阅读说明：本技术 一种白及多糖物质及其制备方法与应用 (Bletilla striata polysaccharide substance and preparation method and application thereof ) 是由 何阳 于 2021-10-20 设计创作，主要内容包括：本发明公开了一种白及多糖物质及其制备方法与应用,属于化妆品技术领域。其制备可包括以下步骤：用低元醇对白及鳞茎进行回流提取,将回流提取液经固液分离后得到的固相物与复合酶和水共同酶解提取,将酶解提取液经固液分离后得到的液相物进行第一次醇沉,收集第一次醇沉后的沉淀；除去上述沉淀中的蛋白质,进行第二次醇沉,收集第二次醇沉后的沉淀,复溶,第一次透析,收集截留于透析装置内的物质,得到白及粗多糖。白及粗多糖进一步采用离子交换和葡聚糖凝胶分别纯化可获得白及多糖。上述白及粗多糖和白及多糖均能够具有一定的抑制炎症因子表达以及缓解紫外线造成的皮肤损伤的作用,并表现出良好的保湿功能,可用于制备相应的化妆品或药物。(The invention discloses a bletilla striata polysaccharide substance and a preparation method and application thereof, belonging to the technical field of cosmetics. The preparation method comprises the following steps: carrying out reflux extraction on the white and the bulbs by using low-alcohol, carrying out enzymolysis extraction on a solid phase substance obtained by carrying out solid-liquid separation on a reflux extracting solution together with a complex enzyme and water, carrying out first alcohol precipitation on a liquid phase substance obtained by carrying out solid-liquid separation on an enzymolysis extracting solution, and collecting a precipitate after the first alcohol precipitation; removing protein in the precipitate, performing second alcohol precipitation, collecting precipitate after the second alcohol precipitation, redissolving, performing first dialysis, and collecting substance trapped in the dialysis device to obtain rhizoma Bletillae crude polysaccharide. The rhizoma bletillae crude polysaccharide is further purified by ion exchange and sephadex respectively to obtain the rhizoma bletillae polysaccharide. The rhizoma bletilla crude polysaccharide and rhizoma bletilla polysaccharide have certain effects of inhibiting inflammatory factor expression and relieving skin injury caused by ultraviolet, show good moisturizing function, and can be used for preparing corresponding cosmetics or medicines.)

1. A preparation method of bletilla striata polysaccharide substances is characterized by comprising the following steps: reflux-extracting white and bulb with low-alcohol to remove small molecular substances, subjecting the reflux extract to solid-liquid separation to obtain solid phase, subjecting the solid phase, complex enzyme and water to enzymolysis extraction, subjecting the liquid phase to first alcohol precipitation, and collecting the precipitate after the first alcohol precipitation;

removing protein in the precipitate, then carrying out second alcohol precipitation, collecting the precipitate after the second alcohol precipitation, redissolving, carrying out first dialysis, and collecting substances trapped in a dialysis device to obtain the crude bletilla polysaccharide.

2. The preparation method according to claim 1, wherein the feed-liquid ratio of the low-alcohol to the common bletilla bulb during the reflux extraction is 1g: 8-12mL, and the concentration of the low-polyhydric alcohol is 92-98 vt%;

preferably, the lower alcohol is ethanol;

preferably, the number of reflux extractions is 2-3;

preferably, each reflux extraction is carried out for 2-3h at 20-30 ℃;

preferably, the bletilla striata bulbs are smashed and sieved bletilla striata bulb powder.

3. The preparation method according to claim 1, wherein in the enzymolysis extraction process, the feed-liquid ratio of the solid phase substance to water is 1g: 8-12 mL; the compound enzyme comprises cellulase and papain, wherein the dosage of the cellulase is 1.2 to 1.8 weight percent of the solid phase substance, and the dosage of the papain is 0.8 to 1.2 weight percent of the solid phase substance;

preferably, the enzymatic extraction is carried out at 52-58 deg.C and pH of 5.2-5.8 for 1.5-2.5 h;

preferably, the number of enzymatic extractions is 2-3.

4. The method of claim 1, wherein removing the protein comprises: mixing a polysaccharide solution obtained by dissolving the precipitate obtained after the first alcohol precipitation with a Sevage reagent, centrifuging after protein denaturation, and removing a protein denaturation layer between a polysaccharide solution layer and a Sevage reagent layer;

preferably, the volume ratio of the polysaccharide solution to the Sevage reagent is 2.5-3.5: 1;

preferably, the centrifugation is to shake the mixture of the polysaccharide solution and the Sevage reagent for 25-35min and then centrifuge for 8-12min at the rotating speed of 3800-4200 r/min;

preferably, before removing the protein, washing the precipitate after the first alcohol precipitation by using ethanol with the concentration of 92-98vt percent.

5. The preparation method according to claim 1, wherein the final concentration of the ethanol used in the first alcohol precipitation and the second alcohol precipitation is 78-82 vt% after the ethanol is added into the liquid phase;

preferably, the liquid phase after the enzymatic extraction is concentrated and then subjected to first alcohol precipitation.

6. The preparation method according to claim 1, further comprising purifying the crude bletilla polysaccharide to obtain bletilla polysaccharide;

preferably, the purification comprises: performing primary purification on the crude bletilla striata polysaccharide by adopting an ion exchange column, wherein the primary purification is to perform gradient elution by sequentially using pure water, 0.08-0.12mol/L NaCl solution, 0.28-0.32mol/L NaCl solution and 0.48-0.52mol/L NaCl solution as eluent; collecting the part with the highest active ingredient content in the final eluate to obtain the primarily purified rhizoma bletilla polysaccharide;

preferably, the volume of each eluent in the primary purification process is 1-2 times the volume of the ion exchange column;

preferably, the ion exchange column is a DEAE-52 cellulose anion exchange column;

preferably, the purifying further comprises: purifying the collected part in the purification step again by using a gel column, wherein an eluant used in the purification process is deionized water, and collecting the part with the maximum content of active ingredients in the eluant;

preferably, the elution volume of the eluent is 1-2 times the volume of the gel column during the repurification;

preferably, the gel column is a Sephadex-G100 gel column;

preferably, the method further comprises performing a second dialysis on the re-purified eluate to obtain the final purified bletilla striata polysaccharide;

preferably, the first dialysis and the second dialysis are both performed in a dialysis device with a molecular weight cut-off of 3400-; wherein, the time of the first dialysis and the second dialysis is 48-72 h;

preferably, after both dialyses, the material trapped in the dialysis device is dried separately.

