greatly improved.
Specifically, according to the preparation method of the dendrobium leaf polysaccharide, the dendrobium leaf powder is soaked in the ethanol water solution with a certain concentration gradient, compared with the method of soaking in the opposite ethanol concentration gradient or without the gradient, the gradient ethanol-precipitated polysaccharide disclosed by the invention can improve the immune improvement effect of the polysaccharide, which is probably caused by different precipitation modes to enable the content and the variety of the remaining flavone in the polysaccharide to be different, in other words, the precipitation mode disclosed by the invention can furthest reserve the flavonoid active substance with the most activity. The method uses the activated carbon to adsorb a great amount of pigment substances such as chlorophyll and the like in the dendrobium leaves, so that the pigment in the leaves can be removed to a great extent, and the removal rate is higher than 99%.
(2) The dendrobium leaf polysaccharide contains part of flavonoid substances with most physiological activity, and the content of pigments is very low, so that the quality of products is not influenced.
(3) The dendrobium leaf polysaccharide prepared by the invention has the effects of improving immunodeficiency and recovering normal health.
Specifically, the dendrobium leaf polysaccharide has an active function similar to dendrobium stem polysaccharide, can achieve the effect of improving the immunity of the organism by improving the structure of intestinal flora, and can be substituted to a certain extent. Compared with the pure dendrobium leaf polysaccharide completely removing flavonoids, the dendrobium leaf polysaccharide prepared by the invention contains part of flavonoids with the most physiological activity, and the flavonoid synergistically helps the polysaccharide to improve the effect of improving immunodeficiency.
The invention simulates the medication condition of patients receiving chemotherapy for a long time to establish an animal model, and the prepared dendrobe leaf polysaccharide can improve intestinal flora through simulation experiment verification so as to achieve the effect of improving immunity, thereby providing theoretical basis for the dendrobe leaf polysaccharide serving as a health care product or a food raw material.
(IV) description of the drawings
The drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. In the drawings:
FIG. 1 is a technical route of traditional dendrobe polysaccharide;
FIG. 2 is a technical route of traditional dendrobe leaf polysaccharide;
FIG. 3 is a technical scheme of the invention.
(V) detailed description of the preferred embodiments
The following examples are set forth in order to provide a thorough understanding of the invention and to provide a further understanding of the invention. However, the present invention is not limited by the following examples.
The various sources of raw materials used in the examples:
5-week-old Balb/c male mice: shanghai Si Laike laboratory animals, Inc.;
dendrobium officinale leaf: collected from Wenzhou Zhejiang;
anhydrous ethanol: 99.5%, MW 46.07, shanghai mclin biochemistry science and technology limited;
activated carbon: 200 mesh, Shanghai Michelin Biochemical technology, Inc.;
papain: 800 mu/mg, Shanghai-derived leaf Biotech Co., Ltd;
cyclophosphamide: 97%, MW 279.1, shanghai ye biotechnology limited;
levamisole hydrochloride: 99%, MW 240.75, shanghai-sourced leaf biotechnology limited;
example 1:
(1) cleaning 100g of dendrobium officinale leaves, drying at 65 ℃, crushing, and then sieving with a 20-mesh sieve;
(2) soaking the above materials in 70% ethanol solution for 8h, 80% ethanol for 8h, and 90% ethanol for 8h, and removing impurities;
(3) drying the soaked sample with ethanol, removing ethanol, adding 30 times volume of water, extracting at 80 deg.C for 3 hr, repeating for 3 times, and mixing extractive solutions;
(4) adding protease into the above extractive solution, performing enzymolysis at 35 deg.C for 3 hr, heating the solution to 95 deg.C, inactivating for 20min, and centrifuging to obtain supernatant;
(5) adding 0.1% of active carbon into the supernatant for decolorization, and centrifuging to obtain supernatant;
(6) concentrating the supernatant, adding 80% ethanol, standing for 12 hr, and centrifuging to obtain precipitate;
(7) dissolving the precipitate in distilled water, and freeze drying to obtain herba Dendrobii leaf polysaccharide.
Example 2:
(1) cleaning 100g of dendrobium officinale leaves, drying at 85 ℃, crushing, and sieving with a 30-mesh sieve;
(2) soaking the materials in 85% ethanol solution for 12h, and soaking in 95% ethanol solution for 12h, and removing impurities;
(3) drying the soaked sample with ethanol, removing ethanol, adding 10 times of water, extracting at 95 deg.C for 2 hr, repeating for 5 times, and mixing extractive solutions;
(4) adding protease into the above extractive solution, performing enzymolysis at 37 deg.C for 1h, heating the solution to 100 deg.C, inactivating for 30min, and centrifuging to obtain supernatant;
(5) adding 0.2% of active carbon into the supernatant for decolorization, and centrifuging to obtain supernatant;
(6) concentrating the supernatant, adding 95% ethanol, standing for 12h, and centrifuging to obtain precipitate;
(7) dissolving the precipitate in distilled water, and freeze drying to obtain herba Dendrobii leaf polysaccharide.
