CN117567657A - Paris polyphylla glucomannan and preparation method and application thereof - Google Patents
Paris polyphylla glucomannan and preparation method and application thereof Download PDFInfo
- Publication number
- CN117567657A CN117567657A CN202311459219.3A CN202311459219A CN117567657A CN 117567657 A CN117567657 A CN 117567657A CN 202311459219 A CN202311459219 A CN 202311459219A CN 117567657 A CN117567657 A CN 117567657A
- Authority
- CN
- China
- Prior art keywords
- paris polyphylla
- glucomannan
- paris
- polyphylla
- mannose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 241000244987 Daiswa polyphylla Species 0.000 title claims abstract description 230
- LUEWUZLMQUOBSB-FSKGGBMCSA-N (2s,3s,4s,5s,6r)-2-[(2r,3s,4r,5r,6s)-6-[(2r,3s,4r,5s,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5s,6r)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](OC3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-FSKGGBMCSA-N 0.000 title claims abstract description 188
- 229920002581 Glucomannan Polymers 0.000 title claims abstract description 186
- 229940046240 glucomannan Drugs 0.000 title claims abstract description 186
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- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 29
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- 150000002703 mannose derivatives Chemical group 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 32
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- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 abstract description 8
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Classifications
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- 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
- A61K31/716—Glucans
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
- A23L33/24—Cellulose or derivatives thereof
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- 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)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
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- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/004—Aftersun preparations
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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
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
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- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0087—Glucomannans or galactomannans; Tara or tara gum, i.e. D-mannose and D-galactose units, e.g. from Cesalpinia spinosa; Tamarind gum, i.e. D-galactose, D-glucose and D-xylose units, e.g. from Tamarindus indica; Gum Arabic, i.e. L-arabinose, L-rhamnose, D-galactose and D-glucuronic acid units, e.g. from Acacia Senegal or Acacia Seyal; Derivatives thereof
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- A—HUMAN NECESSITIES
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- 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 application relates to paris polyphylla glucomannan, a preparation method and application thereof, wherein the paris polyphylla glucomannan has a structure shown in the following general formula I, m and n in the formula I represent the number of repeated units and are natural numbers, m is independently 1 or 2, and n is more than or equal to 30 and less than or equal to 40. The paris polyphylla glucomannan can obviously promote macrophage RAW264.7 to secrete cytokines IL-10 and TNF-alpha, so that the paris polyphylla glucomannan has obvious organism immunity regulating capability, can obviously reduce the cell death rate of skin fibroblasts damaged by hydrogen peroxide, has obvious activity of preventing free radical cell damage, and has the purposes of preparing medicines and/or health-care foods for improving organism immunity and cosmetics for isolating ultraviolet rays and reducing skin damage.
Description
Technical Field
The application relates to the field of cosmetics and medical technology, in particular to paris polyphylla glucomannan and a preparation method and application thereof.
Background
The paris polyphylla is a common name of Paris polyphylla in Paris of Heidae, has long history of medical use, is generally used as a medicament, has the effects of clearing heat and detoxicating, detumescence and relieving pain, cooling liver and arresting convulsion and the like, and is mainly used for treating furuncle and carbuncle swelling, sore throat, snake and insect bite, traumatic injury pain and convulsion. Rhizoma paridis Yunnanensis (Paris polyphylla Smith var. Yunnanensis), also known as Paris polyphylla, is a perennial herb of Paris genus (Paris L.) of Paris of the family Melaninaceae, and is recorded in the pharmacopoeia of the people's republic of China (2020 edition).
The paris polyphylla mainly contains chemical components such as steroid saponins, flavonoids, triterpenes and the like, wherein the steroid saponins are important natural organic compounds with high types and contents of the compounds in the paris polyphylla, and have better biological activity. However, the paris polyphylla serving as a medicinal plant with national characteristics has high use frequency, the material basic research of the traditional efficacy of the paris polyphylla is still to be excavated, and particularly, the paris polyphylla polysaccharide needs to be intensively studied.
Disclosure of Invention
Aiming at the problems in the prior art, the embodiment of the application provides paris polyphylla glucomannan, and a preparation method and application thereof.
In a first aspect, embodiments of the present application provide a paris polyphylla glucomannan having a structure as shown in formula I below:
in the formula I, m and n represent the number of repeating units and are natural numbers, m is independently 1 or 2, and n is more than or equal to 30 and less than or equal to 40.
The novel-structure paris polyphylla glucomannan provided by the embodiment of the application takes beta (1-4) mannose as a main chain, and the polysaccharide chain contains glucose and acetylated mannose. Pharmacological researches show that the paris polyphylla glucomannan with novel structure can remarkably promote macrophage RAW264.7 to secrete cytokines IL-10 and TNF-alpha, and has obvious organism immunoregulation capability. Moreover, the inventor has further studied intensively to find that the paris polyphylla glucomannan can obviously reduce the cell death rate of skin fibroblasts damaged by hydrogen peroxide, has obvious activity of preventing free radical cell damage and can play a role in preventing skin tissues from being damaged by ultraviolet oxidation.
In some embodiments of the present application, the weight average molecular weight of the paris polyphylla dextran is 50kDa to 100kDa.
In some embodiments of the present application, the monosaccharide composition of the paris polyphylla glucomannan includes glucose and mannose, and the molar ratio of glucose to total monosaccharides is 6.5% -9%.
In some embodiments of the present application, the mannose comprises an acetylated mannose substituted with an acetyl group at the 2-or 3-position of the sugar ring, the acetylated mannose comprising 27.5% to 30% by mole of the total monosaccharides.
In some embodiments of the present application, the paris polyphylla glucomannan has a purity of not less than 95%.
In a second aspect, an embodiment of the present application provides a method for preparing a paris polyphylla glucomannan, including:
extracting crude polysaccharide from underground rhizome of paris polyphylla;
extracting a component with the weight average molecular weight of 50 kDa-100 kDa from the crude polysaccharide to obtain paris polyphylla glucomannan.
In some embodiments of the present application, the extracting crude polysaccharide from the underground rhizome of paris polyphylla comprises: pulverizing the underground rhizome of Paris polyphylla, and extracting with warm water or hot water to obtain the crude polysaccharide solution.
In some embodiments of the present application, the extracting the components of the crude polysaccharide having a weight average molecular weight of 50kDa to 100kDa comprises:
Carrying out enzymolysis on the crude polysaccharide, and carrying out fractional alcohol precipitation to obtain the paris polyphylla glucomannan;
refining the components with the weight average molecular weight of 50 kDa-100 kDa in the paris polyphylla glucomannan to obtain the paris polyphylla glucomannan.
In some embodiments of the present application, the enzymatic hydrolysis of the crude polysaccharide, and the fractional alcohol precipitation comprises: and (3) carrying out enzymolysis on the crude polysaccharide by using amylase, boiling to inactivate enzyme, taking clear liquid, grading and carrying out alcohol precipitation, and collecting precipitate.
In some embodiments of the present application, the method of refining comprises one or more of fractional alcohol precipitation, anion exchange chromatography, gel exclusion chromatography, dialysis, and ultrafiltration.
In some embodiments of the present application, the amylase is present at a volume concentration of 0.1% to 0.5%.
In some embodiments of the present application, the temperature of the enzymatic hydrolysis is 60-80 ℃.
In some embodiments of the present application, the time for the enzymatic hydrolysis is 1-3 hours.
In some embodiments of the present application, the time for boiling to inactivate enzymes is from 10 to 15 minutes.
In some embodiments of the present application, the fractional alcohol precipitation comprises: and (3) taking clear liquid for grading alcohol precipitation, enabling the alcohol concentration to reach 35% -45%, 55% -65% and 75% -85% respectively, and collecting precipitation.
In a third aspect, embodiments of the present application provide a cosmetic or pharmaceutical composition comprising an effective amount of the paris polyphylla glucomannan provided in the embodiments of the first aspect of the present application and a cosmetically or pharmaceutically acceptable adjuvant.
In a fourth aspect, the embodiment of the application provides an application of the paris polyphylla glucomannan provided by the embodiment of the first aspect of the application in cosmetics.
In a fifth aspect, embodiments of the present application provide an application of the paris polyphylla glucomannan provided in the embodiments of the first aspect of the present application in preparing a food and/or a drug with modulated immune activity.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments of the present application will be briefly described, and it is possible for a person skilled in the art to obtain other drawings according to these drawings without inventive effort.