7. A bletilla polysaccharide substance prepared by the preparation method of any one of claims 1 to 6;

preferably, the bletilla striata polysaccharide substance comprises at least one of a bletilla striata crude polysaccharide prepared by the preparation method of any one of claims 1 to 5, a primary purified bletilla striata polysaccharide prepared by the preparation method of claim 6, and a final purified bletilla striata polysaccharide prepared by the preparation method of claim 6.

8. The use of bletilla polysaccharide substance as claimed in claim 7, wherein the bletilla polysaccharide substance is used for the preparation of a cosmetic or a pharmaceutical;

preferably, the bletilla polysaccharide substance is used for preparing a skin care product;

preferably, the bletilla polysaccharide substance is used for preparing a skin care product or a medicament for improving or relieving skin damage caused by ultraviolet rays;

preferably, the bletilla polysaccharide substance is used for preparing skin care products or medicaments for inhibiting the expression of inflammatory factors;

preferably, the bletilla polysaccharide substance is used for preparing a moisturizing skin care product.

9. A cosmetic preparation prepared from the bletilla polysaccharide material of claim 7;

preferably, the cosmetic is a skin care product for inhibiting the expression of inflammatory factors, or, for ameliorating or alleviating H₂O₂Skin care products for induced cell damage, or skin care products for moisturizing.

10. A medicament prepared from the bletilla polysaccharide material of claim 7;

preferably, the drug is a drug for inhibiting the expression of an inflammatory factor, or, for ameliorating or alleviating H₂O₂A medicament for induced cell damage.

Technical Field

The invention relates to the technical field of cosmetics, and particularly relates to a bletilla polysaccharide substance and a preparation method and application thereof.

Background

Bletilla striata (Bletilla) is a perennial herb of orchidaceae, and in China, there are 4 populations of Bletilla (b.striata (Thunb.) reichb. f), Bletilla striata (b.formosana (Hayata) Schltr), Bletilla ochracea (Bletilla ochracea Schltr) and Bletilla striata (Bletilla sinensis (Rolfe) Schltr), which are mainly distributed in Yangtze river basin, Qinling mountain area and the like. The plant has been used as a medicine in China for thousands of years, rhizoma bletillae is collected as a medicinal material in Chinese pharmacopoeia, and rhizoma bletillae, rhizoma bletillae and rhizoma bletillae are used as local articles or substitutes. Several bletilla have medicinal value, and the part used as medicine is dry pseudobulb, and has the functions of astringing to stop bleeding, clearing away heat, promoting diuresis, eliminating swelling, promoting granulation, etc.

Polysaccharides are natural macromolecular polymers with molecular weights of tens of thousands or even millions, are widely present in organisms, and carry important biological information such as nucleic acids and proteins in vital activities. In recent years, more and more plant polysaccharides have been isolated and identified, and it has been found that these plant polysaccharides have various biological activities, such as anti-tumor, immunomodulation, etc.

The bletilla polysaccharide separated by different extraction and purification methods has different effects, and the polysaccharide substances in the bletilla have to be further studied.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a preparation method of a bletilla polysaccharide substance, which is simple and feasible and can be industrially produced, and the bletilla polysaccharide substance which can be used in the field of cosmetics and has certain effects of inhibiting the expression of inflammatory factors, relieving skin injury and preserving moisture can be prepared by the method.

The invention also aims to provide a bletilla striata polysaccharide substance prepared by the preparation method.

The invention also aims to provide application of the bletilla striata polysaccharide substance.

The fourth purpose of the invention is to provide a cosmetic which is prepared from the raw materials comprising the bletilla striata polysaccharide substance.

The fifth purpose of the invention is to provide a medicine prepared from the raw materials including the bletilla striata polysaccharide substance.

The application can be realized as follows:

in a first aspect, the present application provides a method for preparing a bletilla polysaccharide substance, comprising the steps of: reflux-extracting white and bulb with low-alcohol to remove small molecular substances, subjecting the reflux extract to solid-liquid separation to obtain solid phase, subjecting the solid phase, complex enzyme and water to enzymolysis extraction, subjecting the liquid phase to first alcohol precipitation, and collecting the precipitate after the first alcohol precipitation;

removing protein in the precipitate, performing second alcohol precipitation, collecting precipitate after the second alcohol precipitation, redissolving, performing first dialysis, and collecting substance trapped in a dialysis device to obtain rhizoma Bletillae crude polysaccharide.

In an alternative embodiment, in the reflux extraction process, the feed-liquid ratio of the low-alcohol to the common bletilla tuber is 1g: 8-12mL, and the concentration of the lower alcohol is 92-98 vt%.

In an alternative embodiment, the lower alcohol is ethanol.

In an alternative embodiment, the number of reflux extractions is 2-3.

In an alternative embodiment, each reflux extraction is carried out at 20-30 deg.C for 2-3 h.

In an alternative embodiment, the bletilla striata bulbs are pulverized and sieved bletilla striata bulb powder.

In an optional embodiment, in the enzymolysis extraction process, the feed-liquid ratio of the solid phase substance to water is 1g: 8-12 mL; the complex enzyme comprises cellulase and papain, wherein the dosage of the cellulase is 1.2 to 1.8 weight percent of the solid phase substance, and the dosage of the papain is 0.8 to 1.2 weight percent of the solid phase substance;

in an alternative embodiment, the enzymatic extraction is carried out at 52-58 deg.C and pH 5.2-5.8 for 1.5-2.5 h.

In an alternative embodiment, the number of enzymatic extractions is 2-3.

In alternative embodiments, removing the protein comprises: and mixing a polysaccharide solution obtained by dissolving the precipitate obtained after the first alcohol precipitation with a Sevage reagent, centrifuging after protein denaturation, and removing a protein denaturation layer between the polysaccharide solution layer and the Sevage reagent layer.