Comparative example 1 Dendrobii caulis leaf polysaccharide alcohol extraction step without gradient
(1) Cleaning 100g of dendrobium officinale leaves, drying at 85 ℃, crushing, and sieving with a 30-mesh sieve;
(2) soaking the materials in 85% ethanol solution for 12h, and soaking in 85% ethanol solution for 12h, and removing impurities;
(3) drying the soaked sample with ethanol, removing ethanol, adding 10 times of water, extracting at 95 deg.C for 2 hr, repeating for 5 times, and mixing extractive solutions;
(4) adding protease into the above extractive solution, performing enzymolysis at 37 deg.C for 1h, heating the solution to 100 deg.C, inactivating for 30min, and centrifuging to obtain supernatant;
(5) adding 0.2% of active carbon into the supernatant for decolorization, and centrifuging to obtain supernatant;
(6) concentrating the supernatant, adding 95% ethanol, standing for 12h, and centrifuging to obtain precipitate;
(7) dissolving the precipitate in distilled water, and freeze drying to obtain herba Dendrobii leaf polysaccharide.
Comparative example 2, the concentration of the dendrobium leaf polysaccharide in the alcohol extraction step is from high to low
(1) Cleaning 100g of dendrobium officinale leaves, drying at 85 ℃, crushing, and sieving with a 30-mesh sieve;
(2) soaking the materials in 95% ethanol solution for 12h, and soaking in 85% ethanol solution for 12h, and removing impurities;
(3) drying the soaked sample with ethanol, removing ethanol, adding 10 times of water, extracting at 95 deg.C for 2 hr, repeating for 5 times, and mixing extractive solutions;
(4) adding protease into the above extractive solution, performing enzymolysis at 37 deg.C for 1h, heating the solution to 100 deg.C, inactivating for 30min, and centrifuging to obtain supernatant;
(5) adding 0.2% of active carbon into the supernatant for decolorization, and centrifuging to obtain supernatant;
(6) concentrating the supernatant, adding 95% ethanol, standing for 12h, and centrifuging to obtain precipitate;
(7) dissolving the precipitate in distilled water, and freeze drying to obtain herba Dendrobii leaf polysaccharide.
Example 3:
(1) cleaning 100g of dendrobium officinale leaves, drying at 50 ℃, crushing, and sieving with a 60-mesh sieve;
(2) soaking the above materials in 60% ethanol solution for 6h, 70% ethanol for 6h, 80% ethanol for 6h, and 90% ethanol for 6h, and removing impurities;
(3) drying the soaked sample with ethanol, removing ethanol, adding 25 times of water, extracting at 75 deg.C for 2 hr, repeating for 3 times, and mixing extractive solutions;
(4) adding protease into the above extractive solution, performing enzymolysis at 40 deg.C for 2 hr, heating the solution to 90 deg.C, inactivating for 15min, and centrifuging to obtain supernatant;
(5) adding 0.15% of active carbon into the supernatant for decolorization, and centrifuging to obtain supernatant;
(6) concentrating the supernatant, adding 85% ethanol, standing for 12 hr, and centrifuging to obtain precipitate;
(7) dissolving the precipitate in distilled water, and freeze drying to obtain herba Dendrobii leaf polysaccharide.
Comparative example 3 extraction of pure polysaccharide from Dendrobium nobile Lindl leaf
(1) Cleaning 100g of dendrobium officinale leaves, drying at 50 ℃, crushing, and sieving with a 60-mesh sieve;
(2) soaking the above materials in 60% ethanol solution for 6h, 70% ethanol for 6h, 80% ethanol for 6h, and 90% ethanol for 6h, and removing impurities;
(3) drying the soaked sample with ethanol, removing ethanol, adding 25 times of water, extracting at 75 deg.C for 2 hr, repeating for 3 times, and mixing extractive solutions;
(4) adding protease into the above extractive solution, performing enzymolysis at 40 deg.C for 2 hr, heating the solution to 90 deg.C, inactivating for 15min, and centrifuging to obtain supernatant;
(5) adding 0.15% of active carbon into the supernatant for decolorization, and centrifuging to obtain supernatant;
(6) concentrating the supernatant, adding 85% ethanol, standing for 12 hr, and centrifuging to obtain precipitate;
(7) dissolving the precipitate in distilled water, and freeze-drying to obtain crude polysaccharide of herba Dendrobii leaf;
(8) redissolving the crude polysaccharide, preparing a solution of 100mg/ml, dialyzing the obtained solution in a dialysis bag, wherein the dialysis medium is deionized water, and the relative molecular weight cutoff is 3500D;
(9) adding 3 times volume of absolute ethyl alcohol into the dialyzed solution, and standing for 24 hours for precipitation;
(10) and centrifuging and collecting the precipitate, and drying in vacuum to obtain the pure dendrobium leaf polysaccharide.