FIG. 1 is a graph showing molecular weight distribution of the glucomannan from Paris polyphylla in the examples of the present application;
FIG. 2 is a liquid chromatography (HPLC) diagram of the composition of Paris polyphylla glucomannan monosaccharides in the examples of the present application;
FIG. 3 is a total ion flow diagram of the methylation of the Yunnan paris polyphylla glucomannan in the examples of the present application;
FIG. 4 is an Infrared (IR) spectrum of the glucomannan of Paris polyphylla in the examples of this application;
FIG. 5 is a Nuclear Magnetic Resonance (NMR) spectrum of Paris polyphylla glucomannan in the examples of the present application, wherein A is 1 H spectrum, B is 13 C spectrum, C is COSY spectrum, D is TOCSY spectrum, E is HSQC spectrumF is HMBC spectrum, G is HSQC-TOCSY spectrum, and H is ROESY spectrum;
fig. 6 shows the effect of paris polyphylla glucomannan on RAW264.7 cell proliferation and cytokine release in the examples of the present application, wherein a is the effect of paris polyphylla glucomannan on macrophage proliferation, B is the effect of paris polyphylla glucomannan on RAW264.4 cell secretion NO, C is the effect of paris polyphylla glucomannan on RAW264.7 cell secretion IL-10, D is the effect of paris polyphylla glucomannan on RAW264.7 cell secretion TNF- α (n=3, "" represents significance compared with the blank group, p represents < 0.05, p represents < 0.01, p represents < 0.001);
fig. 7 shows the protective effect of paris polyphylla glucomannan on L929 cell proliferation and on oxidatively damaged L929 cells, wherein a is the effect of paris polyphylla glucomannan on L929 cell proliferation, B is the effect of paris polyphylla glucomannan on hydrogen peroxide induced L929 cell viability (n=3, "#" indicates significant compared to the blank, "# or #" indicates significant compared to the model, # or # # represents p < 0.05, # or # # represents p < 0.01, # or # # represents p < 0.001).
Detailed Description
Features and exemplary embodiments of various aspects of the present application are described in detail below. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present application. It will be apparent, however, to one skilled in the art that the present application may be practiced without some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the present application by showing an example of the present application. In the drawings and the following description, at least some well-known structures and techniques are not shown in order to avoid unnecessarily obscuring the present application; also, the dimensions of some of the structures may be exaggerated for clarity. Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. In the description of the present application, it is to be noted that the meaning of "plurality" is two or more unless otherwise indicated. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising … …" does not exclude the presence of other like elements in a process, method, article or apparatus that comprises the element.
Paris is a generic name of Paris genus plants of Paris of the family Melanina, and its medical history is long and has been described in ancient books of herbal medicine since the Han dynasty. The rhizome of Paris is loaded into Chinese pharmacopoeia (1977) and the basic plant of Paris polyphylla or Paris polyphylla is specified to have the effects of clearing heat and detoxicating, detumescence and relieving pain, cooling liver and arresting convulsion, and is used for furuncle and carbuncle swelling and pain, sore throat, snake bite, traumatic injury and convulsion.
Paris polyphylla (Paris polyphylla var. Yunnanensis (frank.) hand-mzt.) is also known as Paris polyphylla and is recorded in pharmacopoeia of the people's republic of China (2020 edition). Rhizoma paridis Yunnanensis is generally used as a drug, and has the effects of clearing heat and detoxicating, relieving swelling and pain, cooling liver and arresting convulsion and the like. Is used for treating sore throat, snake and insect bites, convulsion, functional uterine bleeding, neuritis, surgical inflammatory reaction and other diseases, has obvious curative effect, and is the main component of Chinese patent medicines such as paris polyphylla detoxification tincture, gongxuening capsule, yunnan white drug powder, resolving hard mass and pain relieving ointment and the like.
The paris polyphylla mainly contains chemical components such as steroid saponins, flavonoids, triterpenes and the like, wherein the steroid saponins are important natural organic compounds with high types and contents of the compounds in the paris polyphylla, and have better biological activity. However, the paris polyphylla serving as a medicinal plant with national characteristics has high use frequency, the material basic research of the traditional efficacy of the paris polyphylla is still to be excavated, and particularly, the paris polyphylla polysaccharide needs to be intensively studied. Therefore, from the viewpoint of resource development and utilization, research on other components is still required.
Based on the above, the inventor of the application performs systematic chemical and pharmacological researches on polysaccharide of paris polyphylla, reports the paris polyphylla glucomannan with novel structure for the first time, and explores the application of the paris polyphylla glucomannan in the fields of cosmetics, pharmaceutical preparations and foods.
Paris polyphylla glucomannan
An embodiment of a first aspect of the present application provides a paris polyphylla glucomannan having a structure as shown in formula I:
in the formula I, m and n represent the number of repeating units and are natural numbers, m is independently 1 or 2, and n is more than or equal to 30 and less than or equal to 40.
The inventor of the application surprisingly discovers that the Yunnan paris polyphylla glucomannan with a novel structure can be obtained by extracting and purifying the Yunnan paris polyphylla rhizome by using an innovative method, and the inventor discovers that the Yunnan paris polyphylla glucomannan with a novel structure can remarkably promote macrophage RAW264.7 to secrete cytokines IL-10 and TNF-alpha through an activity screening research, so that the Yunnan paris polyphylla glucomannan has obvious organism immunity regulating capability, and the Yunnan paris polyphylla glucomannan has the application of preparing medicines and/or health foods for improving organism immunity. The inventor further researches the pharmacological research, and found that the paris polyphylla glucan can remarkably reduce the cell death rate of skin fibroblasts damaged by hydrogen peroxide, has remarkable activity of preventing free radical cell damage and has remarkable protective effect on skin damaged by oxidation, so that the paris polyphylla glucan has the application of cosmetics for preventing skin from being damaged by oxidation.
In some embodiments, the weight average molecular weight of the paris polyphylla glucomannan is 50kDa to 100kDa.
In some embodiments, the monosaccharide composition of the paris polyphylla glucomannan comprises glucose and mannose, and the molar ratio of the glucose to the total monosaccharides is 6.5% -9%.
In some embodiments, the mannose comprises an acetylated mannose substituted with an acetyl group at the 2-or 3-position of the sugar ring, the acetylated mannose comprising 27.5% to 30% by mole of the total monosaccharides.
In some embodiments, the purity of paris polyphylla glucomannan is not less than 95%.
According to the embodiment of the application, the weight average molecular weight of the novel paris polyphylla glucomannan is 50 kDa-100 kDa, beta (1-4) mannose is taken as a main chain, the polysaccharide chain contains glucose, the glucose accounts for 6.5-9% of the molar ratio of total monosaccharides, the 2 or 3-position part on the sugar ring of mannose is substituted by acetyl, and the molar ratio of the acetylated mannose accounts for 27.5-30% of the total monosaccharides.
Preparation method of paris polyphylla glucomannan
An embodiment of the second aspect of the present application provides a method for preparing a paris polyphylla glucomannan, including the following steps S100 to S200:
s100, extracting crude polysaccharide from underground rhizome of paris polyphylla;
S200, extracting a component with the weight average molecular weight of 50 kDa-100 kDa from the crude polysaccharide to obtain paris polyphylla glucomannan.
According to the embodiment of the application, the Yunnan paris polyphylla glucomannan with novel structure and outstanding activity can be obtained by the preparation method, the weight average molecular weight of the Yunnan paris polyphylla glucomannan is 50 kDa-100 kDa, beta (1-4) mannose is taken as a main chain, the Yunnan paris polyphylla glucomannan contains glucose, the molar ratio of the glucose to total monosaccharides is 6.5-9%, the 2-position or 3-position part of the sugar ring of the mannose is substituted by acetyl to become acetylated mannose, and the molar ratio of the acetylated mannose to the total monosaccharides is 27.5-30%, so the preparation method provided by the embodiment of the second aspect of the application can be used for preparing the Yunnan paris polyphylla glucomannan of the first aspect of the application.
In this embodiment, the underground rhizome of paris polyphylla may be an underground rhizome block of paris polyphylla, or may be powder obtained by pulverizing an underground rhizome block of paris polyphylla.
In some embodiments, the underground rhizome of paris polyphylla is powder obtained by pulverizing underground rhizome blocks of paris polyphylla, and the specific obtaining method is as follows: pulverizing dried rhizoma paridis, extracting the powder with 90-100% ethanol at a ratio of 1 (8-12) and 60-80deg.C for 1-4 hr for 1-2 times to remove liposoluble substances, filtering while hot, collecting filter cake, volatilizing ethanol residue in the filter cake to obtain defatted and depigmented rhizoma paridis residue.