In an alternative embodiment, the volume ratio of polysaccharide solution to Sevage reagent is 2.5-3.5: 1.

in an alternative embodiment, the centrifugation is performed by shaking the mixture of the polysaccharide solution and Sevage reagent for 25-35min, and then centrifuging the mixture for 8-12min at 3800-4200 r/min.

In an alternative embodiment, the method further comprises washing the precipitate after the first alcohol precipitation with ethanol at a concentration of 92-98 vt% before removing the protein.

In an alternative embodiment, the final concentration of ethanol used in the first and second alcohol precipitations is 78-82 vt% after addition to the liquid phase.

In an alternative embodiment, the liquid phase after the enzymatic extraction is concentrated and then subjected to a first alcohol precipitation.

In an optional embodiment, the method further comprises purifying the crude bletilla striata polysaccharide to obtain the bletilla striata polysaccharide.

In an alternative embodiment, the purification comprises: performing primary purification on the crude bletilla striata polysaccharide by adopting an ion exchange column, wherein the primary purification is to perform gradient elution by sequentially using pure water, 0.08-0.12mol/L NaCl solution, 0.28-0.32mol/L NaCl solution and 0.48-0.52mol/L NaCl solution as eluent; collecting the fraction with the highest active ingredient content in the final eluate.

In an alternative embodiment, the volume of each eluent during the primary purification is 1-2 times the volume of the ion exchange column;

in an alternative embodiment, the ion exchange column is a DEAE-52 cellulose anion exchange column.

In an alternative embodiment, the purifying further comprises: and (3) purifying the part collected in the primary purification step again by using a gel column, wherein an eluent used in the secondary purification process is deionized water, and collecting the part with the highest active ingredient content in the eluent.

In an alternative embodiment, the elution volume of the eluent is 1-2 times the volume of the gel column during the repurification.

In an alternative embodiment, the gel column is a Sephadex-G100 gel column.

In an alternative embodiment, the method further comprises subjecting the re-purified eluate to a second dialysis.

In an alternative embodiment, the first dialysis and the second dialysis are both performed in a dialysis device with a molecular weight cut-off of 3400-; wherein, the time of the first dialysis and the second dialysis is 48-72 h.

In an alternative embodiment, after two dialysations, the material trapped in the dialysis device is dried separately.

In a second aspect, the present application provides a bletilla striata polysaccharide material prepared by the method of any one of the preceding embodiments.

In an alternative embodiment, the bletilla striata polysaccharide substance comprises at least one of the crude bletilla striata polysaccharide, the initial purified bletilla striata polysaccharide and the final purified bletilla striata polysaccharide prepared by the above preparation method.

In a third aspect, the present application provides the use of a bletilla polysaccharide material as in the previous embodiments, for example for the preparation of a cosmetic or pharmaceutical product.

In an alternative embodiment, the bletilla polysaccharide material is used to prepare a skin care product.

In an alternative embodiment, the bletilla polysaccharide material is used in the preparation of a skin care product or medicament for ameliorating or alleviating ultraviolet-induced skin damage.

In an alternative embodiment, the bletilla polysaccharide material is used for preparing a skin care product or a medicament for inhibiting the expression of inflammatory factors.

In an alternative embodiment, the bletilla polysaccharide material is used in the preparation of a moisturizing skin care product.

In a fourth aspect, the present application provides a cosmetic, wherein the raw materials for preparing the cosmetic comprise the bletilla striata polysaccharide substance of the previous embodiment.

In an alternative embodiment, the cosmetic is a skin care product for inhibiting the expression of inflammatory factors, or, for ameliorating or alleviating H₂O₂Skin care products for induced cell damage, or skin care products for moisturizing.

In a fifth aspect, the present application provides a medicament prepared from the bletilla striata polysaccharide material of the previous embodiments.

In alternative embodiments, the drug is a drug for inhibiting the expression of an inflammatory factor, or, for ameliorating or alleviating H₂O₂A medicament for induced cell damage.

The beneficial effect of this application includes:

the novel bletilla striata polysaccharide substance is extracted and separated from the dry pseudobulb of the bletilla striata under specific extraction conditions, has certain effects of inhibiting the expression of inflammatory factors and relieving skin damage caused by ultraviolet rays, shows good moisturizing function, can be used for preparing corresponding cosmetics or medicines, and has high use value and application value.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the DEAE-52 ion exchange column chromatography results of the crude polysaccharide SBSP of bletilla striata of example 2;

FIG. 2 shows the gel column chromatography results of DBSP of the initial purification of bletilla striata and polysaccharide in example 2;

FIG. 3 is a graph showing the molecular weight distribution of BSP-1 polysaccharide from the final purified bletilla striata of example 3;

FIG. 4 shows the results of monosaccharide composition measurement of monosaccharide standard and BSP-1 in example 3;

fig. 5 shows the results of the expression of TNF- α (a), IL-6(B), IL-1 β (C), no (d) inflammatory factors by BSP-1 polysaccharide in example 4, where the data are expressed as mean ± sd, and n is 5; # p <0.05 vs. blank group, # p <0.05 vs. model group;

FIG. 6 shows BSP-1 mitigation for H in example 5₂O₂Graph of induced cellular ROS accumulation; the results are expressed as mean ± standard deviation, n ═ 5; wherein, A represents the change of intracellular ROS detected by a flow cytometer, and B represents the relative content of intracellular ROS; # p<0.05, p compared to blank group<0.05, compared to model set;

FIG. 7 is a graph showing the results of BSP-1 effect on intracellular oxidative stress and antioxidant defense enzymes in example 5; the results are expressed as mean ± standard deviation, n ═ 5; the A picture is the influence of BOP-1 on the content of MDA of the Hacat cell, the B picture is the influence of BSP-1 on the content of SOD of the Hacat cell, the C picture is the influence of BSP-1 on the content of GSH of the Hacat cell, and the D picture is the influence of BSP-1 on the content of CAT of the Hacat cell; # p <0.05 vs. blank group, # p <0.05 vs. model group;

FIG. 8 is a graph of the effect of BSP-1 on desiccation damaged HaCaT cells in example 6; the results are expressed as mean ± standard deviation, n ═ 5; the A picture shows the influence of BSP-1 on the content of AQP-3 in HaCat cells, and the B picture shows the influence of BSP-1 on the content of HA in HaCat cells; # p <0.05 compared to blank group, # p <0.05 compared to model group.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The bletilla striata polysaccharide provided by the application and the preparation method and the application thereof are specifically explained below.