Comparative example 4 organic extraction of Dendrobii leaf polysaccharide
(1) Cleaning 100g of dendrobium officinale leaves, drying at 50 ℃, crushing, and sieving with a 60-mesh sieve;
(2) soaking the above materials in 500ml petroleum ether solution for 2h, discarding petroleum ether, repeating the step for 3 times, and volatilizing petroleum ether;
(3) adding 25 times of water into the soaked sample, extracting at 75 ℃ for 2h, repeating for 3 times, and combining the extracting solutions;
(4) concentrating the above extractive solution under reduced pressure, adding sevage solution 3 times in volume, oscillating vigorously for 30min, centrifuging at 4000rpm for 20min, collecting supernatant, repeating for 3 times, and concentrating the extractive solution under reduced pressure;
(5) adding 85% ethanol into the concentrated solution, standing for 12h, and centrifuging to obtain precipitate;
(6) dissolving the precipitate in distilled water, and freeze drying to obtain herba Dendrobii leaf polysaccharide.
Comparative example 5: extraction of dendrobium stem polysaccharide
(1) Cleaning 100g of dendrobium officinale stems, drying at 50 ℃, crushing, and sieving with a 40-mesh sieve;
(2) soaking the above materials in 60% ethanol solution for 6h, 70% ethanol for 6h, 80% ethanol for 6h, and 90% ethanol for 6h, and removing impurities;
(3) drying the soaked sample with ethanol, removing ethanol, adding 25 times of water, extracting at 75 deg.C for 2 hr, repeating for 3 times, and mixing extractive solutions;
(4) adding protease into the above extractive solution, performing enzymolysis at 35 deg.C for 2 hr, heating the solution to 90 deg.C, inactivating for 15min, and centrifuging to obtain supernatant;
(5) adding 0.15% of active carbon into the supernatant for decolorization, and centrifuging to obtain supernatant;
(6) concentrating the supernatant, adding 85% ethanol, standing for 12 hr, and centrifuging to obtain precipitate;
(7) dissolving the precipitate in distilled water, and freeze drying to obtain stem polysaccharide of herba Dendrobii.
Example 4:
construction of an immunodeficiency mouse model and feeding of dendrobium leaf polysaccharide:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, and on days 1-3, i.p. for 3 consecutive days, cyclophosphamide in physiological saline was injected at a dose of 80mg/kg (body weight)/d and an injection volume of 0.3ml, and thereafter every 4 days.
(3) After the 4 th day, the mice were gavaged with the aqueous solution of dendrobii leaf polysaccharide of example 1 every day at a gavage dose of 200mg/kg (polysaccharide/body weight)/d and a gavage volume of 0.2ml, and the weight was weighed before gavage and the status of the mice was observed.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
(5) On day 28, mice were anesthetized and blood was collected and euthanized.
Example 5:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, and on days 1-3, i.p. for 3 consecutive days, cyclophosphamide in physiological saline was injected at a dose of 80mg/kg (body weight)/d and an injection volume of 0.3ml, and thereafter every 4 days.
(3) After the 4 th day, the mice were gavaged with the aqueous solution of dendrobii leaf polysaccharide of example 2 every day at a gavage dose of 200mg/kg (polysaccharide/body weight)/d and a gavage volume of 0.2ml, and the weight was weighed before gavage and the status of the mice was observed.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
(5) On day 28, mice were anesthetized and blood was collected and euthanized.
Comparative example 6:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, and on days 1-3, i.p. for 3 consecutive days, cyclophosphamide in physiological saline was injected at a dose of 80mg/kg (body weight)/d and an injection volume of 0.3ml, and thereafter every 4 days.
(3) After the 4 th day, the mice were gavaged daily with the aqueous solution of dendrobii leaf polysaccharide of example 1 at a gavage dose of 200mg/kg (polysaccharide/weight)/d and a gavage volume of 0.2ml, and the weight was weighed before gavage and the status of the mice was observed.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
(5) On day 28, mice were anesthetized and blood was collected and euthanized.
