CN114106212B - Bletilla ochracea polysaccharide as well as preparation method and application thereof - Google Patents
Bletilla ochracea polysaccharide as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides bletilla ochracea polysaccharide and a preparation method and application thereof, and relates to the technical field of bletilla ochracea polysaccharide. A preparation method of bletilla ochracea polysaccharide comprises the following steps: s1: pulverizing rhizoma Bletillae pseudobulb, and defatting; s2: cold soaking in ethanol, filtering, and collecting residue; s3: mixing the filter residue with complex enzyme, performing ultrasonic treatment in hot water, precipitating with ethanol, centrifuging, and collecting precipitate; s4: washing and deproteinizing the precipitate; s5: precipitating with ethanol, dialyzing, concentrating, and freeze-drying to obtain crude polysaccharide SBOP of rhizoma Bletillae. The invention also provides the bletilla ochracea polysaccharide prepared by the method and application thereof. The invention can prepare the crude polysaccharide SBOP and the micromolecular water-soluble polysaccharide BOP-1, both the crude polysaccharide SBOP and the micromolecular water-soluble polysaccharide BOP-1 can have certain effects of inhibiting the expression of inflammatory factors and relieving skin damage caused by ultraviolet rays, show good moisturizing function and can be used for preparing corresponding cosmetics or medicines.
Description
Technical Field
The invention relates to the technical field of bletilla ochracea polysaccharide, and particularly relates to bletilla ochracea polysaccharide and a preparation method and application thereof.
Background
The skin, the largest organ of the human body, is the first barrier against external attack and irritation. However, in daily life, natural factors such as air pollution and ultraviolet exposure, skin problems such as roughness, sensitivity and fragility bring serious troubles to people, skin care products which adopt synthetic chemicals as raw materials not only consume a large amount of resources, but also generate a series of byproducts, and simultaneously treat symptoms and do not cure the root causes, so that the skin is seriously burdened, and the safety of the skin care products is also a big problem, so that the concept of 'returning to nature, green and environment protection' rapidly spreads the whole cosmetic industry, and the research and development of safe, effective and environment-friendly skin care products are urgently needed.
Polysaccharide is a natural macromolecular polymer with molecular weight of tens of thousands or even millions, widely existing in organisms, and carrying important biological information such as nucleic acid and protein in life 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, immunoregulatory, antioxidant and anti-inflammatory effects. In the aspect of skin protection, plant polysaccharide or fungal polysaccharide has good biological functions in the aspects of preventing water loss of skin, maintaining cuticle and epidermis barriers, inhibiting melanin generation and the like. Therefore, more and more plant polysaccharides are applied to skin protection cosmetic raw materials, and have good and wide development prospects.
Rhizoma bletillae is a traditional Chinese medicine in China, has the effects of tonifying lung, reducing swelling, promoting granulation, stopping bleeding, healing sore and the like, and is used for treating symptoms such as lung injury hemoptysis, incised wound bleeding, ulcer pain, soup fire burn, rhagadia manus et pedis and the like. The Chinese-made common Bletilla pseudobulb includes four kinds of common Bletilla pseudobulb (Bletilla sinensis), small common Bletilla pseudobulb (Bletilla formosana), common Bletilla pseudobulb (Bletilla striata) and common Bletilla pseudobulb (Bletilla ochracea). Folk drug application survey shows that the bletilla ochracea is equivalent to the bletilla striata in terms of drug effect.
The prior art mainly uses the bletilla ochracea pseudobulb to prepare the bletilla ochracea polysaccharide (the structure analysis and the anti-tumor activity research of the bletilla ochracea polysaccharide) by water extraction, alcohol precipitation, repeated freeze thawing and protein removal and decolorization]Shaanxi university 2017, 5.), however, the extracted bletilla ochracea polysaccharide has a molecular weight of 4.9 × 10 5 The molecular weight is large, the water solubility is not easy, and the subsequent application in the production of products is not facilitated.
Disclosure of Invention
The invention aims to provide a preparation method of bletilla ochracea polysaccharide, which can be used for preparing crude bletilla ochracea polysaccharide SBOP and micromolecular water-soluble bletilla ochracea polysaccharide BOP-1, is beneficial to the production of subsequent products, can have certain effects of inhibiting the expression of inflammatory factors and relieving skin damage caused by ultraviolet rays, shows a good moisturizing function, and can be used for preparing corresponding cosmetics or medicaments.
The invention also provides a preparation method of the bletilla ochracea polysaccharide, and the bletilla ochracea polysaccharide comprises SBOP (crude bletilla ochracea polysaccharide) and BOP-1 (small molecular water-soluble bletilla ochracea polysaccharide), has certain effects of inhibiting the expression of inflammatory factors and relieving skin damage caused by ultraviolet rays, shows a good moisturizing function, and can be used for preparing corresponding cosmetics or medicines.
The third purpose of the invention is to provide an application of the bletilla ochracea polysaccharide, namely crude bletilla ochracea polysaccharide SBOP and micromolecular water-soluble bletilla ochracea polysaccharide BOP-1 prepared by the preparation method are applied to skin care products or medicines.
The embodiment of the invention is realized by the following technical scheme:
a preparation method of bletilla ochracea polysaccharide comprises the following steps:
s1: taking rhizoma bletilla pseudobulb, crushing, and performing degreasing pretreatment;
s2: removing polyphenol and flavonoid fat-soluble components from the pretreated bletilla ochracea powder by using an ethanol solution, collecting filter residues and drying;
s3: mixing the filter residue obtained in the step S2 with a complex enzyme, carrying out ultrasonic extraction in hot water, carrying out alcohol precipitation, centrifuging and collecting precipitate to obtain crude bletilla ochracea polysaccharide;
s4: washing and deproteinizing the crude bletilla ochracea polysaccharide;
s5: and carrying out alcohol precipitation, dialysis, concentration and freeze drying on the protein-removed rhizoma bletillae crude polysaccharide solution to obtain rhizoma bletillae crude polysaccharide SBOP.