The application provides a preparation method of a bletilla striata polysaccharide substance, which comprises the following steps: reflux-extracting white and bulb with low-alcohol to remove small molecular substances, subjecting the reflux-extracted solution to solid-liquid separation to obtain solid phase, subjecting the solid phase, complex enzyme and water to enzymolysis extraction, subjecting the liquid phase to first alcohol precipitation, and collecting the precipitate.

Removing protein in the precipitate, performing second alcohol precipitation, collecting precipitate after the second alcohol precipitation, redissolving, performing first dialysis, and collecting substance trapped in a dialysis device to obtain rhizoma Bletillae crude polysaccharide.

In a preferred embodiment, the bletilla striata bulbs are pulverized and sieved to obtain bletilla striata bulb powder. Specifically, the raw materials can be pulverized by a high-speed pulverizer and sieved by a 40-mesh sieve.

In the reflux extraction process, the ratio of the low-alcohol to the bletilla striata bulbs can be 1g: 8-12mL, such as 1g: 8mL, 1g: 9mL, 1g:10mL, 1g: 11mL or 1g: 12mL, etc., preferably 1g:10 mL. The concentration of the lower alcohol may be 92 to 98 vt%, preferably 95 vt%. The above-mentioned lower alcohol may be, for example, methanol or ethanol, and preferably ethanol.

The reflux extraction frequency can be only 1 time, or 2-3 times, and can be increased as required. Each reflux extraction can be carried out at 20-30 deg.C for 2-3 h.

And after reflux extraction, performing solid-liquid separation on the reflux extracting solution in a filtering mode, collecting filter residues and drying in the sun. When the reflux extraction times are multiple times, the reflux extracting solutions extracted each time can be respectively subjected to solid-liquid separation and then combined with filter residues, or the reflux extracting solutions extracted each time can be combined firstly and then subjected to solid-liquid separation in a unified manner, and the filter residues are collected.

And then carrying out enzymolysis extraction on the filter residue, complex enzyme and water together.

In the enzymolysis extraction process, the feed-liquid ratio of solid phase substances (namely filter residues) to water can be 1g: 8-12mL (preferably 1g:10 mL).

In the application, the complex enzyme comprises cellulase and papain, wherein the dosage of the cellulase is 1.2-1.8 wt% (preferably 1.5 wt%) of the filter residue, and the dosage of the papain is 0.8-1.2 wt% (preferably 1 wt%) of the filter residue.

It should be noted that in the complex enzyme used in the present application, the cellulase is a specific enzyme, which can hydrolyze cell walls of bletilla striata bulbs into glucose, so that the contents thereof can be more easily dissolved and extracted. In addition, the cellulase only destroys the cell walls of the bletilla striata bulbs, and cellulose substances are not contained in the substances in the cells of the bletilla striata bulbs, so that the cellulase does not generate adverse effects on the component structures of the contents in the bletilla striata and the bulbs. Compared with other proteases, the papain has higher removal capability on protein substances in white and bulbs, has mild action conditions and does not damage polysaccharide structures.

For reference, the above enzymatic extraction can be carried out at 52-58 deg.C and pH of 5.2-5.8 for 1.5-2.5 h. Preferably, the reaction can be carried out at 55 ℃ and pH5.5 for 2 hours. The times of enzymolysis extraction can be only 1 time, also can be 2-3 times, and can be increased according to the needs. When the times of enzymolysis extraction are multiple times, the liquid phase substances can be combined after solid-liquid separation (such as adopting a centrifugal mode) of the enzymolysis liquid obtained after each extraction, or the liquid phase substances can be collected after the enzymolysis liquid obtained after each extraction is combined and then is subjected to solid-liquid separation (such as adopting a centrifugal mode).

And then carrying out first alcohol precipitation on the liquid phase substance, and collecting the precipitate after the first alcohol precipitation. In this process, the alcohol used is ethanol, which is added to the liquid phase in a final concentration of 78-82 vt% (preferably 80 vt%). In a preferred embodiment, the liquid phase after the enzymatic extraction may be concentrated before the first alcohol precipitation.

Further, the protein in the precipitate after the first alcohol precipitation was removed. It is referred to that the precipitate after the first alcohol precipitation is dissolved (for example, it can be dissolved by distilled water), and the polysaccharide solution obtained by the dissolution is mixed with Sevage reagent, and after the protein denaturation, it is centrifuged to remove the protein denaturation layer located between the polysaccharide solution layer and Sevage reagent layer. The process can effectively remove protein contained in rhizoma Bletillae bulb, and improve extraction purity of polysaccharide substance.

For reference, the volume ratio of the above polysaccharide solution to the Sevage reagent may be 2.5 to 3.5: 1, preferably 3: 1. The centrifugation can be carried out by shaking (vigorous shaking) the mixture of the polysaccharide solution and Sevage reagent for 25-35min (preferably 30min), and then centrifuging at 3800-4200r/min (preferably 4000r/min) for 8-12min (preferably 10 min). After the above operation, the mixture was divided into three distinct layers, the protein layer was located between the polysaccharide solution and the Sevage reagent layer. Preferably, the sugar solution in the upper layer is aspirated and the above-described protein removal operation is repeated until there is no intermediate protein-denatured layer.

In a preferred embodiment, the first precipitation step is further followed by washing (e.g. 3 times) the precipitate with ethanol at a concentration of 92-98 vt% (e.g. 95 vt%).

Further, the protein-removed polysaccharide solution is subjected to a second alcohol precipitation. The alcohol used in the second alcohol precipitation may also be ethanol, which is added to the liquid phase to a final concentration of 78-82 vt% (e.g., 80 vt%). Centrifuging and collecting the precipitate.