Comparative example 7:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, and on days 1-3, i.p. for 3 consecutive days, cyclophosphamide in physiological saline was injected at a dose of 80mg/kg (body weight)/d and an injection volume of 0.3ml, and thereafter every 4 days.
(3) After the 4 th day, the mice were gavaged daily with the aqueous solution of dendrobii leaf polysaccharide of example 2 at a gavage dose of 200mg/kg (polysaccharide/weight)/d and a gavage volume of 0.2ml, and the weight was weighed before gavage and the status of the mice was observed.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
(5) On day 28, mice were anesthetized and blood was collected and euthanized.
Example 6:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, and on days 1-3, i.p. for 3 consecutive days, cyclophosphamide in physiological saline was injected at a dose of 80mg/kg (body weight)/d and an injection volume of 0.3ml, and thereafter every 4 days.
(3) After the 4 th day, the mice were gavaged daily with the aqueous solution of dendrobii leaf polysaccharide of example 3 at a gavage dose of 200mg/kg (polysaccharide/body weight)/d and a gavage volume of 0.2ml, and the weight was weighed before gavage and the status of the mice was observed.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
(5) On day 28, mice were anesthetized and blood was collected and euthanized.
Comparative example 8:
construction of an immunodeficiency mouse model and feeding of dendrobium pure polysaccharide:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, and on days 1-3, i.p. for 3 consecutive days, cyclophosphamide in physiological saline was injected at a dose of 80mg/kg (body weight)/d and an injection volume of 0.3ml, and thereafter every 4 days.
(3) After the 4 th day, the mice were gavaged daily with the aqueous solution of the purified dendrobe polysaccharide of comparative example 3 at a gavage dose of 200mg/kg (polysaccharide/body weight)/d and a gavage volume of 0.2ml, and the body weights were weighed before gavage and the status of the mice was observed.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
(5) On day 28, mice were anesthetized and blood was collected and euthanized.
Comparative example 9:
construction of an immunodeficiency mouse model and feeding of dendrobium organic extracted polysaccharide:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, and on days 1-3, i.p. for 3 consecutive days, cyclophosphamide in physiological saline was injected at a dose of 80mg/kg (body weight)/d and an injection volume of 0.3ml, and thereafter every 4 days.
(3) After the 4 th day, the mice were gavaged daily with the aqueous solution of the organic polysaccharide extracted from dendrobe in comparative example 4, the gavage dose was 200mg/kg (polysaccharide/weight)/d, the gavage volume was 0.2ml, and the weight was weighed before gavage and the status of the mice was observed.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
(5) On day 28, mice were anesthetized and blood was collected and euthanized.
Comparative example 10:
construction of an immunodeficiency mouse model and feeding of dendrobium stem polysaccharide:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, and on days 1-3, i.p. for 3 consecutive days, cyclophosphamide in physiological saline was injected at a dose of 80mg/kg (body weight)/d and an injection volume of 0.3ml, and thereafter every 4 days.
(3) After the 4 th day, the mice were gavaged daily with the aqueous solution of dendrobii caulis stem polysaccharide of comparative example 5 at a gavage dose of 200mg/kg (polysaccharide/body weight)/d and a gavage volume of 0.2ml, and the weight was weighed before gavage and the status of the mice was observed.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
On day 28, mice were anesthetized and blood was collected and euthanized.
Comparative example 11:
construction of an immunodeficiency mouse model and feeding of levamisole hydrochloride:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, and on days 1-3, i.p. for 3 consecutive days, cyclophosphamide in physiological saline was injected at a dose of 80mg/kg (body weight)/d and an injection volume of 0.3ml, and thereafter every 4 days.
(3) After day 4, mice were gavaged with levorotatory mizuo hydrochloride aqueous solution every day at a gavage dose of 40mg/kg (drug/weight)/d and a gavage volume of 0.2ml, and the weight was weighed before gavage and the status of the mice was observed.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
(5) On day 28, mice were anesthetized and blood was collected and euthanized.
Comparative example 12:
construction and water feeding of an immunodeficiency mouse model:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, and on days 1-3, i.p. for 3 consecutive days, cyclophosphamide in physiological saline was injected at a dose of 80mg/kg (body weight)/d and an injection volume of 0.3ml, and thereafter every 4 days.
(3) After day 4, mice were gavaged daily with deionized water at a gavage volume of 0.2ml, and weights were weighed and the status of the mice observed prior to gavage.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
(5) On day 28, mice were anesthetized and blood was collected and euthanized.