Further, the method also comprises the step S6: performing primary purification on crude bletilla ochracea polysaccharide SBOP by an ion exchange column, monitoring the polysaccharide concentration in a chromatographic feed liquid by using a phenol-sulfuric acid method, collecting and concentrating the chromatographic feed liquid with the highest polysaccharide concentration, further purifying by using a gel column, monitoring the polysaccharide concentration in the chromatographic feed liquid by using a phenol-sulfuric acid method, collecting and concentrating the chromatographic feed liquid with the highest polysaccharide concentration, dialyzing, and freeze-drying to obtain bletilla ochracea polysaccharide BOP-1.
Further, the bletilla ochracea crude polysaccharide SBOP is prepared into a polysaccharide SBOP solution with the concentration of 50 to 55mg/ml, the solution is centrifuged for 10 to 15min at the speed of 3500 to 4000 r/min, and the supernatant is filtered by a filter membrane with the diameter of 0.45 mu m, and then the subsequent purification treatment is carried out; performing primary purification by sequentially performing gradient elution with deionized water, 0.05M, 0.1M, 0.2M, 0.3M and 0.5M NaCl solution by using a DEAE-52 ion exchange column, wherein the volume of the eluent in each gradient is 1.5 times of the column volume; further purifying the Sephadex-G50 gel column, and performing gradient elution by using deionized water, wherein the volume of the eluent is 1.5 times of the column volume.
Further, in the step S1, 90-120g of rhizoma bletillae pseudobulbs are taken, ground and sieved by a 40-mesh sieve; taking 12-15g of bletilla ochracea powder, weighing 35-40ml of petroleum ether, degreasing at 50-55 ℃ for 9-10h under reflux, and degreasing all the bletilla ochracea powder in batches.
Further, the specific processing method in step S2 is: carrying out cold-soaking extraction on the pretreated bletilla ochracea powder in 90-95% ethanol solution for 3-4 times according to the material-liquid ratio of 1 (8-12), each time for 20-24h, filtering, collecting filter residues and drying.
Further, the complex enzyme in the step S3 comprises cellulase and neutral protease, and the adding mass percentages of the cellulase and the neutral protease relative to the filter residue are respectively 1.5 to 1.8 percent and 1.2 to 1.5 percent.
Further, the specific processing method in step S3 is: mixing the filter residue obtained in the step S2 with a complex enzyme, and carrying out ultrasonic treatment in hot water at 55 to 65 ℃ for 2 to 3 times according to the material-liquid ratio of 1 (8 to 12), wherein the ultrasonic power is 300 to 400W for 1 to 2h each time; inactivating in a boiling water bath for 15 to 20min after the end, and filtering and combining the filtrate; concentrating the filtrate, precipitating the filtrate with an ethanol solution with the final concentration of 80 to 85 percent, centrifuging and collecting the precipitate to obtain the crude bletilla ochracea polysaccharide.
Further, the specific processing method in step S4 is: mixing and washing bletilla ochracea crude polysaccharide with 90-95% ethanol solution and acetone according to the volume ratio of (1-3) to 1 for 3-4 times; washing, dissolving with distilled water, mixing the bletilla ochracea crude polysaccharide solution with a Sevage reagent according to a volume ratio of (3-5) to 1, violently shaking for 25-30min, and centrifuging for 10-15 min at 3500-4000 r/min; the mixed solution is divided into three layers, the protein denaturation layer is positioned between the rhizoma bletillae crude polysaccharide solution and the Sevage reagent, the rhizoma bletillae crude polysaccharide solution on the upper layer is carefully sucked out, the protein removing step is repeated until no intermediate protein denaturation layer exists, and protein removing treatment is carried out.
Further, the specific processing method in step S5 is: precipitating the crude rhizoma bletillae polysaccharide solution after protein removal with an ethanol solution with the final concentration of 80-85%, centrifuging and collecting the precipitate; redissolving the precipitate with distilled water, dialyzing in a dialysis bag of 3000 to 3500Da for 48 to 72 h, concentrating after dialysis, and freeze-drying to obtain bletilla ochracea crude polysaccharide SBOP.
A rhizoma Bletillae polysaccharide comprises rhizoma Bletillae crude polysaccharide SBOP or micromolecular water soluble rhizoma Bletillae polysaccharide BOP-1; the molecular weight of the micromolecule water-soluble bletilla ochracea polysaccharide BOP-1 is 3000 to 4000Da, and the monosaccharide composition of the micromolecule water-soluble bletilla ochracea polysaccharide is as follows: the molar ratio of mannose to glucose was 1.5.
An application of bletilla ochracea polysaccharide in preparing medicines or skin care products for inhibiting the expression of inflammatory factors and the skin damage caused by ultraviolet rays is disclosed.
Further, the application dosage of the bletilla ochracea polysaccharide is 50 to 150 mu g/ml.
The technical scheme of the embodiment of the invention at least has the following advantages and beneficial effects:
1. the method comprises the steps of extracting by cold soaking with ethanol for multiple times, performing ultrasonic hot water extraction with enzyme to obtain crude bletilla ochracea polysaccharide SBOP, and finally performing monitoring, selective concentration and purification on the crude bletilla ochracea polysaccharide SBOP to obtain the micromolecule water-soluble bletilla ochracea polysaccharide BOP-1.
2. The prepared bletilla ochracea polysaccharide BOP-1 has the molecular weight distribution in the range of 3000Da to 4000Da, has smaller molecular weight, and is beneficial to the production of subsequent products, medicines or skin care products.