Further, the precipitate after the second alcohol precipitation is re-dissolved with distilled water, and loaded into a dialysis device (such as dialysis bag) with cut-off molecular weight of 3400-. Preferably, the first dialysis session is performed with every 3h of deionized water for the first 12h, followed by a change of deionized water in the morning, in the evening.

After the first dialysis, the substances trapped in the dialysis apparatus are concentrated and dried (for example, freeze-dried by a vacuum freeze-dryer) to obtain crude bletilla striata polysaccharide (SBSP in this application indicates crude bletilla striata polysaccharide).

Further, purifying the crude bletilla striata polysaccharide to obtain the purified bletilla striata polysaccharide.

By reference, purification includes: performing primary purification on the crude bletilla striata polysaccharide by adopting an ion exchange column, wherein the primary purification is to perform gradient elution by sequentially using pure water, 0.08-0.12mol/L NaCl solution, 0.28-0.32mol/L NaCl solution and 0.48-0.52mol/L NaCl solution as eluent; collecting the fraction with the highest content of active ingredients in the final eluate to obtain the primarily purified rhizoma Bletillae polysaccharide (DBSP in the present application refers to rhizoma Bletillae crude polysaccharide).

Preferably, the primary purification is performed by gradient elution using pure water, 0.1mol/L NaCl solution, 0.3mol/L NaCl solution and 0.5mol/L NaCl solution as eluents in this order.

In the above-mentioned preliminary purification, the volume of each eluent is 1 to 2 times (preferably 1.5 times) the volume of the ion exchange column. The size of the ion exchange column was 2.6cm (diameter) x 30cm (height of packing).

In the present application, the ion exchange column is preferably a DEAE-52 cellulose anion exchange column. The anion exchange column is suitable for separating various acidic and neutral polysaccharides and mucopolysaccharides. DEAE cellulose anion exchange columns allow the polysaccharide to freely enter its carrier and diffuse rapidly compared to DEAE dextran anion exchange columns and DEAE sepharose anion exchange columns. Compared with DEAE-22 cellulose anion exchange column, DEAE-23 cellulose anion exchange column, DEAE-32 cellulose anion exchange column and DEAE-11 cellulose anion exchange column, the DEAE-52 cellulose anion exchange column has stronger adsorption capacity to protein in white and bulb, and is used for separating and purifying polysaccharide substances of bletilla striata, so that not only can neutral polysaccharide be separated from acidic polysaccharide, but also unclean protein in polysaccharide can be adsorbed on the column, thereby achieving better separation and purification effects.

In the application, the part with the maximum active ingredient content in the final eluent is collected and monitored by adopting a phenol-sulfuric acid method, an elution curve is drawn under the wavelength of 490nm, and the part corresponding to the maximum peak area of the polysaccharide is collected. And then, concentrating the collected part, and freeze-drying to obtain the primarily purified bletilla striata polysaccharide DBSP.

Further, the purification of the present application may further comprise: and (3) purifying the part collected in the primary purification step again by using a gel column, wherein an eluent used in the secondary purification process is deionized water, and collecting the part with the highest active ingredient content in the eluent.

In the above-mentioned re-purification process, the elution volume of the eluent is 1 to 2 times (preferably 1.5 times) the volume of the gel column. The gel column size was 1.6cm (diameter) x 130cm (height of packing).

In the present application, Sephadex-G100 gel column is preferably used as the gel column. The type of the gel column is used specifically based on the molecular weight range of the polysaccharide obtained by the primary purification in the application, so that the polysaccharide can be effectively separated according to the size of the molecular weight.

Similarly, the part with the highest content of active ingredients in the eluate can be collected by monitoring with phenol-sulfuric acid method, and collecting the main absorption peak. Subsequently, the above-collected fractions are concentrated (which may be carried out using a rotary evaporator).

Further, the eluate after the second purification is subjected to a second dialysis to obtain a final purified bletilla striata polysaccharide (herein BSP-1 denotes the final purified bletilla striata polysaccharide). Through determination and comparison, the finally purified bletilla polysaccharide BSP-1 is a novel polysaccharide extracted from bletilla for the first time.

For reference, the second dialysis can be performed in a dialysis device (e.g., dialysis bag) having a molecular weight cutoff (e.g., 3500 Da). The second dialysis time can also be 48-72 h. Preferably, the deionized water is changed every 3 hours before the first 12 hours of the second dialysis process, and then changed every morning, noon and evening.

In an alternative embodiment, after two dialyzings, the material trapped in the dialysis device is dried separately (e.g., freeze-dried with a freeze-dryer).

Accordingly, the application provides a bletilla striata polysaccharide substance prepared by the preparation method.

The rhizoma bletillae polysaccharide substance comprises at least one of the rhizoma bletillae crude polysaccharide, the initial purified rhizoma bletillae polysaccharide and the final purified rhizoma bletillae polysaccharide prepared by the preparation method. That is, the bletilla striata polysaccharide substance may refer to the bletilla striata crude polysaccharide (SBSP), the primary purified bletilla striata polysaccharide (DBSP) or the final purified bletilla striata polysaccharide (BSP-1) referred to in the present application, and does not exclude the mixture including any two or three of SBSP, DBSP and BSP-1.

In addition, the application also provides application of the bletilla striata polysaccharide substance, for example, the bletilla striata polysaccharide substance can be used for preparing cosmetics or medicines.

The cosmetic can be skin care product, and other types of cosmetic.

In some preferred embodiments, the bletilla polysaccharide substance can be used for preparing skin care products or medicaments for improving or relieving skin damage caused by ultraviolet rays, and also can be used for preparing skin care products or medicaments for inhibiting the expression of inflammatory factors; can also be used for preparing skin care products with moisturizing effect.

Correspondingly, the application also provides a cosmetic, and the preparation raw materials of the cosmetic comprise the bletilla striata polysaccharide substance. The cosmetic is skin care product for inhibiting inflammatory factor expression, or improving or relieving H₂O₂Skin care products for induced cell damage, or skin care products for moisturizing.