Comparative example 13:
normal mouse model construction and water feeding:
(1) grouping 5-week-old Balb/c male mice into groups, wherein each group comprises 10 mice, the room temperature is 20-25 ℃, the air humidity is 50-70%, the mice are fed with water and food freely for 1 week, and the illumination is 12 h/d;
(2) mice were weighed and labeled, day 1-3, and were injected intraperitoneally with a physiological saline solution at an injection volume of 0.3ml for 3 consecutive days, and thereafter every 4 days.
(3) After day 4, mice were gavaged daily with distilled water in a gavage volume of 0.2ml, and the weight was weighed before gavage and the status of the mice was observed.
(4) On day 27, mouse feces were collected for intestinal flora analysis.
(5) On day 28, mice were anesthetized and blood was collected and euthanized.
Example 7:
mouse serum immunoglobulin IgA index determination
(1) Taking the blood of the mice in the examples and the comparative examples, and naturally coagulating the blood in a 1.5ml centrifuge tube for 30min at room temperature;
(2) centrifuging the blood at 3000rpm for 20min, and collecting supernatant as serum;
(3) the sera were analyzed using a mouse immunoglobulin enzyme linked immunoassay kit (Elisa).
The results of the experiments are shown in the following table:
TABLE 1 serum immunoglobulin A index of differently treated mice
| IgA concentration (μ g/ml) | |
| Comparative example 13 | 301.74 |
| Comparative example 12 | 207.85 |
| Example 4 | 283.55 |
| Example 5 | 280.98 |
| Comparative example 6 | 262.74 |
| Comparative example 7 | 257.66 |
| Example 6 | 282.31 |
| Comparative example 8 | 266.05 |
| Comparative example 9 | 253.21 |
| Comparative example 10 | 285.15 |
| Comparative example 11 | 289.50 |
Immunoglobulins are organic components of the immune system and play an important role in humoral immunity, reflecting to some extent whether the immune system of the body is powerful or not. The experimental result shows that the concentration of the immunoglobulin A in the serum of the mouse in the comparative example 12 is obviously reduced compared with that in the comparative example 13, and is only 68.89%, so that the immunodeficient mouse model constructed by the invention is proved to have corresponding representativeness. After the dendrobium leaf polysaccharide in the example 4 is fed, the immunoglobulin A index of a mouse is restored to 283.55 mug/ml, the immunoglobulin A index is improved by 36.42 percent compared with that of a model, the immunoglobulin A index is only reduced by 6.03 percent compared with that of a normal mouse, and compared with a mouse fed with levamisole hydrochloride in the comparative example 11, the immunoglobulin A index has no significant difference, and the dendrobium leaf polysaccharide prepared by the method has the effects of improving immunodeficiency and restoring normal health. Compared with the immunoglobulin A index of a mouse fed with the dendrobium stem polysaccharide, the immunoglobulin A index of the mouse fed with the dendrobium stem polysaccharide has no significant difference, and the dendrobium stem polysaccharide and the dendrobium leaf polysaccharide have similar treatment effects and can be substituted to a certain extent. Comparative example 8 the pure dendrobium nobile leaf polysaccharide improves the immunoglobulin A index to 266.05 mug/ml, which proves that the pure dendrobium nobile leaf polysaccharide also has the effect of improving immune diseases, but compared with example 4, the immune recovery effect is reduced by 23.12 percent, probably because the polysaccharide in example 4 contains part of flavonoid substances with physiological activity, the flavonoid substances synergistically help the polysaccharide to improve the treatment effect, and the polysaccharide prepared by the invention is proved to have higher application value. In comparative examples 6 and 7, the dendrobium leaf polysaccharide is extracted by adopting opposite ethanol concentration gradients or no gradient, although the dendrobium leaf polysaccharide also has the immune recovery effect, the recovery effect is reduced by 7.34% and 9.13% compared with the polysaccharide in example 4, which shows that the polysaccharide is precipitated by using the ethanol in a gradient manner, so that the immune improvement effect can be improved, and the polysaccharide is probably caused by different precipitation modes so as to ensure that the content and the variety of the remaining flavone in the polysaccharide are different.