3. The crude bletilla ochracea polysaccharide SBOP and the bletilla ochracea polysaccharide BOP-1 prepared by the method have good anti-inflammatory effect, antioxidant effect and moisturizing effect, and are beneficial to application in medicines or skin care products.
Drawings
FIG. 1 is a graph showing the results of monitoring the chromatography feed liquid provided in example 1; in the figure: a DEAE-52 ion exchange column chromatography monitoring result chart of the SBOP of the A-rhizoma bletillae crude polysaccharide, a gel column chromatography monitoring result chart of a peak 1 of a DEAE-52 ion exchange column of the SBOP of the B-rhizoma bletillae crude polysaccharide, and a gel column chromatography monitoring result chart of a peak 2 of a DEAE-52 ion exchange column of the SBOP of the C-rhizoma bletillae crude polysaccharide;
FIG. 2 is a graph showing the results provided in Experimental example 1;
FIG. 3 is a graph showing the results provided in Experimental example 2;
FIG. 4 is a graph showing the results provided in Experimental example 4;
FIG. 5 is a graph showing the results provided in Experimental example 5;
FIG. 6 is a graph showing the results provided in Experimental example 6; in the figure: A-TNF-alpha expression result graph, B-IL-6 expression result graph, C-IL-1 beta expression result graph and D-NO expression result graph;
FIG. 7 is a graph showing the results provided in Experimental example 7; in the figure: a-graph of results of intracellular ROS level changes, B-graph of results of intracellular ROS relative content;
FIG. 8 is a graph showing the results provided in Experimental example 7; in the figure: A-MDA content result graph, B-SOD content result graph, C-GSH content result graph, and D-CAT content result graph;
FIG. 9 is a graph showing the results provided in Experimental example 8; A-AQP-3 content result chart and B-HA content result chart.
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 ochracea polysaccharide provided by the embodiment of the invention and the preparation method and the application thereof are specifically explained below.
Example 1
The embodiment provides a preparation method of bletilla ochracea polysaccharide, which comprises the following steps:
s1: taking 90g of rhizoma bletillae pseudobulb, crushing and sieving by a 40-mesh sieve; taking 12g of bletilla ochracea powder, weighing 35ml of petroleum ether, degreasing and refluxing for 9h at 50 ℃, and completely degreasing the bletilla ochracea powder in batches;
s2: cold soaking the pretreated rhizoma bletilla in 95% ethanol solution according to the material-liquid ratio of 1;
s3: mixing a complex enzyme in the filter residue obtained in the step S2, wherein the complex enzyme comprises cellulase and neutral protease, and the adding mass percentages of the cellulase and the neutral protease relative to the filter residue are respectively 1.5% and 1.2%; carrying out ultrasonic treatment in hot water at 55 ℃ for 2 times according to the material-liquid ratio of 1; inactivating in boiling water bath for 15min, filtering, and mixing filtrates; concentrating the filtrate, precipitating with 80% ethanol solution, centrifuging, and collecting precipitate to obtain rhizoma Bletillae crude polysaccharide;
s4: mixing and washing rhizoma bletillae crude polysaccharide with 95% ethanol solution and acetone according to the volume ratio of 1; washing, dissolving with distilled water, mixing the rhizoma bletilla crude polysaccharide solution with a Sevage reagent according to a volume ratio of 3; the mixed solution is divided into three layers, the protein denaturation layer is positioned between the rhizoma bletillae crude polysaccharide solution and the Sevage reagent, the rhizoma bletillae crude polysaccharide solution on the upper layer is carefully sucked out, the protein removing step is repeated until no intermediate protein denaturation layer exists, and protein removing treatment is carried out;
s5: precipitating the crude rhizoma bletilla polysaccharide solution with protein removed with 80% ethanol solution, centrifuging, and collecting precipitate; redissolving the precipitate with distilled water, dialyzing in a 3500Da dialysis bag for 48 h, concentrating after dialysis, and freeze-drying to obtain crude bletilla ochracea polysaccharide SBOP;
s6: preparing crude rhizoma bletilla polysaccharide SBOP into 50mg/ml polysaccharide SBOP solution, centrifuging at 4000 r/min for 10min, taking supernatant, filtering through a 0.45 mu M filter membrane, performing primary purification through a DEAE-52 ion exchange column (2.6 multiplied by 60 cm), sequentially adopting deionized water, 0.05M, 0.1M, 0.2M, 0.3M and 0.5M NaCl solution to perform gradient elution, wherein the volume of eluent of each gradient is 1.5 times of the column volume, drawing an elution curve at 490nm wavelength, monitoring the polysaccharide concentration in the chromatographic feed liquid by using a phenol-sulfuric acid method, collecting and concentrating the feed liquid of peak 1 and peak 2 with the highest polysaccharide concentration in the feed liquid in figure 1A, further purifying through a Sephadex-G50 gel column (1.8 multiplied by 150 cm), performing gradient elution by using deionized water, wherein the volume of the eluent is 1.5 times of the column volume, monitoring the polysaccharide concentration in the chromatographic feed liquid by using a phenol-sulfuric acid method, as shown in figures 1B and 1C, and collecting a symmetrical peak of the feed liquid in the elution peak in the single peak in the elution feed liquid in the figure 1A; as can be seen from fig. 1C, the feed liquid collected as peak 2 in fig. 1A was eluted to obtain 3 asymmetric peaks, indicating that peak 2 contains a relatively impure polysaccharide and has a low concentration; therefore, the feed liquid of peak 1 is collected and concentrated, dialyzed for 72 h in a dialysis bag of 1000Da, and freeze-dried after the dialysis is finished to obtain the bletilla ochracea polysaccharide BOP-1.