Correspondingly, the application also provides a medicament, and the preparation raw materials of the medicament comprise the bletilla striata polysaccharide substance of the previous embodiment. The above drugs are drugs for inhibiting the expression of inflammatory factors, or for ameliorating or alleviating H₂O₂A medicament for induced cell damage.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Preparation of bletilla crude polysaccharide SBSP

Step (1): taking 1.0g of Bletilla striata (Bletilla striata) bulb, crushing by a high-speed crusher, sieving by a 40-mesh sieve to obtain Bletilla striata powder, extracting by refluxing with 95 vt% ethanol water solution with the material-liquid ratio of 1g:10mL for 3 times, filtering, collecting filter residue and drying in the sun.

Step (2): performing enzymolysis extraction on the filter residue in the step (1) twice under the conditions of 55 ℃, material-liquid ratio of 1g:10mL, pH5.5 and enzymolysis time of 2h by adopting an enzyme-assisted water extraction method, wherein a complex enzyme consisting of cellulase and papain is added in the enzymolysis process, wherein the dosage of the cellulase is 1.5 wt% of the weight of the filter residue, the dosage of the papain is 1 wt% of the weight of the filter residue, and merging the filtrate after centrifugation.

And (3): concentrating the filtrate in the step (2), carrying out first alcohol precipitation by using ethanol with the final concentration of 80 vt%, centrifuging and collecting the precipitate.

And (4): the precipitate in step (3) was washed 3 times with 95 vt% ethanol.

And (5): dissolving the precipitate washed in the step (4) with distilled water, and mixing the polysaccharide solution and Sevage reagent according to a volume ratio of 3:1, shaking vigorously for 30min, and centrifuging at 4000r/min for 10 min. The mixture was seen to be divided into three distinct layers, the protein layer being intermediate the polysaccharide solution and Sevage reagent. Carefully suck out the supernatant sugar solution, and repeat the above protein removal step until there is no intermediate protein denaturation layer.

And (6): and (4) carrying out secondary alcohol precipitation on the polysaccharide solution from which the protein is removed in the step (5) through 80 vt% ethanol, and centrifuging to obtain a precipitate.

And (7): redissolving the precipitated distilled water obtained in the step (6), filling the redissolved precipitated distilled water into a dialysis bag with the molecular weight cutoff of 3500Da for carrying out first dialysis for 72h (the deionized water needs to be changed every 3h in the first 12h, and then the deionized water needs to be changed every morning, noon and evening), concentrating after the dialysis is finished, and carrying out freeze drying by a vacuum freeze dryer to obtain crude bletilla polysaccharide SBSP.

Example 2

Purification of crude bletilla polysaccharide SBSP

Step (1): preparing 50mg/mL polysaccharide solution from crude bletilla polysaccharide SBSP, centrifuging at 4000r/min for 10min, primarily purifying by using a DEAE-52 ion exchange column (2.6 multiplied by 30cm), and carrying out gradient elution by using deionized water, 0.1mol/L NaCl solution, 0.3mol/L NaCl solution and 0.5mol/L NaCl solution in sequence, wherein the elution volume of each gradient is 1.5 times of the column volume. Monitoring with phenol-sulfuric acid method, drawing elution curve at 490nm wavelength to obtain four polysaccharide parts (as shown in figure 1), the first polysaccharide part has the largest peak area, which indicates that the peak is the most abundant part of the four parts, so the first peak is collected, concentrated, and freeze-dried to obtain the initial purified rhizoma bletilla polysaccharide (DBSP).

Step (2): preparing the primarily purified bletilla striata polysaccharide (DBSP) in the step (1) into 30mg/mL, further purifying 5mL of the primarily purified bletilla striata polysaccharide (DBSP) by a Sephadex-G100(1.6 multiplied by 130cm) column through a Sephadex column, eluting with deionized water, wherein the elution volume is 1.5 times of the column volume, collecting by an automatic collector, and monitoring by a phenol-sulfuric acid method. Collecting the main absorption peaks, concentrating with rotary evaporator, performing second dialysis for 72h with dialysis bag with molecular cut-off of 3500Da (the first 12h requires changing deionized water every 3h, and then changing deionized water every morning, evening), and freeze-drying with freeze-dryer to obtain the final purified rhizoma Bletillae polysaccharide (BSP-1) (as shown in FIG. 2).

Example 3

Structural identification and analysis of polysaccharide BSP-1

(1) Molecular weight determination of polysaccharide BSP-1

Measuring molecular weight distribution by using a high-efficiency gel permeation chromatography system, wherein a chromatographic column is a Shodex OH-pak SB-804HQ column (8mm multiplied by 300 mm); the detector is a parallax detector RID and a Diode Array Detector (DAD); the column temperature is 35 ℃; the sample volume is 30 mu L; the mobile phase is 0.1mol/L NaCl; the flow rate was 0.5 mL/min. D series dextran standard products (the molecular weights are respectively D0-180Da, D1-2.7KDa, D2-5.25KDa, D3-9.75KDa, D4-13.05KDa, D5-36.8KDa, D6-64.65KDa, D7-135.35KDa, D8-300.6KDa and D2000-2000KDa) with different molecular weights are prepared into standard solution with the concentration of 1mg/ml, after the standard solution is measured by the high-efficiency gel permeation chromatography system, a retention time is horizontal coordinates, the weight average molecular weight Mw is vertical coordinates to make a standard curve, and after the standard curve is corrected by GPC software, the weight average molecular weight of the polysaccharide BSP-1 is 15670Da, the number average molecular weight is 10800Da, the polydispersity number (Mw/Mn) is 1.45, and the molecular weight distribution range of the polysaccharide BSP-1 is shown.

As shown in FIG. 3, the distribution curve of molecular weight integral (Ht%) shows that the sum of all polysaccharide fragments with molecular weight ≤ 1000Da in polysaccharide BSP-1 is about 0% of all polysaccharide fragments, and the mass percentage of polysaccharide fragments with smaller molecular weight is accumulated with increasing molecular weight, and all polysaccharide fragments with molecular weight ≤ 40000Da in polysaccharide BSP-1 are about 100% of all polysaccharide fragments.