Specifically, the concentration of ethanol is preferably 95%, the feed-to-liquid ratio is preferably 1:10, the temperature is preferably 70 ℃, the extraction time is preferably 2h, and the extraction times are preferably 2 times.
In some embodiments, extracting crude polysaccharide from the underground rhizome of paris polyphylla comprises: extracting the residue of the defatted rhizoma paridis underground rhizome with hot water or warm water to obtain crude polysaccharide solution.
Specifically, the water temperature for extraction is 75-85 ℃, the ratio of extracted liquid to extracted liquid is 1 (8-12), and the ratio can be selected to be 1:10; the extraction time is 2-4 h, the extraction times are 1-2 times, and 2 times are optional.
In some embodiments, extracting components of the crude polysaccharide having a weight average molecular weight of 50kDa to 100kDa includes the following steps S210 to S220:
s210, carrying out enzymolysis on crude polysaccharide, and grading and alcohol precipitation to obtain the paris polyphylla glucomannan.
In this embodiment, the enzymatic hydrolysis crude polysaccharide includes: the solution of crude polysaccharide is enzymatically hydrolyzed with amylase to remove the starch polysaccharide therein.
Specifically, the enzymolysis temperature is 60-80 ℃, the enzymolysis time is 1-3 h, the concentration of amylase is 0.1% -0.5% (v/v), and the concentration is optional 0.2%.
In an embodiment of the present application, the fractional alcohol precipitation includes: boiling the crude polysaccharide solution after enzymolysis to inactivate enzyme, centrifuging, collecting clear liquid, grading, precipitating with ethanol, and collecting precipitate.
Specifically, the supernatant-taking fractionation alcohol precipitation includes the following steps S211 to S214:
s211, adding 95% ethanol into the clear liquid after enzyme deactivation under stirring until the final concentration of the ethanol is 35% -45%, and centrifuging to obtain 35% -45% alcohol sediment and 35% -45% alcohol sediment supernatant;
s212, adding 95% ethanol into the 35% -45% ethanol precipitation supernatant under stirring until the final concentration of the ethanol is 55% -65%, centrifuging to obtain 55% -65% ethanol precipitation and 55% -65% ethanol precipitation supernatant, washing 55% -65% ethanol precipitation for 3-5 times by using 95% ethanol, adding water for redissolution, and freeze-drying;
s213, adding 95% ethanol into the 55% -65% ethanol precipitation supernatant under stirring until the final concentration of the ethanol is 75% -85%, centrifuging to obtain 75% -85% ethanol precipitation and ethanol precipitation supernatant, washing 75% -85% ethanol precipitation 3-5 times by using 95% ethanol, adding water for re-dissolving, and freeze-drying;
and S214, combining the products obtained after the two freeze-drying to obtain the paris polyphylla glucomannan.
Specifically, the final alcohol concentration in step S211 may be selected to be 40%, the final alcohol concentration in step S212 may be selected to be 60%, and the final alcohol concentration in step S213 may be selected to be 80%.
S220, refining a component with the weight average molecular weight of 50 kDa-100 kDa in the crude paris polyphylla glucomannan to obtain paris polyphylla glucomannan.
In the embodiment of the application, the method for refining the paris polyphylla glucomannan comprises one or more of anion exchange column chromatography, gel exclusion chromatography, dialysis and ultrafiltration.
Specifically, the crude paris polyphylla glucomannan can be redissolved by adding deionized water, insoluble matters are removed by centrifugation, optionally, the purification is carried out by alcohol precipitation and fractionation, gel exclusion chromatography, dialysis or ultrafiltration, the fraction or trapped fluid or permeate containing the paris polyphylla glucomannan is collected, and if the paris polyphylla glucomannan contains salt, the purified paris polyphylla glucomannan is obtained by direct vacuum freeze drying or vacuum concentration and then alcohol precipitation and vacuum drying after desalting.
As will be appreciated by those skilled in the art, for Gel exclusion chromatography, gel materials such as Sephadex series, polyacrylamide Bio-Gel P series and Gel fillers formed by cross-linking them are reasonably selected according to the molecular weight of the glucomannan material in the polysaccharide component of the glucomannan of Paris polyphylla, and then the packing, loading and sequential elution with saline or non-saline elution solutions and collection of fractions are performed according to the actual properties of each filler. Combining the effluent peaks, concentrating the effluent or not, loading into dialysis bag for dialysis or ultrafiltration membrane for dialysis or ultrafiltration for desalting, collecting desalted retentate, and vacuum freeze drying or vacuum reduced pressure drying to obtain purified rhizoma paridis glucomannan. Or purifying polysaccharide component of Paris polyphylla by dialysis or ultrafiltration, and selecting ultrafiltration membrane with proper molecular weight for tangential flow ultrafiltration interception. For example, the aqueous solution of the paris polyphylla polysaccharide component is fully ultrafiltered by an ultrafiltration membrane bag with the molecular weight smaller than that of paris polyphylla glucomannan, permeate is collected to remove substances with small molecular weight, then the aqueous solution is fully dialyzed or ultrafiltered by a dialysis bag or ultrafiltration membrane bag with the molecular weight larger than that of paris polyphylla glucomannan, retentate is collected, concentrated and then vacuum freeze-dried or vacuum reduced-pressure dried to obtain the refined paris polyphylla glucomannan.
Cosmetic or pharmaceutical composition
Embodiments of the third aspect of the present application provide a cosmetic or pharmaceutical composition comprising an effective amount of paris polyphylla glucomannan, including paris polyphylla glucomannan according to embodiments of the first aspect of the present application or paris polyphylla glucomannan obtained according to the preparation method of embodiments of the second aspect of the present application, and a cosmetically or pharmaceutically acceptable adjuvant.
In some embodiments, the effective amount of paris polyphylla glucomannan in the cosmetic is 0.01% -10%.
In some embodiments, the effective amount of the paris polyphylla glucomannan in the pharmaceutical composition is 0.01% -10%.
In some embodiments, the excipients include one or more of excipients, carriers, diluents, film formers.
Optionally, the excipient comprises one or more combinations of starch, carbomer, glycerol, ethylparaben, triethanolamine, polysorbate 80, propylene glycol.
Optionally, the carrier comprises one or more of a capsule, a hydrogel patch, purified water.
Optionally, the diluent comprises one or more of glycerol, propylene glycol, butylene glycol, purified water.
Optionally, the film forming agent comprises one or more combinations of polyvinylpyrrolidone, trimethylsiloxysilicate, polyvinylpyrrolidone, polyvinyl alcohol, acrylic copolymers.
In some embodiments, the cosmetic dosage form includes a mask, cream, ointment, barrier.
In some embodiments, the dosage form of the pharmaceutical composition includes lyophilized powder, oral liquid, capsule, pellet, tablet.
Application of paris polyphylla glucomannan in cosmetics
Embodiments of the fourth aspect of the present application provide a cosmetic use of paris polyphylla glucomannan.
According to the embodiment of the application, the paris polyphylla glucomannan has the effect of reducing the death of the mouse skin fibroblasts with oxidative damage and can protect the skin with ultraviolet oxidative damage. Therefore, the paris polyphylla glucomannan can be applied to cosmetics with the effect of preventing ultraviolet oxidation injury.
Application of paris polyphylla glucomannan in preparation of food and/or medicine with immunocompetence regulated
Embodiments of the fifth aspect of the present application provide for the use of paris polyphylla glucomannan in the preparation of a food and/or pharmaceutical product with modulated immune activity.
According to the embodiment of the application, the paris polyphylla glucomannan can activate macrophages, stimulate the macrophages to release immune related active substances such as cell immune factors IL-10, TNF-alpha and the like, and has remarkable immune regulation activity, so that the paris polyphylla glucomannan can be applied to preparing food and/or medicines with immune activity regulation.
The embodiment of the application also provides a structure analysis method of the paris polyphylla glucomannan, which comprises the following steps:
(1) Molecular weight measurement: taking a paris polyphylla glucomannan sample, and measuring by adopting a high-efficiency gel exclusion chromatography-differential detector detection method.