Example 2
The present embodiment provides S1: taking 120g of bletilla ochracea pseudobulb, crushing and sieving by a 40-mesh sieve; taking 15g of bletilla ochracea powder, weighing 40ml of petroleum ether, degreasing and refluxing at 55 ℃ for 10h, and completely degreasing the bletilla ochracea powder in batches;
s2: cold-soaking pretreated rhizoma bletilla powder in 95% ethanol solution at a material-liquid ratio of 1;
s3: mixing a complex enzyme with the filter residue obtained in the step S2, wherein the complex enzyme comprises cellulase and neutral protease, and the adding mass percentages of the cellulase and the neutral protease relative to the filter residue are respectively 1.8% and 1.5%; carrying out ultrasonic treatment in hot water at 65 ℃ for 3 times, each time for 2 hours, according to the material-liquid ratio of 1; inactivating in boiling water bath for 20min, filtering, and mixing filtrates; concentrating the filtrate, precipitating with 85% ethanol solution, centrifuging, and collecting precipitate to obtain rhizoma Bletillae crude polysaccharide;
s4: mixing and washing crude rhizoma bletillae polysaccharide with 95% ethanol solution and acetone according to the volume ratio of 3; washing, dissolving with distilled water, mixing the rhizoma bletilla crude polysaccharide solution with a Sevage reagent according to a volume ratio of 5; the mixed solution is divided into three layers, the protein denaturation layer is positioned between the rhizoma bletillae crude polysaccharide solution and the Sevage reagent, the rhizoma bletillae crude polysaccharide solution on the upper layer is carefully sucked out, the protein removing step is repeated until no intermediate protein denaturation layer exists, and protein removing treatment is carried out;
s5: precipitating the crude rhizoma bletilla polysaccharide solution with protein removed with 85% ethanol solution, centrifuging, and collecting precipitate; redissolving the precipitate with distilled water, dialyzing in a 3000Da dialysis bag for 72 h, concentrating after dialysis, and freeze-drying to obtain crude bletilla ochracea polysaccharide SBOP;
s6: preparing crude polysaccharide SBOP of bletilla ochracea into 55mg/ml polysaccharide SBOP solution, centrifuging at 3500r/min for 15min, taking supernatant, filtering through a 0.45 mu M filter membrane, carrying out primary purification through a DEAE-52 ion exchange column (2.6 multiplied by 60 cm), sequentially carrying out gradient elution through deionized water, naCl solutions of 0.05M, 0.1M, 0.2M, 0.3M and 0.5M, wherein the volume of eluent of each gradient is 1.5 times of the column volume, monitoring the polysaccharide concentration in the chromatographic feed liquid by using a phenol-sulfuric acid method, collecting and concentrating the chromatographic feed liquid with the highest polysaccharide concentration, further purifying through a Sephadex-G50 gel column (1.8 multiplied by 150 cm), carrying out gradient elution through deionized water, wherein the volume of eluent is 1.5 times of the column volume, monitoring the polysaccharide concentration in the chromatographic feed liquid by using a phenol-sulfuric acid method, collecting and concentrating the chromatographic feed liquid with the highest polysaccharide concentration, dialyzing for 72 h in a 1000Da dialysis bag, and carrying out freeze drying to obtain the bletilla ochracea polysaccharide-1 BOP after dialysis.
Example 3
The present embodiment provides S1: taking 100g of bletilla ochracea pseudobulb, crushing and sieving by a 40-mesh sieve; taking 14g of rhizoma bletillae powder, weighing 38ml of petroleum ether, degreasing and refluxing at 55 ℃ for 10h, and completely degreasing the rhizoma bletillae powder in batches;
s2: cold soaking and extracting pretreated rhizoma bletillae powder in a 95% ethanol solution for 4 times according to the material-liquid ratio of 1;
s3: mixing a complex enzyme with the filter residue obtained in the step S2, wherein the complex enzyme comprises cellulase and neutral protease, and the adding mass percentages of the cellulase and the neutral protease relative to the filter residue are respectively 1.6% and 1.4%; carrying out ultrasonic treatment in hot water at 60 ℃ for 3 times according to the material-liquid ratio of 1; inactivating in boiling water bath for 18min, filtering, and mixing filtrates; concentrating the filtrate, precipitating with 85% ethanol solution, centrifuging, and collecting precipitate to obtain rhizoma Bletillae crude polysaccharide;
s4: mixing and washing rhizoma bletillae crude polysaccharide with 90% ethanol solution and acetone according to the volume ratio of 2; washing, dissolving with distilled water, mixing the bletilla ochracea crude polysaccharide solution with a Sevage reagent according to a volume ratio of 4; the mixed solution is divided into three layers, the protein denaturation layer is positioned between the rhizoma bletillae crude polysaccharide solution and the Sevage reagent, the rhizoma bletillae crude polysaccharide solution on the upper layer is carefully sucked out, the protein removing step is repeated until no intermediate protein denaturation layer exists, and protein removing treatment is carried out;
s5: precipitating the crude rhizoma bletilla polysaccharide solution with protein removed with 80% ethanol solution, centrifuging, and collecting precipitate; redissolving the precipitate with distilled water, dialyzing in a dialysis bag of 3500Da for 60 h, concentrating after dialysis, and freeze-drying to obtain crude rhizoma Bletillae polysaccharide SBOP;
s6: preparing crude bletilla ochracea polysaccharide SBOP into 50mg/ml polysaccharide SBOP solution, centrifuging at 4000 r/min for 12min, taking supernatant, filtering by a 0.45 mu M filter membrane, carrying out primary purification by a DEAE-52 ion exchange column (2.6 multiplied by 60 cm), sequentially carrying out gradient elution by deionized water, naCl solutions of 0.05M, 0.1M, 0.2M, 0.3M and 0.5M, wherein the volume of eluent of each gradient is 1.5 times of the column volume, monitoring the polysaccharide concentration in the chromatographic feed liquid by a phenol-sulfuric acid method, collecting and concentrating the chromatographic feed liquid with the highest polysaccharide concentration, further purifying by a Sephadex-G50 gel column (1.8 multiplied by 150 cm), carrying out gradient elution by deionized water, wherein the volume of the eluent is 1.5 times of the column volume, monitoring the polysaccharide concentration in the chromatographic feed liquid by a phenol-sulfuric acid method, collecting and concentrating the chromatographic feed liquid with the highest polysaccharide concentration, dialyzing for 72 h in a 1000Da dialysis bag, and carrying out freeze drying to obtain bletilla ochracea polysaccharide-1 BOP after dialysis.
