CN117384308A - Radix tetrastigme polysaccharide prepared based on Phellinus fermentation and application thereof - Google Patents
Radix tetrastigme polysaccharide prepared based on Phellinus fermentation and application thereof Download PDFInfo
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- CN117384308A CN117384308A CN202311332245.XA CN202311332245A CN117384308A CN 117384308 A CN117384308 A CN 117384308A CN 202311332245 A CN202311332245 A CN 202311332245A CN 117384308 A CN117384308 A CN 117384308A
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- polysaccharide
- galactose
- radix tetrastigme
- fermentation
- thdp2
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Abstract
The invention discloses a radix tetrastigme polysaccharide prepared based on Phellinus fermentation and application thereof. The radix tetrastigme polysaccharide consists of more than 99 percent of polysaccharide by weight percent; the polysaccharide consists of galactose, mannose, fucose and 3-O-methyl-galactose, and the molar ratio of the galactose to the mannose is 8.5-11.0:1.5-3:0.8-1.5:1.0. the radix tetrastigme polysaccharide is obtained by extracting, separating and purifying radix tetrastigme from Phellinus linteus fermentation. The radix tetrastigme polysaccharide has remarkable anti-tumor activity and intestinal flora structure regulating effects, can be directly used as an anti-tumor and/or intestinal flora structure regulating functional product, and can also be used for preparing the anti-tumor and/or intestinal flora structure regulating functional product.
Description
Technical Field
The invention belongs to the technical field of natural products and polysaccharides, and particularly relates to a radix tetrastigme polysaccharide prepared based on Phellinus linteus fermentation and application thereof.
Background
Radix tetrastigme (Tetrastigma hemsleyanum Diels et Gilg) is a plant of genus cliff of family Vitaceae, and is also referred to as an alias: gold thread hoist, school name: the climbing vine of the cliff is a traditional conventional green herbal medicine in areas such as Zhejiang, fujian and the like, and has the best medicinal effect on underground tuberous roots and fruits. Modern pharmacological researches have shown that radix tetrastigme has the effects of resisting inflammation, easing pain, relieving fever, resisting tumor and virus, regulating immunity and the like. Radix tetrastigme is often used as a dietary supplement because of its various health care effects of relieving fever, anti-hyperplasia, anti-inflammatory, etc.
The radix tetrastigme contains active ingredients such as polysaccharide, flavone, polyphenol and the like and nutritional ingredients such as starch, protein and the like, and in the traditional active ingredient extraction and separation process, the ingredients such as starch, protein, irritant ingredients and the like can be extracted together, so that the treatment steps in separation are increased, and the content of the active ingredients in the final product is low, and the activity is low.
The existing radix tetrastigme polysaccharide is obtained by adopting a traditional extraction and separation method. The Chinese patent ZL201711200506.7 discloses a preparation method and application of radix tetrastigme polysaccharide, which are implemented sequentially by taking radix tetrastigme leaves as raw materials according to the following steps: (1) hot water leaching; suction filtering and collecting filtrate; vacuum concentration; ethanol is added into the concentrated solution; and collecting the precipitate, namely the radix tetrastigme crude polysaccharide. (2) dissolving the crude polysaccharide in water, and adding Sevag reagent; centrifuging to remove the protein layer and the organic layer after stirring, and repeating the steps for a plurality of times until no protein appears; (3) Dissolving the deproteinized polysaccharide in deionized water, and performing DEAE-52 ion exchange chromatography and Sephadex G-100 Sephadex gel chromatography to obtain radix Apioris Fortunei polysaccharide single component. The obtained radix tetrastigme polysaccharide can obviously reduce blood sugar and blood fat of an organism, improve the enzyme activity level of antioxidant enzymes in the organism, has a certain repairing effect on islets, and has good medicinal value and market value. If more active substances such as active polysaccharide with application value, safety and environmental protection can be developed based on the radix tetrastigme, the method has positive significance.
Disclosure of Invention
The invention discovers that more than 90% of the radix tetrastigme tuberous root is viscous high polymer and starch with high molecular weight, so that active polysaccharide in the radix tetrastigme is difficult to release. However, starch is a rich carbon source for Phellinus linteus fungus fermentation and can be fully utilized in the food processing process.
Based on the findings, the invention provides a radix tetrastigme polysaccharide prepared based on Phellinus fermentation and application thereof. According to the invention, researches show that the new radix tetrastigme polysaccharide obtained based on Phellinus fermentation has excellent in-vitro anti-tumor effect and effect of adjusting intestinal flora structure, and can be used in various fields such as food, health products, medicines, daily chemical products and the like. Based on the above results, the present invention has been completed.