Example 8:
mouse organ index calculation
(1) After the mice of the above examples and comparative examples were euthanized, organs such as thymus and spleen were dissected;
(2) rinsing the organ in PBS (pH 7.4), and sucking off water;
(3) weighing the above organs and calculating an organ index, wherein organ index is organ mass/body weight
The results of the experiments are shown in the following table:
TABLE 2 mouse organ index
| Index of thymus | Spleen index | |
| Comparative example 13 | 1.50*10-3 | 6.82*10-3 |
| Comparative example 12 | 2.34*10-4 | 4.17*10-3 |
| Example 4 | 1.42*10-3 | 6.66*10-3 |
| Example 5 | 1.39*10-3 | 6.56*10-3 |
| Comparative example 6 | 1.21*10-3 | 6.31*10-3 |
| Comparative example 7 | 1.15*10-3 | 6.24*10-3 |
| Example 6 | 1.41*10-3 | 6.58*10-3 |
| Comparative example 8 | 1.32*10-3 | 6.59*10-3 |
| Comparative example 9 | 1.17*10-3 | 6.14*10-3 |
| Comparative example 10 | 1.39*10-3 | 6.62*10-3 |
| Comparative example 11 | 1.48*10-3 | 6.97*10-3 |
The experimental results showed that similar to example 7, the mouse immune organ index in comparative example 12 was significantly decreased, and the thymus index and spleen index were only 2.34 × 10-4And 4.17 x 10-3Far below normal mice, demonstrating a serious defect in their immune system. In example 4 and comparative example 10, after the dendrobium leaf polysaccharide and the dendrobium stem polysaccharide are both fed for a long time, the atrophy of immune organs of mice is improved, the results are similar to those in comparative example 11, the mice are enabled to recover normal levels, and the polysaccharide provided by the invention is proved to have feasibility of recovering immune diseases.
Example 9:
determination of short chain fatty acids in mouse feces
(1) Taking 0.2g of the mouse feces of the examples and the comparative examples in a sterile centrifuge tube;
(2) adding 2ml of PBS solution into the centrifuge tube, wherein the pH value of the PBS is 7.4;
(3) fully oscillating and shaking the centrifugal tube uniformly, standing for 3min, and repeating the step for 3 times;
(4) centrifuging the liquid at 8000rpm for 3min, and collecting supernatant;
(5) the clear solution was passed through a 0.22 μm aqueous filter and then detected by gas chromatography.
The results of the experiments are shown in the following table:
TABLE 3 short chain fatty acid content in mouse feces
| Acetic acid (mu mol/g) | Propionic acid (mu mol/g) | Butyric acid (μmol/g) | Valeric acid (mu mol/g) | |
| Comparative example 13 | 74.32 | 6.78 | 30.25 | 3.22 |
| Comparative example 12 | 33.25 | 6.54 | 12.31 | 1.01 |
| Example 4 | 97.18 | 7.03 | 46.98 | 2.89 |
| Example 5 | 89.65 | 6.57 | 33.59 | 2.97 |
| Comparative example 6 | 76.64 | 5.99 | 31.42 | 2.08 |
| Comparative example 6 | 77.38 | 6.24 | 32.51 | 2.31 |
| Example 7 | 91.09 | 7.04 | 28.59 | 2.44 |
| Comparative example 7 | 90.10 | 6.35 | 40.79 | 2.68 |
| Comparative example 8 | 96.99 | 6.61 | 46.25 | 2.97 |
| Comparative example 9 | 71.19 | 6.03 | 30.33 | 2.22 |
| Comparative example 10 | 74.37 | 6.69 | 38.17 | 1.95 |
| Comparative example 11 | 79.32 | 8.21 | 27.22 | 3.03 |
There is increasing evidence that intestinal flora is involved in the development or cure of various diseases, and that metabolites of intestinal microorganisms play a crucial role in the regulation of certain physiological activities, wherein short chain fatty acids represent to some extent the state of the intestinal flora. The experimental results show that the content of total short-chain fatty acids in the comparative example 12 is reduced to 53.11 mu mol/g, which shows that the activity of the flora in the intestinal tract of the model animal is inhibited, the content of fermentation metabolites is lower, while the content of the short-chain fatty acids in the comparative example 11 is basically restored to the level in the comparative example 13, and the fact that the metabolic disturbance of the intestinal flora caused by immunodeficiency can be restored by the drug treatment is proved. In examples 4, 5 and 6 and comparative examples 6, 7, 8 and 10, the total content of short-chain fatty acids is greatly increased, wherein the content of examples 4, 5 and 6 and comparative examples 8, 9 and 10 is even higher than that of comparative example 13, which proves that the polysaccharide can be fermented and utilized by intestinal flora, promotes the amplification of beneficial bacteria therein, relieves the negative effects brought by immune diseases, and has the application value of improving immunity.