Experimental example 1
Determination of molecular weight of bletilla ochracea polysaccharide BOP-1
Determining the molecular weight distribution of the bletilla ochracea polysaccharide BOP-1 prepared in the example 1 by adopting a high-efficiency gel permeation chromatography system; the chromatographic column is Shodex OH-pakSB-804 HQ column (8 mm multiplied by 300 mm), the detector is a parallax detector (RID) and a Diode Array Detector (DAD) which are used together, the column temperature is 35 ℃, the sample injection amount is 30 mu l, the mobile phase is 0.1M NaCl, and the flow rate is 0.5mL/min; standard curves are made by using dextran standards (D0, 1, 2,3,4, 5, 6, 7 and 2000) with different molecular weights, GPC software is used for correcting the standard curves, and then the weight average molecular weight (Mw) of the bletilla ochracea polysaccharide BOP-1 is 3992Da, the number average molecular weight (Mn) is 3187Da and the polydispersity (Mw/Mn) is 1.25, which shows that the molecular weight distribution range of the bletilla ochracea polysaccharide BOP-1 is narrow, and the result is shown in figure 2.
As shown in fig. 2, the total of all polysaccharide fragments with a molecular weight of 1000Da or less in the balata jeponensis BOP-1 is about 0% of all polysaccharide fragments, the mass percentage of polysaccharide fragments with a smaller molecular weight is accumulated with the increase of the molecular weight, and all polysaccharide fragments with a molecular weight of 11000Da or less in the balata jeponensis BOP-1 are about 90% or more of all polysaccharide fragments.
As shown in a molecular weight differential distribution curve (dw/dlogM diagram) in FIG. 2, in the bletilla ochracea polysaccharide BOP-1, the polysaccharide fragment with the molecular weight within the range of 3000Da-4000Da enclosed by a virtual frame occupies the largest mass fraction, and the polysaccharide fragments with the molecular weight less than 1000Da and greater than 11000Da occupy the smallest mass fraction, which indicates that the molecular weight of the polysaccharide BOP-1 is distributed within the range of 3000Da-4000 Da.
Experimental example 2
Monosaccharide composition determination of bletilla ochracea polysaccharide BOP-1
Hydrolyzing the bletilla ochracea polysaccharide BOP-1 prepared in example 1 and each monosaccharide standard with 2ml of 2M TFA solution at 110 deg.C for 4h, cooling to room temperature, adjusting pH to neutrality with 2M NaOH, and analyzing by high performance liquid chromatography with Agilent Zorbax SB-C18 (150 mm. Times.4.6 mm), column temperature of 35 deg.C, and mobile phase A of 15% acetonitrile +85% KH 2 PO 4 NaOH buffer (pH 6.9), mobile phase B40% acetonitrile +60% KH 2 PO 4 NaOH buffer (pH 6.9), flow rate 1ml/min, elution gradient: 0,0% by weight B,25min;25% by weight B,30min;100% of A. The results are shown in FIG. 3.
As can be seen from fig. 3, the monosaccharide composition of the bletilla ochracea polysaccharide BOP-1 prepared in example 1 was obtained by measuring the molar ratio based on the retention time of standard monosaccharides and an external standard method: the molar ratio of mannose to glucose was 1.5.
Experimental example 3
Infrared spectrometry of bletilla ochracea polysaccharide BOP-1
The bletilla ochracea polysaccharide BOP-1 prepared in example 1 was prepared into KBr pellets and scanned by Bruker Tensor27 infrared spectrometer at 4000-400cm -1 Obtaining an infrared absorption spectrogram according to the spectral signals in the range. The results are shown in Table 1.
The BOP-1 is 3385cm in the amount of rhizoma Bletillae polysaccharide -1 Wide and strong absorbance bonds appear at the position, which are all caused by O-H stretching vibration; 2877 cm -1 And 1370 cm -1 The absorption peak is classified as C-H stretching vibration and bending vibration of pyranose; 1726 cm -1 And 1242 cm -1 The absorption peak is caused by C = O valence vibration and C-O vibration of O-acetyl; 1056 cm of -1 The absorption peak at (a) represents the presence of the pyranoside; in addition, 873 cm -1 The weak absorption peak at (a) is due to the mannose residue.
Experimental example 4
GC-MS methylation analysis of bletilla ochracea polysaccharide BOP-1
Weighing 5mg of bletilla ochracea polysaccharide BOP-1 prepared in example 1, dissolving the sample in 1ml of dimethyl sulfoxide (DMSO), adding 50mg of potassium hydroxide powder (KOH), magnetically stirring until the KOH is dissolved, adding 0.9ml of methyl iodide, carrying out ultrasonic reaction for 1h under the condition of nitrogen sealing, adding 2ml of water after the reaction to decompose the methyl iodide to terminate the reaction, and extracting by adopting dichloromethane to obtain methylated polysaccharide; adding 1mL of 2M TFA solution to dissolve the methylated polysaccharide, performing acid hydrolysis on the methylated polysaccharide at 110 ℃ for 4h, adjusting the pH to 10 by using 2M KOH solution, adding 25 mg of sodium borohydride, stirring in a water bath at 50 ℃ for reduction for 2h, adding glacial acetic acid to stop reaction, and freeze-drying; analysis was performed by GC-MS using an 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 rises to 280 ℃ at a speed of 5 ℃/min, and the temperature is kept for 20 min; the flow rate of the column is 1.0 mL/min, the temperature of a sample inlet is 250 ℃, the pressure in front of the column is 100 kPa, the split ratio is 10; MS conditions: the ionization mode EI comprises 70 electron energy, 290 ℃ of transmission line, 230 ℃ of ion source, 150 ℃ of quadrupole rod and 50-600 mass range. And comparing with a CCRC spectrum database to judge the type of the glycosidic bond. The results are shown in FIG. 4.