(2) Monosaccharide composition analysis: taking a Paris polyphylla glucomannan sample, carrying out acid hydrolysis to obtain monosaccharide, carrying out derivatization on the monosaccharide by 1-phenyl-3-methyl-5-pyrazolone (PMP), using octadecylsilane chemically bonded silica gel as a chromatographic column of a filler, and analyzing the monosaccharide composition of Paris polyphylla glucomannan by a high performance liquid chromatograph.
(3) Methylation analysis: taking a paris polyphylla glucomannan sample, carrying out polysaccharide methylation by using methyl halide reagents such as methyl iodide and the like under alkaline conditions, reducing by using sodium borohydride after hydrolysis under alkaline conditions, carrying out acetylation on methylated sugar alcohol by using anhydride, carrying out gas chromatograph-mass spectrometer (GC-MS) analysis after extraction, and judging the glycosidic bond connection mode of paris polyphylla glucomannan.
(4) And (3) infrared spectrum analysis: and taking a paris polyphylla glucomannan sample, fully drying, and measuring the infrared spectrum of the sample on an infrared spectrometer by adopting a solid potassium bromide tabletting method.
(5) Nuclear magnetic resonance analysis: dissolving rhizoma paridis Yunnanensis glucomannan sample in heavy water, vacuum freeze drying, repeating three times of heavy water exchange, dissolving in heavy water, and detecting nuclear magnetic resonance spectrum including one dimension 1 H and 13 c-spectrum and two dimensions 1 H- 1 H COSY、 1 H- 1 H TOCSY、 1 H- 1 H ROESY、 1 H- 13 C HSQC、 1 H- 13 C HSQC-TOCSY、 1 H- 13 Correlation spectra such as chmbc.
(6) And (3) comprehensive data analysis: analyzing the analysis data of the steps comprehensively, and analyzing the chemical structure of the paris polyphylla glucomannan.
According to the structural analysis method, structural analysis is carried out on the paris polyphylla glucomannan provided by the embodiment of the application, and the result shows that the paris polyphylla glucomannan only shows one chromatographic peak on HPGPC gel chromatography, the weight average molecular weight is about 50 kDa-100 kDa, and the monosaccharide composition shows that the paris polyphylla glucomannan consists of mannose or contains a small amount of glucose; methylation analysis showedBeta (1-4) glycosidic bond exists in the paris polyphylla glucomannan; infrared spectrum analysis shows that the carbonyl absorption peak of acetyl and the hydroxyl absorption peak on the sugar ring have pyranose absorption signal peaks in the fingerprint region which all accord with the absorption peak of glucomannan; can be clearly attributed to the paris polyphylla glucomannan according to nuclear magnetic resonance spectrum data 1 H and 13 and C.
In summary of the above data, the paris polyphylla glucomannan provided in the examples of the present application has the following structural characteristics shown in formula I: mannose beta (1-4) glycosidic bond is mainly, and partial mannose is accompanied by 2 or 3-site acetylation.
In the formula I, m and n represent the number of sugar repeating units and are natural numbers, m is independently 1 or 2, and n is more than or equal to 30 and less than or equal to 40.
Examples
The invention is illustrated by the following specific examples. It should be noted that the embodiments described below are exemplary only for explaining the present application and are not to be construed as limiting the present application. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: preparation of Paris polyphylla glucomannan
Extraction of paris polyphylla crude polysaccharide
S10, weighing 300g of dried and crushed paris polyphylla underground rhizome powder, adding 3L of 95% ethanol, extracting for 2 times at 80 ℃ for 2 hours each time, filtering while the paris polyphylla underground rhizome powder is hot, collecting a filter cake, and volatilizing ethanol remained in the filter cake;
s20, adding 3L deionized water into the filter cake, extracting for 2 hours at 80 ℃, centrifuging at 4000rpm for 15 minutes after the extraction is finished, and taking a supernatant; extracting the residue repeatedly, and mixing the supernatants;
s30, adding 0.2% (v/v) alpha-amylase into the supernatant and enzyme at 70 DEG CSolution 2h, I 2 The KI solution is developed without starch detection, the enzyme is boiled and deactivated for 10min, and the solution is centrifuged at 4000rpm for 15min, and the supernatant is taken;
s40, adding 95% ethanol into the supernatant until the final concentration of the ethanol is 40%, centrifuging at 4000rpm for 15min, and obtaining 40% ethanol sediment and 40% ethanol supernatant; adding 95% ethanol into 40% ethanol supernatant under stirring until the final concentration of ethanol is 60%, centrifuging at 4000rpm for 15min to obtain 60% ethanol precipitate and 60% ethanol supernatant, washing 60% ethanol precipitate with 95% ethanol for 3 times, adding water, redissolving, and lyophilizing; adding 95% ethanol into 60% ethanol supernatant under stirring until the final concentration of ethanol is 80%, centrifuging at 4000rpm for 15min to obtain 80% ethanol precipitate and 80% ethanol supernatant, washing 80% ethanol precipitate with 95% ethanol for 3 times, adding water, redissolving, and lyophilizing;
S50, combining the twice freeze-dried alcohol sediments to obtain paris polyphylla crude polysaccharide, wherein the total weight of the paris polyphylla crude polysaccharide is 5.27g after freeze-drying;
refined paris polyphylla glucomannan
S60, weighing 400mg of paris polyphylla crude polysaccharide, adding 10mL of deionized water for dissolution, centrifuging at 4000rpm for 15min, filtering the supernatant by a filter membrane of 0.45 mu M, slowly and uniformly injecting the filtered supernatant into the top end of a DEAE52 cellulose chromatographic column, eluting by water, 0.1M NaCl solution, 0.3M NaCl solution and 0.5M NaCl solution in sequence, controlling the flow rate to be 1mL/min, collecting 5mL of the supernatant by each tube, and detecting the polysaccharide content by a phenol-sulfuric acid method. And drawing an elution curve of the polysaccharide component by taking the light absorption value as an ordinate and the tube number as an abscissa. Combining the eluents belonging to the same eluting peak, collecting the sample with the largest eluting curve, washing the polysaccharide in the part with water, concentrating under reduced pressure, and vacuum freeze drying to obtain refined rhizoma paridis dextran 300mg.
Example 2: structural analysis of Paris polyphylla glucomannan
Test method
1. Molecular weight and distribution thereof
The molecular weight and distribution of the paris polyphylla glucomannan prepared in example 1 were analyzed by high performance gel exclusion chromatography-differential detector detection (HPGPC-RID).
Chromatographic instrument: agilent technologies 1260series high performance liquid chromatograph;
Chromatographic conditions: shodex OHPak SB-804HQ (8 mm. Times.300 mm) column, column temperature of 35deg.C, differential detector, mobile phase of 0.1M NaCl, flow rate of 0.5mL/min;
the measuring process comprises the following steps: 5mg of paris polyphylla glucomannan samples or dextran reference substances with known molecular weight are respectively taken, a mobile phase is added to prepare a solution with the concentration of 5mg/mL, the solution is filtered by a microporous membrane with the concentration of 0.2 mu m, and 30 mu L of filtrate is analyzed by a high performance liquid chromatograph and a chromatogram is recorded. The data are processed by GPC software, a standard curve is drawn, the data are substituted into an equation, and the molecular weight is calculated.
2. Analysis of monosaccharide composition
Taking 1mL of monosaccharide standard solution and 1mL of sample solution, placing the monosaccharide standard solution and the sample solution into a hydrolysis tube, adding 1mL of 4M trifluoroacetic acid solution, uniformly mixing, and carrying out oven hydrolysis for 4h at 110 ℃. After the hydrolysis reaction was completed, the hydrolysate was dried by rotary evaporation using a rotary evaporator concentrator to ensure complete removal of TFA. After drying, 200. Mu.L of deionized water was added for dissolution, then 200. Mu.L of 0.6M NaOH solution and 400. Mu.L of 0.5M PMP-methanol solution were sequentially added, uniformly mixed, and the mixture was subjected to derivatization reaction in an oven at 70℃for 1 hour, cooled to room temperature, neutralized with 200. Mu.L of 0.6M hydrochloric acid, subjected to chloroform extraction for 3 times (2 mL each time), centrifuged (10000 rpm,10 min) and filtered through a microporous membrane of 0.2 μm for liquid chromatography.
Chromatographic conditions:
chromatograph: agilent technologies 1260series high performance liquid chromatograph;
chromatographic column: hadesil C18-Bio (250 mm. Times.4.6 mm, id.5 μm); column temperature: 25 ℃;
mobile phase: a:0.1M phosphate buffer; b: acetonitrile, a/b=82/18;
eluting: the flow rate is 1mL/min, the sample injection amount is 10 mu L, and the time is 60min;
and (3) detection: DAD detector, wavelength 250nm.