Example 10:
determination of flavone content in dendrobium polysaccharide
(1) 1g of each of the dendrobe leaf powder, the polysaccharide samples in example 2 and comparative examples 1,2,3 and 4 is taken and tightly wrapped by filter paper;
(2) placing the paper bag in a Soxhlet extractor, adding petroleum ether, and refluxing for 3h until the extractive solution is colorless;
(3) placing the precipitate in a centrifuge tube, adding 20ml of 75% ethanol, and performing ultrasonic extraction for 30 min;
(4) centrifuging the above extractive solution at 6000rpm for 5min, collecting supernatant 2mL, placing into 10mL volumetric flask, sequentially adding 5% sodium nitrite 0.6mL, standing for 5min, 10% aluminum nitrate 0.6mL, standing for 5min, and 1mol/L sodium hydroxide 6mL, standing for 5min, and adding 80% ethanol to desired volume;
(5) shaking and standing the solution for 15min, measuring absorbance value Ai at the wavelength of 510nm, and substituting into a standard curve for calculation to obtain flavone content. Calculated according to the following formula:
total flavone extraction rate (%) - (C × n × V0 × 10)-3)/M×100％
In the formula: c, concentration of total flavonoids in sample solution (mg/mL)
n: dilution factor
V0 volume constant (mL) of sample solution
M; sample Mass (g)
The experimental results are as follows:
| flavone content (mg/g) | |
| Dendrobium leaves | 5.340 |
| Example 2 | 1.049 |
| Comparative example 1 | 1.497 |
| Comparative example 2 | 0.769 |
| Comparative example 3 | 0.013 |
| Comparative example 4 | 0.887 |
Experimental results show that flavonoids in the dendrobium pure polysaccharide are almost completely removed, while flavonoids in the dendrobium leaf polysaccharide still contains 19.64% of the total amount, and the flavonoids can provide physiological activity for the polysaccharide in an auxiliary way. The content of flavone in the polysaccharide extracted by the traditional organic reagent method is 15.44 percent lower than that of the polysaccharide, but the polysaccharide has better immune recovery effect according to the analysis of the results in the examples 7 and 8, which is probably because the extraction method provided by the invention retains more active flavonoid substances. And different ethanol settlement concentration gradients are adopted, so that the flavone content in the obtained polysaccharide is different, if ethanol with gradient concentration is not adopted, the flavone content in the polysaccharide is higher than that in the embodiment, and if the ethanol with gradient concentration is adopted, more flavonoids are removed, but according to the result analysis in the embodiment 7 and the embodiment 8, the immune recovery effect of the polysaccharide in the comparative example 1 and the comparative example 2 is relatively weaker, which probably means that different settlement modes can keep different flavonoid molecular structures, and the mode adopted by the invention can keep the most active flavonoids.
Example 11:
determination of pigment in dendrobium polysaccharide
(1) Taking 1g of each of the dendrobium leaf powder, the polysaccharide samples in example 2 and comparative example 4, placing the dendrobium leaf powder and the polysaccharide samples in the comparative example 4 into a mortar, adding 5ml of 95% ethanol, and grinding the mixture into homogenate;
(2) adding 45ml of ethanol into the homogenate and continuously grinding;
(3) standing the homogenate in the dark for 24 hours;
(4) the homogenate was centrifuged, and the supernatant was examined for absorbance at 665nm and 645 nm.
Total pigment content ═ 20.0 a665+8.02A645)/20
The experimental results are as follows:
| pigment content (mg/g) | |
| Dendrobium leaves | 2.894 |
| Example 2 | 0.022 |
| Comparative example 4 | 0.018 |
Experimental results show that compared with the traditional organic reagent extraction method, the dendrobium leaf polysaccharide extraction method provided by the invention can also remove pigments in leaves to a great extent, the removal rate is higher than 99%, and the product quality is not influenced.
Claims (9)
1. A preparation method of dendrobium officinale leaf polysaccharide comprises the following steps:
(1) cleaning Dendrobium officinale leaves, drying and crushing;
(2) soaking the material treated in the step (1) in an ethanol water solution with the volume concentration of 60-95% for extraction to remove impurities, wherein the extraction times are 2-6 times, and the concentration of the ethanol water solution used in the next extraction is higher than that of the ethanol water solution used in the previous extraction, so as to obtain a soaked sample;
(3) removing ethanol in the soaked sample obtained in the step (2) completely, and adding water for extraction to obtain a water extract;
(4) adding protease into the water extract obtained in the step (3) for enzymolysis reaction, inactivating, and centrifuging to obtain supernatant;
(5) adding activated carbon into the supernatant obtained in the step (4) for decoloring, and centrifuging to obtain a supernatant;
(6) concentrating the supernatant obtained in the step (5), adding 80-95 vol% ethanol water, standing overnight, and centrifuging to obtain precipitate;
(7) and (4) dissolving the precipitate obtained in the step (6) in distilled water, and freeze-drying to obtain the dendrobium officinale leaf polysaccharide.
2. The method of claim 1, wherein: in the step (2), the concentration of the ethanol aqueous solution used in the next extraction is at least 5% higher than that of the ethanol aqueous solution used in the previous extraction.