The invention also provides a preparation method of the radix tetrastigme polysaccharide, which has the advantages of simple operation and easy control, and is suitable for industrial mass production.
The invention also provides the application of the radix tetrastigme polysaccharide prepared based on Phellinus linteus fermentation, which has remarkable anti-tumor activity and intestinal flora structure regulating effect, can be directly used as an anti-tumor and/or intestinal flora structure regulating functional product, and can also be used for preparing an anti-tumor and/or intestinal flora structure regulating functional product.
The Phellinus linteus fermentation of the present invention has two main effects: on one hand, starch inactive substances in the radix tetrastigme are subjected to enzymolysis by biological enzymes generated by inoculating Phellinus linteus fermentation, so that the release and extraction of active polysaccharide are facilitated; on the other hand, the active radix tetrastigme polysaccharide with a specific structure is obtained.
A radix Apioris Fortunei polysaccharide prepared by Phellinus Linteus fermentation comprises polysaccharide with weight percentage of more than 99%; the polysaccharide consists of galactose, mannose, fucose and 3-O-methyl-galactose, wherein the molar ratio of galactose, mannose, fucose and 3-O-methyl-galactose is 8.5-11.0:1.5-3:0.8-1.5:1.0.
in order to achieve the better inventive effect, the following preferences are made:
the galactose is alpha-galactose and beta-galactose, and more preferably alpha-D-galactose and beta-D-galactose.
The mannose is α -mannose, and more preferably α -D-mannose.
The fucose is alpha-fucose, and more preferably alpha-L-fucose.
The 3-O-methyl-galactose is alpha-3-O-methyl-galactose, and more preferably alpha-3-O-methyl-D-galactose.
The structural units of the polysaccharide are preferably (1-2) linked alpha-D-galactose (alpha-D-Galp) residues and (1-6) linked alpha-D-mannose (alpha-D-Manp) residues in the main chain structure; the C-6 position of an alpha-D-galactose residue with a (1-2) linkage is substituted by beta-D-galactose (beta-D-Galp) end group, the C-2 position of two (1-6) continuous alpha-D-mannose residues with a first branched chain consisting of (1-6) linked alpha-3-O-methyl-D-galactose (alpha-3-O-Me-Galp) residue and beta-D-galactose end group, and an alpha-L-fucose (alpha-L-Fucp) end group.
Further preferably, the main chain structure is formed by sequentially connecting an alpha-D-galactose residue connected with (1-2) and an alpha-D-mannose residue connected with (1-6).
The three branches on the main chain of the radix tetrastigme polysaccharide of the invention have various different variation combinations, the beta-D-galactose end group can replace the C-6 position of any (1-2) connected alpha-D-galactose residue on the main chain, the first branch and one alpha-L-fucose (alpha-L-Fucp) end group can be arranged in any order on the C-2 position of two (1-6) connected alpha-D-mannose residues in sequence, for example, the three branches can have structural units shown in figure 5a, and the structural units shown in figure 5a are only used as an example of one arrangement order of the two end groups and the first branch, and are not used for limiting the arrangement order of the two end groups and the first branch.
In FIG. 5a Galp is galactopyranose, manp is mannopyranose and Fucp is fucopyranose. The structure shown in FIG. 5a is a repeating unit, and the number of repeating units is determined according to the molecular weight of the radix tetrastigme polysaccharide.
Optionally, the weight average molecular weight of the radix tetrastigme polysaccharide prepared based on Phellinus linteus fermentation is preferably 10kDa-15kDa, more preferably 12kDa-13kDa, and most preferably 12.3kDa-12.8kDa.
The radix tetrastigme polysaccharide prepared based on Phellinus linteus fermentation can be obtained by extracting, separating and purifying from Phellinus linteus fermented radix tetrastigme by adopting a preparation method of water-soluble polysaccharide, and is preferably obtained by extracting, separating and purifying from Phellinus linteus fermented radix tetrastigme with hot water. Comprising the following steps: the method comprises the steps of obtaining crude polysaccharide of the radix tetrastigme by Phellinus linteus fermentation radix tetrastigme-water extraction and alcohol precipitation and deproteinizing by a Sevage reagent, purifying by anion exchange chromatography and gel filtration chromatography, and freeze-drying to obtain the radix tetrastigme polysaccharide prepared based on Phellinus linteus fermentation. The specific technical scheme is as follows:
a preparation method of radix tetrastigme polysaccharide prepared based on Phellinus fermentation comprises the following steps:
(1) Preparing fermented radix tetrastigme crude polysaccharide: fermenting radix tetrastigme tubers by Phellinus, extracting the fermentation product with water, precipitating with ethanol, removing protein, and lyophilizing supernatant to obtain radix tetrastigme crude polysaccharide (number is F-THDP) fermented by Phellinus;
(2) Purifying: and (3) carrying out column chromatography filled with diethylaminoethyl cellulose (DEAE cellulose) ion exchanger on the aqueous solution of the crude polysaccharide (F-THDP) of radix tetrastigme obtained in the step (1) after fermentation of the phellinus igniarius, collecting eluent eluted by 0.15mol/L-0.3mol/LNaCl aqueous solution, carrying out gel filtration chromatography, collecting eluent containing polysaccharide, dialyzing and freeze-drying to obtain the radix tetrastigme polysaccharide (F-THDP 2) after fermentation of the phellinus igniarius.