3. Methylation analysis
Methylation reaction: the dry sodium hydroxide powder (120 mg) was taken and 1mL of anhydrous DMSO was added to dissolve sodium hydroxide sufficiently to obtain 120mg/mL of an alkaline DMSO solution. Weighing 3-5 mg of paris polyphylla glucomannan, placing the polyglucose into a 10mL spiral tube, adding 1mL of DMSO solution to dissolve a polysaccharide sample, adding 1mL of NaOH/DMSO solution after the sample is dissolved, sealing and carrying out ultrasonic treatment for 10min, adding 200 mu L of methyl iodide, carrying out ultrasonic light-shielding reaction for 10min, adding 200 mu L of methyl iodide again, carrying out reaction for 10min, and finally adding 400 mu L of methyl iodide, and carrying out reaction for 30min. After the reaction is finished, adding 1mL of deionized water to quench the reaction, adding chloroform according to the volume of 1:1 to extract for 3 times, combining organic phases, washing the organic phases with deionized water for 3 times, and finally drying the organic phases under reduced pressure.
Acid hydrolysis: to the dried sample was added 2mL of 2M TFA solution, and the tube was capped and hydrolyzed with acid at 120℃for 2h.
And (3) reduction: spin-drying the above hydrolysate, dissolving in 1mL of 2M NH 4 OH, and 1mL of freshly prepared 1M NaBD was added 4 Solution (in 2M NH 4 OH as solvent), the sample was sonicated for 1 minute and incubated at room temperature for 2.5 hours. After the reaction was completed, 400. Mu.L of acetic acid was added to remove the excess reducing agent, and the sample was evaporated to dryness under reduced pressure by a rotary evaporator.
Acetylation: to the above sample, 1mL of pyridine and 1mL of acetic anhydride were added to the test tube, and the tube was sealed at 100℃for 1.5 hours. After cooling, the reaction was quenched by addition of 1mL of water, extracted 3 times with 3mL of dichloromethane, washed 3 times with organic phase, and finally the organic phase was dried by spinning, dissolved across the membrane by addition of 1mL of chromatographic grade chloroform, and GC-MS analysis was performed.
GC conditions: DB-5MS quartz capillary column (30 m x 0.25mm x 0.25 μm); column temperature: the initial temperature is 80 ℃, the temperature is kept for 1min, the temperature is programmed to be 5 ℃/min to 250 ℃ and the temperature is kept for 5min; the column flow rate is 1.5mL/min; the temperature of the sample inlet is 270 ℃; the split ratio is 10:1; the carrier gas is high-purity helium, and the sample injection amount is 1 mu L.
MS conditions: ionization mode EI; electron energy 70eV; the temperature of the transmission line is 290 ℃; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; the mass range is 50-600; and comparing the result with a CCRC spectrum database so as to judge the type of the glycosidic bond.
4. Infrared spectroscopic analysis
According to the fourth part of 2020 edition of Chinese pharmacopoeia, a solid tabletting method is adopted: taking 2mg of sample, vacuum drying at 40deg.C for 24 hr, tabletting with KBr, and performing Fourier transform on the sample by using Tensor 27 mid-infrared spectrometerLines 4000-400 cm -1 Scanning and recording a spectrogram.
5. Nuclear magnetic resonance analysis
Dissolving 20mg of Paris polyphylla glucomannan in 0.5mL D 2 O, freeze-drying, D 2 After three O exchanges, the lyophilized sample was dissolved in 0.5mL D 2 O (99.9 atom% D, containing 0.05% by weight of the internal standard 3- (trimethyllyl) -propionic-2, 3-d) 4 acid sodium salt) was measured using a Bruker 800MHz NMR spectrometer 1 H/ 13 C NMR spectrum and two-dimensional spectrum 1 H- 1 HCOSY、 1 H- 1 H TOCSY、 1 H- 1 H ROESY、 1 H- 13 C HMBC、 1 H- 13 C HSQC-TOCSY 1 H- 13 C HSQC is analyzed and processed by adopting MestReNova and Topspin software.
Experimental results
The properties of the paris polyphylla glucomannan in the embodiment of the application are as follows: white or white-like solid, odorless, and moisture-absorbing.
The molecular weight and the distribution analysis result are shown in figure 1, and the analysis result of the high-efficiency gel exclusion chromatography shows that the purified paris polyphylla glucomannan extracted from paris polyphylla rhizome has only one chromatographic peak, the weight average molecular weight is 92kDa, and the polydispersity coefficient is 6.85; through the analysis of multiple batches of samples, the weight average molecular weight of the paris polyphylla glucomannan is 50 kDa-100 kDa.
The analysis result of the monosaccharide composition is shown in figure 2, and the result shows that the HPLC chart after the paris polyphylla glucomannan is derived before the PMP column shows that the paris polyphylla glucomannan contains mannose and a small amount of glucose, which indicates that the paris polyphylla glucomannan mainly consists of mannose and contains a small amount of glucose, and the molar ratio of glucose to total monosaccharides is 6.5% -9% after multi-batch analysis.
The methylation analysis results are shown in FIG. 3, and the results show that the paris polyphylla glucomannan has beta (1-4) mannose glycosidic bond, a small amount of beta (1-4) glucose glycosidic bond, beta (1-4) mannose glycosidic bond and beta (1-4) glucose glycosidic bond.
The infrared spectrum analysis results are shown in FIG. 4, which shows that，3400～3430cm -1 The characteristic absorption of the stretching vibration of the hydroxyl O-H on the sugar ring; 2920-2890 cm -1 Stretching and vibrating the last methine C-H of the sugar ring; 1730-1740 cm -1 Carbonyl stretching vibration characteristic absorption peak of acetyl; 1000-1060 cm -1 C-O-C stretching vibration on the sugar ring; 810-890 cm -1 Is the configurational characteristic absorption of beta type monosaccharide.
The Nuclear Magnetic Resonance (NMR) detection results are shown in FIG. 5, and are detailed 1 H and 13 the assignment of the C NMR signals is shown in Table 1.
Nuclear magnetic resonance results indicate that, in NMR 1 In the H spectra, the terminal proton chemical shifts are the terminal proton signals of non-reducing terminal beta-D-Glc- (1 → (abbreviated as D), →4) -beta-D-Man- (1 → (abbreviated as A/E), acetyl-containing in the 3-position →4) -3-O-acetyl-beta-D-Man- (1 → (abbreviated as C), reducing terminal beta-D-Man (abbreviated as F), acetyl-containing in the 2-position →4) -2-O-acetyl-beta-D-Man- (1 → (abbreviated as B) and reducing terminal alpha-D-Man (abbreviated as G) at 4.53, 4.76/4.73, 4.82, 4.91, 4.95 and 5.19ppm, respectively, the molar ratio of residues A, B, C, D, E, F and G can be obtained as 6.1:3.0:2.0:1.0:3.2:0.8:0.6, wherein residues B and C are mannose residues with acetyl groups substituted at the 2-and 3-positions, respectively, so that the molar ratio of the sugar residues substituted by acetyl groups to the glucose residues to the total monosaccharides can be obtained by calculating the molar ratio of the residues, and the molar ratio of the sugar residues in polysaccharide extracted in multiple batches is calculated to obtain 27.5% -30% of the molar ratio of the mannose residues with acetyl groups substituted at the 2-and 3-positions to the total monosaccharides and 6.5% -9% of the molar ratio of the glucose residues to the total monosaccharides in NMR 13 In the C spectrum, the chemical shift is a carbonyl carbon signal of sugar ring end group at 95-105 ppm and an acetyl carbonyl carbon signal at 175-177 ppm.