As shown in FIG. 4, there are 4 chromatographic peaks with higher response values between 18 and 22min, and the chromatographic peaks marked with an "x" in the figure represent non-sugar substances, and the specific attribution is detailed in Table 2. Comparing MS diagrams corresponding to the spectrum peaks with literature and CCRC standard diagrams to obtain various sugar alcohol acetate derivatives which can be assigned to BOP-1; the major fragment ion peaks of the chromatographic peaks at 19.62min and 19.72min were 71,87, 101,118, 129, 145, 161, 205, assigned to 2,3,4, 6-tetra-O-methyl-1, 5-di-O-acetyl-mannitol and 2,3,4, 6-tetra-O-methyl-1, 5-di-O-acetyl-glucitol, respectively, indicating that there are structural fragments of Man- (1 → and Glc- (1 → in the sugar chain; the main fragment ion peaks at 21.67min and 21.78 min were 71,87, 99,101, 117, 129, 145, 161, 233 and 71,87, 101, 117, 129, 145, 161, 233, which were assigned to 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, suggesting that the sugar chain contained structural fragments of → 1) -Man- (4 → and → 1) -Glc- (4 → a, the molar ratios of which were 1.12.
Experimental example 5
Preparation before experiment:
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 rapid thawing, rapidly transferring the cells into a 15mL centrifuge tube in a biological safety cabinet, adding 4mL of complete culture medium, blowing and beating uniformly, and centrifuging for 3min at 1000 rmp; after the supernatant was decanted, 2mL of medium was added to the centrifuge tube, blown evenly, transferred to a cell culture dish and the medium was replenished10mL, shake well, place in 5% CO 2 And culturing the cells in a cell culture box at 37 ℃. Observing the cell state on time, carrying out cell passage when the cell grows to about 80-90% in a culture dish, counting by using a cell counter when the cell is transmitted to the third generation, observing the cell survival rate and state, and carrying out subsequent experiments when the cell survival rate is more than 95% after the cell density is adjusted.
MTT cell viability assay:
the cell density of RAW264.7 was adjusted to 1.0X 10 5 Each well is provided with 200 mul of fishplate per well, the fishplate bar grows in a 96-well plate in an adherence way, after 24 hours of culture, the bletilla ochracea polysaccharide BOP-1 prepared in the embodiment 1 is respectively diluted into 150 mul/ml, 100 mul/ml and 50 mul/ml by using culture media, 200 mul of bletilla ochracea polysaccharide BOP-1 with different concentrations are respectively added into cells of each well, and each concentration is respectively made into 5 parallel groups; simultaneously setting a blank group as that 200 mul of culture medium is added into each hole of the cells; after 20 h of incubation, 100. Mu.L of MTT solution with a concentration of 0.5mg/mL was added to each well, and after 4h of incubation, 100. Mu.L of DMSO solution was added to each well and shaken well for 10min in the dark using a cell shaking plate apparatus, and the absorbance of each well was measured at 570 nm using a microplate reader. The results are shown in figure 5, the cell viability of the bletilla ochracea polysaccharide BOP-1 is above 90% at the concentrations of 150 mug/ml, 100 mug/ml and 50 mug/ml, which indicates that the BOP-1 has no toxicity to RAW264.7 cells and has a remarkable proliferation effect on RAW264.7 cells at the concentration of 150 mug/ml. Expressed as mean ± standard deviation, n =5, # p<0.05, compared to the blank group.
Experimental example 6
Preparation before experiment:
establishing an LPS-induced RAW264.7 cell inflammation model:
the cell density of RAW264.7 was adjusted to 1.0X 10 5 Culturing for 24h, adding 200 μ l LPS solution into each well cell to make LPS final concentration 1000ng/ml, and culturing in 5% CO 2 And incubating for 24h in a cell culture box at 37 ℃ to form an LPS-induced RAW264.7 cell inflammation model.
Determination of NO and inflammatory factor content:
the bletilla ochracea polysaccharide BOP-1 prepared in the example 1 is diluted into 150 mug/ml, 100 mug/ml and 50 mug/ml with culture medium respectively, 200 mug/l is added into each hole of an LPS induced RAW264.7 cell inflammation model respectively, and each concentration is respectively made into 5 parallel groups; meanwhile, setting a blank group as that 200 mul of culture solution is added into normal RAW264.7 cells, and setting a model group as that 200 mul of culture solution is added into an LPS (LPS) induced RAW264.7 cell inflammation model; after 24h of culture, collecting cell supernatant, respectively measuring the NO amount of each group by using a Biyun day total NO detection kit, and respectively measuring the TNF-alpha, IL-1 beta and IL-6 amount of each group by using a Biyun day corresponding ELISA kit. Results are shown in figure 6, expressed as mean ± standard deviation, n =5, # p <0.05, compared to the blank group; * p <0.05, compared to model group.