3. The method of claim 2, wherein: in the step (2), the concentration of the ethanol aqueous solution used in the next extraction is at least 10% higher than that of the ethanol aqueous solution used in the previous extraction.
4. The method according to any one of claims 1 to 3, wherein: in the step (1), the drying temperature is 50-90 ℃; the particle size of the crushed particles is 20-60 meshes.
5. The method according to any one of claims 1 to 3, wherein: in the step (3), the ratio of the mass of the added water to the mass of the soaked sample after the ethanol is removed is 1:10-1:60, the extraction temperature is 60-95 ℃, the extraction time is 1-6h, the extraction times are 2-5 times, and finally the water extracting solutions are combined.
6. The method according to any one of claims 1 to 3, wherein: in the step (4), the protease is papain, the enzymolysis temperature is 30-50 ℃, the enzymolysis time is 2-5h, and the inactivation mode is to keep the temperature at 80-100 ℃ for 15-60 min.
7. The method according to any one of claims 1 to 3, wherein: in the step (5), active carbon is added to make the mass concentration of the active carbon be 0.1-0.2%.
8. The dendrobium officinale leaf polysaccharide prepared by the preparation method according to claim 1.
9. The use of the dendrobium officinale leaf polysaccharide of claim 8 in preparing food or health care products with the efficacy of improving immunodeficiency.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170274008A1 (en) * | 2016-03-24 | 2017-09-28 | Hong Kong Baptist University | Use of polysaccharide in Dendrobium officinale for anti-fatigue |
| CN107286269A (en) * | 2017-08-11 | 2017-10-24 | 上海海洋大学 | Two kinds of preparation method and applications with immunoregulatory dendrobium candidum leaf polyose |
| CN107474151A (en) * | 2017-08-29 | 2017-12-15 | 霍山县长冲中药材开发有限公司 | A kind of processing method for obtaining dendrobium polysaccharide from the stem of noble dendrobium with alcohol deposition method |
| CN107880149A (en) * | 2017-12-29 | 2018-04-06 | 贺州市星辉科技有限公司 | A kind of extraction process of Dendrobium officinale polysaccharide |
| CN108840956A (en) * | 2018-05-04 | 2018-11-20 | 山西大学 | A kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide |
| CN110092846A (en) * | 2019-05-30 | 2019-08-06 | 遵义医科大学 | A method of extracting Dendrobium officinale polysaccharide |
| AU2020101156A4 (en) * | 2020-06-26 | 2020-07-30 | Guangzhou Boteng Trading Ltd. | A Dendrobium Extract, A Method For Preparing It And Its Application |
-
2021
- 2021-05-13 CN CN202110523365.2A patent/CN113354748B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170274008A1 (en) * | 2016-03-24 | 2017-09-28 | Hong Kong Baptist University | Use of polysaccharide in Dendrobium officinale for anti-fatigue |
| CN107286269A (en) * | 2017-08-11 | 2017-10-24 | 上海海洋大学 | Two kinds of preparation method and applications with immunoregulatory dendrobium candidum leaf polyose |
| CN107474151A (en) * | 2017-08-29 | 2017-12-15 | 霍山县长冲中药材开发有限公司 | A kind of processing method for obtaining dendrobium polysaccharide from the stem of noble dendrobium with alcohol deposition method |
| CN107880149A (en) * | 2017-12-29 | 2018-04-06 | 贺州市星辉科技有限公司 | A kind of extraction process of Dendrobium officinale polysaccharide |
| CN108840956A (en) * | 2018-05-04 | 2018-11-20 | 山西大学 | A kind of method of Bi-aqueous extraction purifying Dendrobium officinale polysaccharide |
| CN110092846A (en) * | 2019-05-30 | 2019-08-06 | 遵义医科大学 | A method of extracting Dendrobium officinale polysaccharide |
| AU2020101156A4 (en) * | 2020-06-26 | 2020-07-30 | Guangzhou Boteng Trading Ltd. | A Dendrobium Extract, A Method For Preparing It And Its Application |
Non-Patent Citations (2)
| Title |
|---|
| YANG, KE等: "Physicochemical characterization of polysaccharide from the leaf of Dendrobium officinale and effect on LPS induced damage in GES-1 cell", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| ZHANG, MIN等: "Isolation of polysaccharides from Dendrobium officinale leaves and anti-inflammatory activity in LPS-stimulated THP-1 cells", 《CHEMISTRY CENTRAL JOURNAL》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114617796A (en) * | 2022-03-31 | 2022-06-14 | 华南农业大学 | Preparation method of anti-aging dendrobium nobile leaf polysaccharide mask |
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