The proton signal at position 4 of 1, 4-beta-D-mannose (A/E residue) is 3.83ppm and the chemical shift of its corresponding C-4 is 79.4/80.3ppm, which is significantly shifted to the lower field relative to the corresponding chemical shift of unsubstituted mannose, indicating that the hydroxyl groups at positions 1 and 4 of mannosyl A/E are substituted, the C-4 chemical shift (higher than A residue) of E residue is slightly different from H-1 chemical shift (lower than A residue) compared to A residue, possibly due to the attachment of E residue to acetyl-containing B and C residues. The proton signal at position 2 of 1, 4-beta-D-mannose (residue B) containing acetyl is 5.52ppm, the proton signal at position 4 is 3.82ppm, the chemical shift of C-2 is 74.5ppm, the chemical shift of C-4 is 79.5ppm, the shift of 2 and 4 carbon relative to unsubstituted mannose is significantly lower, and the combination of the previous literature shows that the hydroxyl at position 2 of mannose B is substituted by acetyl and the hydroxyl at position 4 is connected with other residues. The proton signal at 3 position of 1, 4-beta-D-mannose containing acetyl at 3 position is 5.11ppm, the proton signal at 4 position is 4.04ppm, and the chemical shift of C-3 and C-4 corresponding to the proton signal is 76.3ppm, the chemical shift of hydrogen carbon at 3 position and 4 position relative to unsubstituted mannose is obviously shifted to a low field, which indicates that 3-hydroxyl of mannose C is substituted by acetyl, and 4-hydroxyl is connected with other residues.
1 H and 13 c remote correlation HMBC spectra show that C-1 (A1) of mannose A and H-4 (A4, B4, G4) of mannose groups A, B and G have correlation signals, C-1 (B1) of mannose groups B and H-4 (E4) of mannose groups E have correlation signals, C-1 (C1) of mannose groups C and H-4 (A4) of mannose groups A have correlation signals, C-1 (E1) of mannose groups E and H-4 (A4) of mannose groups A and H-4 (C4) of mannose groups C have correlation signals. Due to the too small glucose ratio in the paris polyphylla glucomannan, only NMR is caused 1 The H spectrum shows the terminal proton signal of the glucose residue (residue D), whereas NMR 13 No signal was found for the glucose residues in the C spectrum, but it was seen by methylation analysis that glucose was linked to other residues in the form of beta (1.fwdarw.4) glycosidic linkages. Thus, in combination with the above analysis, the linkage pattern of mannose groups was found to be: mannose groups (A/E) form a main chain with beta (1.fwdarw.4) glycosidic linkages, and mannose groups (B and C) substituted with acetyl groups at positions 2 and 3 and glucose residues (residue D) are attached to mannose groups interspersed in the main chain with beta (1.fwdarw.4) glycosidic linkages.
From the above data, the novel chemical structure of the novel Yunnan paris polyphylla glucomannan is shown in the following formula I:
in the formula I, m and n represent the number of sugar repeating units and are natural numbers, m is independently 1 or 2, and n is more than or equal to 30 and less than or equal to 40.
The structure is characterized in that: (1) The paris polyphylla glucomannan is novel in structure extracted from rhizome of paris polyphylla; (2) the monosaccharide connection mode of the paris polyphylla glucomannan is as follows: beta (1-4) mannose glycosidic bond is taken as main chain.
Table 1: paris polyphylla glucomannan 1 H/ 13 Assignment of C NMR
Example 3: immunomodulatory test of Paris polyphylla glucomannan
Test article and reagent
Paris polyphylla glucomannan (PMM): the paris polyphylla glucomannan prepared according to example 1;
DMEM high sugar medium, 0.25% pancreatin EDTA solution: shanghai source culture Biotech stock Co.Ltd;
penicillin, streptomycin, fetal bovine serum: biological Industries company;
phosphate buffered saline PBS: wuhan Seville Biotechnology Co., ltd;
bacterial Lipopolysaccharide (LPS): sigma Co., USA;
nitric oxide detection kit: a Biyun Tian biotechnology;
TNF-alpha, IL-10 detection kit: euphoria technology limited;
sterilizing water: and (5) homemade in a laboratory.
The mouse macrophage cell line RAW264.7 is purchased from the cell resource center of the national academy of sciences of life.
Experimental method
1. Solution preparation
Sample mother liquor: weighing 10mg of paris polyphylla glucomannan, dissolving to 10mg/mL with sterilized water, preserving at 4 ℃, diluting to a final concentration of 200 mug/mL with a cell culture medium before use, and filtering and sterilizing with a 0.2 mu m microporous filter membrane for later use.
Lipopolysaccharide LPS: 1mg/mL stock solution was prepared with sterile PBS, stored at 4℃and diluted with cell culture medium to a final concentration of 1. Mu.g/mL before the experiment, and filtered through a 0.22 μm microporous filter membrane.
RAW264.7 cell culture and passage
Cell resuscitation: the macrophages RAW264.7 frozen in liquid nitrogen were removed and rapidly placed in a 37 ℃ thermostatic water bath to allow complete thawing of the cells in a short period of time. The thawed cells were transferred to a 15mL centrifuge tube containing 8mL DMEM medium, centrifuged at 800rpm for 5min, and the supernatant was discarded. Adding 2mL of DMEM medium containing 1% (v/v) penicillin-streptomycin diab and 10% (v/v) fetal bovine serum into a centrifuge tube, gently blowing the precipitated cells until the precipitated cells are completely suspended to obtain a cell suspension, transferring the cell suspension into a T25 culture flask, washing the centrifuge tube with 4mL of medium to ensure that the precipitated cells are completely transferred into the T25 culture flask, keeping about 6mL of medium in the culture flask, observing the cell density under an inverted microscope, and placing CO 2 Culturing in a constant temperature incubator. Culture conditions: 5% CO 2 Culturing at 37deg.C under saturated humidity, and changing liquid the next day.
Cell passage: after resuscitating, cells were subcultured after growing to 90% confluence (90% of the surface area of the flask was covered with cells), the old medium was discarded, washed 2 times with sterile PBS, 2mL of DMEM medium was added, gently swirled and the adherent cells were inoculated into a new flask (typically passaged at a 1:2 ratio) after being completely suspended, the medium in the flask was kept at about 6mL, and the cell density was observed under an inverted microscope. Culture conditions: 5% CO 2 Culturing at 37deg.C under saturated humidity, and changing liquid the next day.
In the subsequent cell experiments,the resuscitated cell RAW264.7 was used as the experimental object, and the culture conditions were 5% CO when not explicitly described 2 Culturing in a constant temperature incubator at 37 ℃ under saturated humidity.
3. Effects on RAW264.7 cell proliferation
RAW264.7 cells were grown at 1X 10 4 Each well was plated into a 96-well plate for 24 hours. Fresh medium containing each concentration of Paris polyphylla glucomannan (0, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL, 80. Mu.g/mL, 160. Mu.g/mL, 320. Mu.g/mL, 640. Mu.g/mL) was added, and the culture was continued for 24 hours at each well of 150. Mu.L. mu.L of 5mg/mL MTT solution is added into each well, the culture is continued for 4 hours at 37 ℃, the culture medium is discarded, 150 mu.L of DMSO solution is added into each well, the mixture is mixed by shaking, and the absorbance A value at 490nm is measured on an enzyme-labeled instrument. The cell viability was calculated as follows:
4. effect on NO Release by RAW264.7 cells
Taking RAW264.7 cells in logarithmic growth phase at 2×10 5 The cells/well were inoculated into 24-well plates, cultured for 24 hours, and fresh medium (low concentration: 50. Mu.g/mL, medium concentration: 100. Mu.g/mL, high concentration: 200. Mu.g/mL) containing each concentration of Paris polyphylla glucomannan was added thereto, and 150. Mu.L of LPS was used as a positive control per well. And (3) taking a culture supernatant after culturing for 24 hours, and immediately detecting the NO content, wherein the specific operation is described in the instruction of the NO content detection kit.
ELISA method for detecting influence on secretion level of macrophage cell factor
Taking RAW264.7 cells in logarithmic growth phase at 2×10 5 The cells/well were inoculated into 24-well plates, cultured for 24 hours, and fresh medium (low concentration: 50. Mu.g/mL, medium concentration: 100. Mu.g/mL, high concentration: 200. Mu.g/mL) containing each concentration of Paris polyphylla glucomannan was added thereto, and 150. Mu.L of LPS was used as a positive control per well. And (5) continuously culturing for 24 hours, collecting culture supernatant, and preserving at-80 ℃ for later use. And detecting the contents of TNF-alpha and IL-10 in the culture solution according to the instruction of ELISA kit.
Experimental results
1. Effects on macrophage proliferation
Referring to FIG. 6-A, compared with the normal control group, the Yunnan paris polyphylla glucomannan can remarkably promote RAW264.7 cell proliferation at the concentration of 10-160 mug/mL, the cell proliferation is most remarkable at the concentration of 160 mug/mL, and the concentration of 320-640 mug/mL has no effect of promoting cell proliferation.