As can be seen from fig. 6, the LPS-induced RAW264.7 cell inflammatory factor expression model group has significantly higher NO synthesis amount and inflammatory factor expression, and has significant difference (p < 0.05) compared with the blank group, while the croaker and polysaccharide BOP-1 dry prognosis polysaccharide group has significantly inhibited NO synthesis amount and inflammatory factor expression, and is dose-dependent and reduced; the high-dose bletilla ochracea polysaccharide BOP-1 has good inhibition effects on NO, TNF-alpha, IL-6 and IL-1 beta, wherein the inhibition effect on IL-6 is most obvious; therefore, the bletilla ochracea polysaccharide BOP-1 can effectively inhibit the expression of inflammatory factors, so that the anti-inflammatory effect is achieved.
Experimental example 7
Preparation before experiment:
recovery and culture of Hacat cells: taking out Hacat cells from a liquid nitrogen tank, quickly thawing the Hacat cells in a water cup at about 37 ℃, quickly transferring the Hacat cells to 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 supernatant, adding 2mL of culture medium into the centrifuge tube, blowing uniformly, transferring into cell culture dish, supplementing 10mL of culture medium, shaking uniformly, and placing in 5% CO 2 And culturing the cells in a cell culture box at 37 ℃. Observing the cell state on time, carrying out cell passage when the cell grows to about 80-90% in a culture dish, counting by using a cell counter when the cell is transmitted to the third generation, observing the cell survival rate and state, and adjusting the cell density to be thinThe cell survival rate is more than 95 percent, and subsequent experiments can be carried out.
And (3) measuring the active oxygen level and the activity of the antioxidant enzyme:
the Hacat cell density was adjusted to 1.0X 10 at a non-toxic dose 5 Each cell per ml, 200 mul of each well is connected with a 96-well plate for adherent growth, after 24 hours of culture, 200 mul of 150 mul/ml, 100 mul/ml and 50 mul/ml of bletilla ochracea polysaccharide BOP-1 are respectively added into cells of each well, and each concentration is respectively made into 5 parallel groups; after 24H of culture, the old culture solution is discarded, PBS solution is added for washing for 1 to 2 times, and 0.7mM of H is added into each hole 2 O 2 200 mul of solution; meanwhile, a blank group is set as a normal Hacat cell, and a model group is set as a Hacat cell to which 0.7mM H is directly added 2 O 2 200 mul of solution; collecting cell supernatant after 24h of culture, and measuring the reactive oxygen species level in the cells by using a cell Reactive Oxygen Species (ROS) analysis kit of Biyuntian; the results are shown in fig. 7, with the results expressed as mean ± standard deviation, n =5. Meanwhile, collecting cell supernatant, and respectively measuring the contents of MDA, SOD, GSH and CAT in cells by using a detection kit for Malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and Catalase (CAT) of Biyuntian; the results are shown in FIG. 8, where the results are expressed as mean. + -. Standard deviation, n =5, # p<0.05, compared to blank; * p is a radical of formula<0.05, compared to the model set.
As can be seen from FIG. 7, model group H 2 O 2 Treatment resulted in a robust increase in the mean fluorescence intensity of DCF-DA in Hacat cells over the blank (p)<0.05 I.e. ROS levels are steadily increased over the blank group; the ROS level of the experimental group pretreated by the bletilla ochracea polysaccharide BOP-1 is obviously reduced compared with that of the model group (p)<0.05 And is reduced in a dose-dependent manner; this indicates that the bletilla ochracea polysaccharide BOP-1 can reduce ROS levels and thereby reduce H 2 O 2 Causing aging of Hacat cells.
As can be seen from FIG. 8, the Malondialdehyde (MDA) content as a marker of oxidative stress was found in the model group H 2 O 2 The treated Hacat cells showed a significant increase (p) over the blank<0.05 Whereas the experimental group pretreated by the bletilla ochracea polysaccharide BOP-1 significantly inhibited the increase of the MDA content (p)<0.05 And dose-dependently decreased; SOD,The content measurement of GSH and CAT shows that the model group H 2 O 2 SOD, GSH and CAT contents (p) in treated Hacat cells were significantly reduced compared to the blank<0.05 In addition, the experiment group pretreated by the bletilla ochracea polysaccharide BOP-1 obviously increases the contents (p) of SOD, GSH and CAT in cells in a dose-dependent mode<0.05 And in a dose-dependent increase; this indicates that bletilla ochracea polysaccharide BOP-1 can reduce H 2 O 2 Has effects in relieving oxidative stress of cells and increasing the content of antioxidase, i.e. rhizoma Bletillae polysaccharide BOP-1 has antioxidation effect on cells, thereby reducing H 2 O 2 Causing aging of Hacat cells.
Experimental example 8
And (3) testing the moisturizing activity:
hacat cell density was adjusted to 3.0X 10 5 Culturing 2mL of cell sap in a 6-pore plate for 24 hours per well, sucking out the cell sap to establish a drying model, setting the drying air speed to be 0.6m/s, respectively adding 2mL of culture medium containing 150 mu g/mL, 100 mu g/mL and 50 mu g/mL of bletilla ochracea polysaccharide BOP-1 into each well after drying for 25min, and setting 5 parallel groups per concentration; meanwhile, setting a blank group as a normal Hacat cell, and directly adding 2mL of culture medium into a cell drying model as a model group; continuously culturing the cells for 24h, collecting cell supernatant, determining the content of aquaporin AQP-3 by using an aquaporin 3 (AQP-3) elisa detection kit, and determining the content of HA by using a Hyaluronic Acid (HA) detection kit; the results are shown in FIG. 9, where the results are expressed as mean. + -. Standard deviation, n =5, # p<0.05, compared to blank; * p is a radical of<0.05, compared to the model set.
As can be seen from FIG. 9, the AQP-3 and HA content of the model group of the dry injured cells is significantly reduced compared with that of the blank group (p < 0.05); in the experimental group treated by the bletilla ochracea polysaccharide BOP-1, the contents of AQP-3 and HA in cells are obviously improved (p is less than 0.05) compared with the contents in the model group, and the contents are increased in a dose-dependent manner; the result shows that the bletilla ochracea polysaccharide BOP-1 can promote the secretion of aquaporins AQP-3 and hyaluronic acid HA by HaCat cells with dry damage, and physiological regulators such as AQP-3, HA and the like are helpful for keeping the moisture balance of the skin, so that the bletilla ochracea polysaccharide BOP-1 can be inferred to have the moisturizing effect.