(2) Monosaccharide composition determination of polysaccharide BSP-1

Hydrolyzing polysaccharide BSP-1 and each monosaccharide standard substance by 2mol/L TFA 2mL at 110 ℃ for 4h, cooling to room temperature, adjusting the pH value to be neutral by using 2mol/L NaOH, and then analyzing by adopting high performance liquid chromatography, wherein a chromatographic column is Agilent Zorbax SB-C18(150mm multiplied by 4.6mm), and the column temperature is 35 ℃; the mobile phase A is 15% acetonitrile + 85% KH₂PO₄NaOH buffer (pH 6.9), mobile phase B40% acetonitrile + 60% KH₂PO₄NaOH buffer (pH 6.9); the flow rate is 1 mL/min; elution gradient: 0-25min, 0-25% B; 25-30min, 100% A. The results of the monosaccharide composition measurements of the monosaccharide standard and BSP1 are shown in fig. 4. And (3) determining the molar ratio according to the retention time of the standard monosaccharide and an external standard method to obtain the mannose-to-glucose ratio of the BSP-1 monosaccharide composition to be 2: 1.

(3) Infrared spectrometry of polysaccharide BSP-1

Polysaccharide BSP-1 was prepared into KBr pellets and scanned by a Bruker Tensor 27 infrared spectrometer at 4000-^-1Obtaining an infrared absorption spectrogram according to the spectral signals in the range.

TABLE 1 FT-IR assignment of BSP-1

The FT-IR assignments for BSP-1 are shown in Table 1, with the results shown: BSP-1 at 3385cm^-1Wide and strong absorbance bonds appear at the positions, which are caused by O-H stretching vibration; 2877cm^-1And 1373cm^-1Absorbing C-H stretching vibration and bending vibration which are classified as pyranose; 1726cm^-1And 1242cm^-1The absorption of (b) is caused by C ═ O valence vibration and C — O vibration of the O-acetyl group. 1056cm^-1The absorption peak of (a) represents the presence of pyranoside. Further, according to the conventional document, 873cm^-1The weak peak of (a) is due to mannose residues.

(4) GC-MS methylation analysis of polysaccharide BSP-1

Weighing a polysaccharide sample, dissolving 5mg in 1mL of dimethyl sulfoxide (DMSO), adding 50mg of potassium hydroxide powder (KOH), magnetically stirring until the KOH is dissolved, adding 0.9mL of iodomethane, carrying out ultrasonic reaction for 1h under the condition of nitrogen sealing, adding 2mL of water after the reaction to decompose the iodomethane to terminate the reaction, and extracting by using dichloromethane to obtain the methylated polysaccharide. The methylated product was dissolved by adding 1mL of a 2mol/L TFA solution, and the methylated polysaccharide was subjected to acid hydrolysis at 110 ℃ for 4 h. Then, adjusting the pH value to 10 by using 2mol/L KOH solution, adding 25mg of sodium borohydride, stirring and reducing in a water bath at 50 ℃ for 2 hours, adding glacial acetic acid to stop the reaction, and freeze-drying. Analysis was performed by GC-MS, HP-5MS quartz capillary column (50 mm. times.0.25 mm); column temperature: the initial temperature is 150 ℃, the temperature is programmed to rise to 200 ℃ at a speed of 2 ℃/min, then the temperature is raised to 280 ℃ at a speed of 5 ℃/min, and the temperature is kept for 20 min; the column flow rate is 1.0 mL/min; the temperature of a sample inlet is 250 ℃; the front column pressure is 100 kPa; the split ratio is 10: 1; the carrier gas was high purity helium, and the sample size was 1 μ L. MS conditions: an ionization mode EI; electron energy 70; the transmission line temperature is 290 ℃; the ion source temperature is 230 ℃; the temperature of the quadrupole rods is 150 ℃; the mass range is 50-600; and comparing with a CCRC spectrum database to judge the type of the glycosidic bond.

TABLE 2 GC-MS signal assignment of BSP-1 methylated products

Methylated sugar	Sugar chain type	Retention time	Molar ratio of	Ion fragment (m/z)
					2,3,4,6-Me4-Glc	terminal Manp	19.62	1.01	71,87,101,118,129,145,161,205
2,3,4,6-Me4-Man	terminal Glcp	19.72	1.00	71,87,101,118,129,145,161,205
					2,3,6-Me3-Glc	1,4-linked Manp	21.67	2.47	71,87,99,101,117,129,145,161,233
2,3,6-Me3-Man	1,4-linked Glcp	21.78	2.19	71,87,101,117,129,145,161,233

As shown in Table 2, each of the sugar alcohol acetate derivatives of BSP-1 was 2,3,4, 6-tetra-O-methyl-1, 5-di-O-acetyl-mannitol, 2,3,4, 6-tetra-O-methyl-1, 5-di-O-acetyl-glucitol, 2,3, 6-tri-O-methyl-1, 4, 5-tri-O-acetyl-mannitol and 2,3, 6-tri-O-methyl-1, 4, 5-tri-O-acetyl-glucitol, respectively, in a molar ratio of 1.01:1.00:2.47: 2.19.

The sugar chain may contain the following structural fragments:

[Glc-1→4-β-D-Man-1→4-β-D-Man-1→4-β-D-Glc-1→4-β-D-Man-1→4-β-D-Glc-1→4-β-D-Man→]。

example 4

Evaluation of anti-inflammatory Activity of polysaccharide BSP-1

(1) Recovery and culture of RAW264.7 cells: taking out RAW264.7 cells from a liquid nitrogen tank, putting the cells into a water cup at about 37 ℃ for quick thawing, quickly transferring the cells into a 15mL centrifuge tube in a biological safety cabinet, adding 4mL of complete culture medium, uniformly blowing and beating, and centrifuging for 3min at 1000 rmp. Pouring out the supernatant, adding 2mL of culture medium into the centrifuge tube, blowing uniformly, transferring into a cell culture dish, and culturingSupplementing nutrient medium to 10mL, shaking, and placing in cell culture box (5% CO)₂Cell culture was carried out at 37 ℃. Observing the cell state on time, and carrying out cell passage when the cells grow to be about 80-90% in the culture dish. When the cell density reaches the third generation, a cell counter is used for counting, the cell survival rate and the cell state are observed, and subsequent experiments can be carried out when the cell survival rate is more than 95% after the cell density is adjusted.