2. Effect on macrophage NO Release
Referring to fig. 6-B, compared with the normal control group, the paris polyphylla glucomannan can significantly promote the release of macrophage NO, and the promoting amount of NO is positively correlated with the concentration of paris polyphylla glucomannan, which indicates that paris polyphylla glucomannan has better immune activity and is dose-dependent.
3. Effect on macrophage secretion of TNF- α, IL-10
Referring to FIG. 6-C, the paris polyphylla glucomannan has a remarkable promoting effect on the secretion of IL-10 by macrophages, the quantity and effect relationship are obvious, the greater the capacity of promoting the macrophages to secrete IL-10 is as the concentration of the paris polyphylla glucomannan is increased, and the maximum quantity of the IL-10 secreted by the macrophages is reached when the concentration reaches 200 mug/mL. As shown in fig. 6-D, the paris polyphylla glucomannan has a remarkable promotion effect on the secretion of TNF-alpha by macrophages, and the stronger the capacity of promoting the secretion of TNF-alpha by the macrophages is as the concentration of the paris polyphylla glucomannan is increased, the paris polyphylla glucomannan is suggested to have the immunopotentiating activity.
Example 4: influence of Paris polyphylla glucomannan on the survival rate of oxidatively damaged skin fibroblastsTest article and reagent
Paris polyphylla glucomannan (PMM): the paris polyphylla glucomannan prepared according to example 1;
DMEM high sugar medium, 0.25% pancreatin EDTA solution: shanghai source culture Biotech stock Co.Ltd;
penicillin, streptomycin, fetal bovine serum: biological Industries company;
phosphate buffered saline PBS: wuhan Seville Biotechnology Co., ltd;
thiazole blue (MTT): beijing Soy Bao technology Co., ltd;
Sterilizing water: self-making in a laboratory;
mouse skin fibroblasts L929: purchased from the cell resource center of the national academy of sciences of life sciences.
Experimental method
1. Solution preparation
Sample mother liquor: weighing rhizoma paridis dextran 10mg, dissolving with sterilized water to 10mg/mL, filtering with 0.22 μm microporous membrane, sterilizing, and preserving at 4deg.C.
MTT mother liquor: prepared into 5mg/mL mother liquor by sterile PBS, preserved in dark at-20 ℃, diluted by serum-free cell culture medium to a final concentration of 0.5mg/mL and filtered by a 0.22 mu m microporous filter membrane before experiments.
2. Cell culture and passage
Cell resuscitation: mouse fibroblast L929 frozen in liquid nitrogen was removed and rapidly placed in a 37℃thermostat water bath to allow complete thawing of the cells in a short period of time. The thawed cells were transferred to a 15mL centrifuge tube containing 4mL DMEM medium, centrifuged at 1000rpm for 5min, and the supernatant was discarded. 2mL of DMEM medium containing 1% (v/v) penicillin-streptomycin diabody and 10% (v/v) fetal bovine serum was added to the centrifuge tube, and the pelleted cells were gently swirled until they were completely suspended to obtain a cell suspension. Transferring the cell suspension to a 10cm dish, washing the centrifuge tube with 2mL of culture medium to ensure complete transfer of the precipitated cells to the dish, maintaining about 10mL of culture medium in the dish, observing cell density under an inverted microscope, and placing CO 2 Culturing in a constant temperature incubator. Culture conditions: 5% CO 2 Culturing at 37deg.C under saturated humidity, and changing liquid the next day.
Cell passage: after resuscitating and culturing the cells, carrying out cell subculture when the cells grow to 90% confluence (90% area of the surface of a culture bottle is covered by the cells), discarding the old culture medium, washing with sterile PBS for 2 times, adding 2mL of DMEM culture medium, gently blowing the cells and fully suspending the adherent cells, inoculating the cells into a new culture bottle, keeping about 10mL of the culture medium in a culture dish, and observing the cell density under an inverted microscope.
3. Effect on L929 cell proliferation
L929 cells were grown at 2X 10 4 The cells/well were plated in 96-well plates and cultured for 24 hours, and fresh medium containing each concentration of Paris polyphylla glucomannan (0, 50. Mu.g/mL, 250. Mu.g/mL, 500. Mu.g/mL) was added to each well, 200. Mu.L of each well was used, and the culture was continued for 24 hours. And (3) discarding the prior culture medium, adding 100 mu L of 0.5mg/mL MTT solution into each hole, continuously culturing for 4 hours at 37 ℃, discarding the culture medium, adding 150 mu L of LDMSO solution into each hole, shaking and mixing uniformly, measuring the absorbance A value at 490nm on an enzyme-labeled instrument, and calculating the cell survival rate. The cell viability was calculated as follows:
4. for H 2 O 2 Effect of induced L929 cell viability
The cells in each well of the 96-well plate are inoculated with 20000 cells, after the cells are cultured for 24 hours, 100 mug/mL, 200 mug/mL and 500 mug/mL of paris polyphylla glucomannan samples are added for treatment for 24 hours, the culture solution is not sucked out, a hydrogen peroxide solution with the final concentration of 800 mug per well is added for 2 hours, the hydrogen peroxide solution is sucked out, and after the MTT (0.5 mg/mL) solution diluted by a serum-free solution is added for 4 hours, the cell viability is measured at 490 nm. Cells without hydrogen peroxide solution were used as control, only hydrogen peroxide solution was used as model, and hydrogen peroxide and polysaccharide were used as experimental, 3 replicates per group.
Experimental results
1. Effect on L929 cell proliferation
Referring to fig. 7-a, compared with the normal control group, the paris polyphylla glucomannan has no significant proliferation or proliferation inhibition effect on L929 cells, indicating that paris polyphylla glucomannan is a safe dose at a concentration of 50-500 μg/mL.
2. For H 2 O 2 Effect of induced L929 cell viability
Referring to fig. 7-B, the survival rate of the hydrogen peroxide-induced model group cells was significantly reduced compared to the normal control group, with only about 25%, indicating that hydrogen peroxide was able to cause severe oxidative damage to L929 cells. Compared with the model group, the paris polyphylla glucomannan group can remarkably increase the cell survival rate, which suggests that paris polyphylla glucomannan can protect skin tissues damaged by oxidation.
Example 6: preparation of paris polyphylla glucomannan freeze-dried powder injection
1. Material
The yunnan paris polyphylla glucomannan prepared according to example 1, pharmaceutical grade sodium chloride.
2. Prescription of prescription
| Material name | Dosage of |
| Paris polyphylla glucomannan | 25g |
| Sodium chloride | 2g |
| Water for injection | 500mL |
| Is co-manufactured into | 1000 pieces |
3. Preparation process
Weighing the prescription amount of the paris polyphylla glucomannan and sodium chloride, adding the water for injection to the full amount, stirring to dissolve completely, and sterilizing by an intermittent hot-pressing method. Adding 0.3% of medicinal active carbon, stirring for 20min, removing carbon with Buchner funnel and 3.0 μm microporous membrane, and filtering to remove heat source. Filtering with microporous membrane of 0.22 μm after the content is qualified, packaging in vial penicillin bottles, each bottle containing 0.5mL, half-pressing, freeze-drying in a freeze-drying oven according to a set freeze-drying curve, pressing, discharging from the oven, capping, inspecting with eyes, and packaging to obtain the final product.
And (3) freeze-drying: feeding the sample into a box, cooling the partition plate to-40 ℃, and keeping the temperature for 4 hours; the cold trap was cooled to-50℃and a vacuum was started to 250 μbar. Sublimation is started: heating to-20deg.C for 1 hr at uniform speed, and maintaining for 3 hr; heating to-10 ℃ at constant speed for 3 hours, maintaining for 8 hours, and maintaining 100-250 mu bar in vacuum; and drying: heating to-5 ℃ for 2 hours, maintaining for 2 hours, and maintaining 150-200 mu bar in vacuum; heating to 10 ℃ for 0.5h, maintaining for 2h, and maintaining 80-100 mu bar in vacuum; raising the temperature to 40 ℃ for 0.5h, keeping the temperature for 4h, and vacuumizing to the minimum.
Example 7: preparation of paris polyphylla glucomannan capsule
1. Material
A paris polyphylla glucomannan, food or pharmaceutical grade starch prepared according to example 1.
2. Prescription of prescription
| Material name | Dosage of |
| Paris polyphylla glucomannan | 100g |
| Starch | 500g |
| Is co-manufactured into | 5000 granules |
3. Preparation process
Weighing the prescription amount of the paris polyphylla glucomannan and the starch, stirring to completely mix, adding a proper amount of talcum powder, granulating by an ethanol wet method, sieving, drying, and filling into a No. 2 capsule shell, wherein each capsule body is filled with 20mg of paris polyphylla glucomannan, so as to obtain the paris polyphylla glucomannan capsule.