In addition, experiments prove that the crude bletilla ochracea polysaccharide SBOP prepared by the method also has the anti-inflammatory, antioxidant and moisturizing effects.
In conclusion, the bletilla ochracea crude polysaccharide SBOP is prepared by a specific method, and then the small molecular water-soluble bletilla ochracea polysaccharide BOP-1 is prepared by purification, wherein the bletilla ochracea crude polysaccharide SBOP and the small molecular water-soluble bletilla ochracea polysaccharide BOP-1 have good anti-inflammatory effect, antioxidant effect and moisturizing effect, and are beneficial to application in medicines or skin care products.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by 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.
Claims (6)
1. The preparation method of bletilla ochracea polysaccharide is characterized by comprising the following steps:
s1: taking rhizoma bletilla pseudobulb, crushing, and performing degreasing pretreatment;
s2: removing polyphenol and flavonoid fat-soluble components from pretreated rhizoma bletilla ochracea powder by using an ethanol solution, collecting filter residues and drying;
s3: mixing the filter residue obtained in the step S2 with a complex enzyme, carrying out ultrasonic extraction in hot water, carrying out alcohol precipitation, centrifuging and collecting precipitate to obtain crude bletilla ochracea polysaccharide;
s4: washing and deproteinizing the crude bletilla ochracea polysaccharide;
s5: carrying out alcohol precipitation, dialysis, concentration and freeze drying on the crude bletilla ochracea polysaccharide solution subjected to protein removal to obtain bletilla ochracea crude polysaccharide SBOP;
the specific processing method of the step S3 is as follows: mixing the filter residue obtained in the step S2 with a complex enzyme, and carrying out ultrasonic treatment in hot water at 55-65 ℃ for 2-3 times according to the material-liquid ratio of 1 (8-12), wherein the ultrasonic power is 300-400W for 1-2h each time; inactivating in a boiling water bath for 15 to 20min after the end, and filtering and combining the filtrate; concentrating the filtrate, precipitating the filtrate with an ethanol solution with the final concentration of 80-85%, centrifuging and collecting the precipitate to obtain crude bletilla ochracea polysaccharide;
the complex enzyme in the step S3 comprises cellulase and neutral protease, and the adding mass percentages of the cellulase and the neutral protease relative to the filter residue are respectively 1.5 to 1.8% and 1.2 to 1.5%;
the specific processing method of the step S5 is as follows: carrying out alcohol precipitation on the bletilla ochracea crude polysaccharide solution after protein removal by using an ethanol solution with the final concentration of 80-85%, and centrifuging to collect precipitate; redissolving the precipitate with distilled water, dialyzing in a dialysis bag of 3000-3500 Da for 48-72 h, concentrating after dialysis, and freeze-drying to obtain crude bletilla ochracea polysaccharide SBOP;
step S6: performing primary purification on crude bletilla ochracea polysaccharide SBOP by a DEAE-52 ion exchange column, performing gradient elution by sequentially adopting deionized water, 0.05M, 0.1M, 0.2M, 0.3M and 0.5M NaCl solutions, collecting chromatography feed liquid with the highest polysaccharide concentration and concentrating, further purifying by a Sephadex-G50 gel column, performing gradient elution by adopting deionized water, collecting chromatography feed liquid with the highest polysaccharide concentration and concentrating, dialyzing, and performing freeze drying to obtain bletilla ochracea polysaccharide BOP-1;
the bletilla ochracea polysaccharide BOP-1 is small molecular water-soluble bletilla ochracea polysaccharide BOP-1, the molecular weight of the bletilla ochracea polysaccharide BOP-1 is 3000 to 4000Da, and the monosaccharide composition of the bletilla ochracea polysaccharide is as follows: the molar ratio of mannose to glucose was 1.5.
2. The preparation method of bletilla ochracea polysaccharide as claimed in claim 1, wherein the specific treatment method in the step S2 is as follows: carrying out cold-soaking extraction on the pretreated bletilla ochracea powder in 90-95% ethanol solution for 3-4 times according to the material-liquid ratio of 1 (8-12), each time for 20-24h, filtering, collecting filter residues and drying.
3. The preparation method of bletilla ochracea polysaccharide according to claim 1, wherein the specific treatment method in the step S4 is as follows: mixing and washing bletilla ochracea crude polysaccharide with 90-95% ethanol solution and acetone according to a volume ratio of (1-3) to 1 for 3-4 times; washing, dissolving with distilled water, mixing a bletilla ochracea crude polysaccharide solution with a Sevage reagent according to a volume ratio of (3 to 5) to 1, violently shaking for 25 to 30min, and centrifuging for 10 to 15min at a speed of 3500 to 4000 r/min; the mixed solution is divided into three layers, the protein denaturation layer is positioned between the rhizoma bletillae crude polysaccharide solution and the Sevage reagent, the rhizoma bletillae crude polysaccharide solution on the upper layer is carefully sucked out, the protein removing step is repeated until no intermediate protein denaturation layer exists, and protein removing treatment is carried out.
4. Bletilla ochracea polysaccharide prepared by the preparation method according to any one of claims 1 to 3.
5. The application of the bletilla ochracea polysaccharide as claimed in claim 4 in preparing medicines or skin care products for inhibiting the expression of inflammatory factors.
6. The application of bletilla ochracea polysaccharide as claimed in claim 5, wherein the application dosage of the bletilla ochracea polysaccharide is 50 to 150 μ g/ml.
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