(2) MTT cell viability assay: the cell density was adjusted to 1.0X 10⁵One per mL, the plate was attached to wall in 96 wells, 200. mu.L per well, 24h later, bletilla striata polysaccharide BSP-1 was diluted with medium to 200, 100 and 50 (. mu.g/mL), 5 parallel groups each, and cultured for 20 h. Adding 100 mu L of MTT solution with the concentration of 0.5mg/mL, adding 100 mu L of DMSO solution after 4h, fully shaking for 10min in a dark place by using a cell plate shaking instrument, and measuring the light absorption value of each hole at 570nm by using a microplate reader.

(3) Determination of NO and inflammatory factor content: the inhibition effect of bletilla striata polysaccharide BSP1 on excessive NO induced by LPS and inflammatory factor production under 150 mug/mL, 100 mug/mL and 50 mug/mL is determined according to the method of the instruction of the determination kit.

As shown in fig. 5, the expression of proinflammatory factors in the RAW264.7 cell model group induced by LPS is obviously increased, and has a significant difference (p <0.05) compared with the blank group, while the expression of inflammatory factors is obviously inhibited in the polysaccharide group subjected to polysaccharide BSP-1 stem prognosis; the high-dose polysaccharide BSP-1 has better inhibition effect on TNF-alpha, IL-6 and IL-1 beta, wherein the inhibition effect on IL-6 is most obvious; therefore, it is speculated that the polysaccharide BSP-1 can effectively inhibit the expression of inflammatory factors, thereby achieving the anti-inflammatory effect.

Example 5

Polysaccharide BSP-1 protection H₂O₂Induced cell injury assay

The recovery and cell viability of the HaCaT cells were determined as in examples 4(1) and (2). Culturing HaCaT cell in a nontoxic dose for 24H, adding polysaccharide BSP-1 of 150 μ g/mL, 100 μ g/mL and 50 μ g/mL, and culturing with HaCaT cell for 24H, discarding old culture solution, washing with PBS solution for 1-2 times, and adding H of 0.7mmol/L₂O₂Culturing in solution for 24h to induce cell aging, and finally determining the activity of intracellular active oxygen and antioxidase according to the instruction in the kit.

Reactive Oxygen Species (ROS) are free radicals produced mainly by mitochondria and are closely related to normal physiological functions and human diseases. The production and clearance of ROS is in a state of homeostasis to maintain normal physiological function. However, when the level of reactive oxygen species produced by the cell exceeds the self-scavenging capacity, oxidative stress occurs, resulting in a series of chronic diseases. To study the effect of BSP-1 on Hacat intracellular ROS, we first detected intracellular ROS levels by flow cytometry using a DCFH-DA fluorescent probe. As shown in FIG. 6, APAP treatment H compared to the control group₂O₂The cells cause an increase in the ROS content (p) in Hacat cells<0.05). However, BSP-1 significantly inhibited H in a dose-dependent manner compared to the model group₂O₂Induced intracellular ROS accumulation (p)<0.05)。

Malondialdehyde (MDA) is an indicator of lipid peroxidation, and superoxide dismutase content (SOD) and Catalase (CAT) are two important antioxidant enzymes that protect cell integrity during biological oxidation. Glutathione (GSH) is considered the first barrier against oxidative stress. To determine the effect of BSP-1 on oxidative stress and antioxidant enzymes in Hacat cells, we examined changes in Hacat intracellular MDA, GSH, CAT and SOD. As shown in FIG. 7, H is compared with that of the control group₂O₂Significant induction of intracellular MDA increase and intracellular GSH, CAT and SOD decrease (p)<0.05). Notably, BSP-1 pretreatment significantly induced a decrease in intracellular MDA and an increase in intracellular GSH, CAT and SOD (p) in a dose-dependent manner<0.05). Shows that BSP-1 inhibits the accumulation of intracellular ROS by regulating intracellular oxidative stress kinase and antioxidant defense enzyme so as to protect Hacat cells from H₂O₂Induced damage.

Example 6

Moisturizing Activity of polysaccharide BSP-1

Diluting HaCaT cells with good growth state to 3 × 10⁵Adding the cells/mL into a 6-well plate according to the volume of 2mL per well for culturing for 24hThen, the cell sap is sucked out to establish a drying model, the drying air speed is set to be 0.6m/s, after drying is carried out for 25min, culture media respectively containing 150 mu g/mL, 100 mu g/mL and 50 mu g/mL polysaccharide BSP-1 are added to continue culturing the cells for 24h, and finally cell culture supernatant is collected to measure the contents of aquaporin AQP-3 and hyaluronic acid HA according to the kit steps.

As can be seen from FIG. 8, at higher experimental concentrations, after the HaCat cells subjected to dry damage are treated with polysaccharide BSP-1 with different concentrations, the content of AQP-3 is higher, and the AQP-3 is increased in a dose-dependent manner, so that the AQP content is remarkably improved compared with that of a model group. Similarly, the HA content also increases with the increase of the polysaccharide concentration, and HAs a significant difference compared with the model group, which indicates that the polysaccharide BSP-1 can promote the secretion of aquaporin AQP-3 and hyaluronic acid HA by HaCat cells after drying treatment. Physiological regulators such as AQP-3 and HA help the skin to keep water balance, so whether the sample HAs the moisturizing effect can be judged according to the content of AQP-3 and HA secreted by HaCat cells of the immortalized stratum corneum of a human.

In conclusion, the polysaccharide BSP-1 of bletilla striata is extracted and separated from the dry pseudobulb of bletilla striata, and pharmacological experiments show that the prepared polysaccharide BSP-1 can inhibit the expression of inflammatory factors and skin damage caused by ultraviolet rays in a dose-dependent manner, shows a good moisturizing function, and can be applied to preparing skin care cosmetics.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.