Example 8: preparation of paris polyphylla glucomannan oral liquid
1. Material
The method of example 1 gave a caramel flavor as the flavor of paris polyphylla glucomannan, food or pharmaceutical grade flavoring.
2. Prescription of prescription
| Material name | Dosage of |
| Paris polyphylla glucomannan | 25g |
| Sucrose | 0.15g |
| Caramel essence | 0.025g |
| Purified water | 1000mL |
| Is co-manufactured into | 500 counts |
3. Preparation process
Weighing the prescription amount of the paris polyphylla glucomannan, the sucrose and the caramel essence, adding purified water for complete dissolution, filtering with a microporous filter membrane of 0.22 mu m, filling the filtrate according to the amount of 2mL per bottle by an oral liquid filling machine, sealing and sterilizing to obtain the product.
Example 9: preparation of Paris polyphylla glucomannan gel
1. Material
A paris polyphylla glucomannan, cosmetic grade or food grade material prepared according to example 1.
2. Prescription of prescription
| Material name | Dosage of |
| Paris polyphylla glucomannan | 50g |
| Carbomer 940 | 60g |
| Glycerol | 500g |
| Hydroxy-phenyl ethyl ester | 5g |
| Triethanolamine salt | 50g |
| Polysorbate 80 | 20g |
| Propylene glycol | 500mL |
| Purified water | 10L |
3. Preparation process
Weighing the formula amount of paris polyphylla glucomannan, and adding 5L of water for dissolving for standby; uniformly scattering carbomer 940 powder with a prescription amount on the surface of glycerin, fully wetting the carbomer, adding 4L of water to fully swell the carbomer, adding a polysaccharide solution which is dissolved in advance, and uniformly stirring and mixing to obtain a mixed solution I; dissolving ethylparaben in propylene glycol, and slowly adding into the mixed solution I to obtain a mixed solution II; and then the polysorbate 80 and the triethanolamine with the prescription amount are dissolved in 1L of water, added into the mixed solution II under the stirring condition, and canned after being stirred uniformly, the paris polyphylla glucomannan gel is obtained.
Example 10: preparation of paris polyphylla glucomannan facial cleanser
1. Material
The paris polyphylla glucomannan, other cosmetic grade or food grade materials prepared according to example 1.
2. Formulation of
0.5g of paris polyphylla glucomannan, 3.0g of glycerin, 3.0g of butanediol, 0.6g of propylene glycol, 0.1g of EDTA sodium, 0.3g of guar gum, 2.0g of zinc dioxide and C 12 ～C 15 Alcohol benzoate 4.0g, C 12 ～C 20 Alkyl glucoside 3.0g, C 14 ～C 22 Alcohol 0.5g, cetostearyl alcohol 1.2g, nipagin ester 0.2g, sodium stearate 0.3g, dimethiconol 0.5g, polysorbate 0.2g, deionized water 60g.
3. Preparation process
Weighing the formula amount of paris polyphylla glucomannan, and adding purified water for complete dissolution; taking glycerol, butanediol, propylene glycol, EDTA sodium, guar gum, zinc dioxide, sodium stearate, and C 12 ～C 15 Alcohol benzoate, C 12 ～C 20 Alkyl glucosides, C 14 ～C 22 Adding purified water into alcohol and cetostearyl alcohol, and mixing; mixing the nipagin ester, the polydimethylsiloxane alcohol and the polysorbate with purified water. Mixing the above prepared solutions, stirring, and canning.
Example 11: preparation of paris polyphylla glucomannan mask
1. Material
The paris polyphylla glucomannan prepared according to example 1, other cosmetic grade or food grade raw materials.
2. Formulation of
5.0g of paris polyphylla glucomannan, 3.0g of xanthan gum, 3.0g of sodium hyaluronate, 0.3g of propylene glycol, 0.5g of carbomer, 0.1g of nipagin ester, 0.25g of PPG-10 methyl glucose ether, 1.0g of glyceroglycoside and 200g of deionized water.
3. Preparation process
S10: adding deionized water, xanthan gum, sodium hyaluronate, propylene glycol, carbomer and nipagin ester into an emulsifying pot, heating to 70deg.C under stirring, maintaining the temperature after reaching the temperature, continuously stirring until the solution is transparent and uniform, maintaining the temperature for 20 min, and cooling to 40deg.C.
S20: ph=7.4±0.2 was adjusted.
S30: continuously cooling to 35 ℃, adding PPG-10 methyl glucose ether, paris polyphylla glucomannan and glyceroglycosides, continuously stirring uniformly, and discharging after inspection is qualified. Coating with non-woven fabric substrate.
While the present application has been described with reference to a preferred embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the present application. In particular, the technical features mentioned in the respective embodiments may be combined in any manner as long as there is no structural conflict. The present application is not limited to the specific embodiments disclosed herein, but encompasses all technical solutions falling within the scope of the claims.
Claims (11)
1. The paris polyphylla glucomannan is characterized by having a structure shown in the following general formula I:
in the formula I, m and n represent the number of repeating units and are natural numbers, m is independently 1 or 2, and n is more than or equal to 30 and less than or equal to 40.
2. The paris polyphylla glucomannan according to claim 1, wherein the weight average molecular weight of the paris polyphylla glucomannan is 50kDa to 100kDa.
3. The paris polyphylla glucomannan according to claim 1, wherein the monosaccharide composition of paris polyphylla glucomannan comprises glucose and mannose, and the molar ratio of glucose to total monosaccharides is 6.5% -9%.
4. The paris polyphylla glucomannan according to claim 3, wherein the mannose comprises an acetylated mannose substituted with acetyl at the 2-or 3-position of the sugar ring, and the molar ratio of the acetylated mannose to the total monosaccharides is 27.5% -30%.
5. The preparation method of the paris polyphylla glucomannan is characterized by comprising the following steps:
extracting crude polysaccharide from underground rhizome of paris polyphylla;
extracting a component with the weight average molecular weight of 50 kDa-100 kDa from the crude polysaccharide to obtain the paris polyphylla glucomannan.
6. The method for preparing the paris polyphylla glucomannan according to claim 5, wherein,
The extracting of crude polysaccharide from the underground rhizome of paris polyphylla comprises the following steps: extracting underground rhizome of paris polyphylla with warm water or hot water to obtain solution of the crude polysaccharide; and/or
The extracting of the components with the weight average molecular weight of 50 kDa-100 kDa in the crude polysaccharide comprises the following steps:
carrying out enzymolysis on the crude polysaccharide, and carrying out fractional alcohol precipitation to obtain the paris polyphylla glucomannan;
refining the components with the weight average molecular weight of 50 kDa-100 kDa in the paris polyphylla glucomannan to obtain the paris polyphylla glucomannan.
7. The method for preparing the paris polyphylla glucomannan according to claim 6, wherein,
the enzymolysis of the crude polysaccharide, and the fractional alcohol precipitation comprises: performing enzymolysis on the crude polysaccharide with amylase, boiling to inactivate enzyme, collecting clear liquid, grading, precipitating with ethanol, and collecting precipitate; and/or
The refining method comprises one or more of fractional alcohol precipitation, anion exchange chromatography, gel exclusion chromatography, dialysis and ultrafiltration.
8. The method for preparing the paris polyphylla glucomannan according to claim 7, wherein the volume concentration of the amylase is 0.1-0.5%, and/or
The enzymolysis temperature is 60-80 ℃, and/or
The enzymolysis time is 1-3 h, and/or
The boiling and enzyme deactivation time is 10-15 min, and/or
The fractional alcohol precipitation comprises: and (3) taking clear liquid for grading alcohol precipitation, enabling the alcohol concentration to reach 35% -45%, 55% -65% and 75% -85% respectively, and collecting precipitation.
9. A cosmetic or pharmaceutical composition comprising an effective amount of paris polyphylla glucomannan comprising a paris polyphylla glucomannan according to any one of claims 1 to 4 or a preparation method according to any one of claims 5 to 8 and a cosmetically or pharmaceutically acceptable adjuvant.
10. Use of the paris polyphylla glucomannan according to any one of claims 1 to 4 in cosmetics.
11. Use of the paris polyphylla glucomannan according to any one of claims 1 to 4 for preparing a food and/or a drug with modulated immune activity.
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