In order to achieve a better inventive effect, it is preferable that:
phellinus linteus (Sanghuangporus sanghuang) is selected from Phellinus linteus, and can be commercially available products.
In step (1), it includes: slicing radix tetrastigme tubers, sterilizing, cooling, inoculating Phellinus, fermenting and culturing, extracting a fermentation product with water at 90-100 ℃ to obtain an aqueous extract, concentrating to obtain a concentrated solution, removing proteins from a precipitate obtained by alcohol precipitation of the concentrated solution by using a Sevage reagent, and freeze-drying a supernatant to obtain crude radix tetrastigme polysaccharide fermented by Phellinus.
The fermentation culture conditions are suitable for Phellinus, and the preferred fermentation culture conditions are as follows: the temperature is 24-28 ℃, the humidity is 50-65%, and the culture time is 18-25 days.
The fermentation product is extracted by water, the water is used as an extraction solvent, the dosage is not strictly limited, and the water accounting for 2-6 times of the weight of the fermentation product can be selected. The fermentation product is preferably extracted with water at a temperature of 90℃to 100℃for a period of at least 2 hours.
The alcohol precipitation step is carried out using alcohol precipitation reagents commonly used in the art, preferably ethanol or aqueous ethanol solutions. The ethanol aqueous solution is ethanol aqueous solution with volume percentage concentration of more than or equal to 90 percent.
The Sevage reagent adopts Sevage reagent commonly used in the field, preferably adopts chloroform and n-butanol, wherein the volume ratio of chloroform to n-butanol is preferably 4:1.
In step (2), the conditions of the column chromatography packed with the DEAE cellulose ion exchanger are preferably: the elution is carried out by gradually increasing the concentration of the NaCl aqueous solution from 0 to 0.7 mol/L.
The DEAE-cellulose ion exchanger may be selected from commercially available products such as DEAE cellulose-52 ion exchanger, etc.
The gel filtration chromatography adopts polyacrylamide sephadex column filtration chromatography, and can be commercially available products such as Sephacryl S series (SephacrylS-100) and the like.
The conditions of the gel filtration chromatography are preferably: elution was performed with 0.05mol/L phosphate buffer and 0.15mol/L aqueous NaCl solution, wherein the volume ratio of phosphate buffer to aqueous NaCl solution was 2-3:1.
The preparation method of the phosphate buffer solution is according to a method commonly used in the art, for example, reference is made to the Chinese pharmacopoeia.
The dialysis is performed by using a dialysis bag with the aperture of 5000Da-8000Da so as to remove small molecules.
The radix tetrastigme polysaccharide F-THDP2 prepared based on Phellinus linteus fermentation has good anti-tumor activity. For example: the inhibition rate of F-THDP2 on MCF-7, A549 and HeLa is 51.63-57.51% when the concentration is 0.6mg/mL, and the inhibition rate of F-THDP2 on the three tumor cells is obviously enhanced compared with that of unfermented radix tetrastigme polysaccharide THDP2.
Compared with the action of unfermented radix tetrastigme polysaccharide THDP2 and a blank control group in the mice, the invention can remarkably increase the number of intestinal beneficial bacteria such as lactobacillus and bifidobacterium and reduce the number of intestinal pathogenic bacteria such as colibacillus, thereby regulating the intestinal flora structure and enhancing the intestinal microbial peristalsis. The radix tetrastigme polysaccharide F-THDP2 prepared based on Phellinus linteus fermentation has the effects of regulating the structural activity of intestinal flora (promoting the proliferation of intestinal probiotics such as lactobacillus and bifidobacterium, inhibiting the proliferation of intestinal pathogenic bacteria such as escherichia coli) and enhancing the peristaltic activity of intestinal microorganisms.
Therefore, the radix tetrastigme polysaccharide F-THDP2 prepared based on Phellinus linteus fermentation can be directly used as an anti-tumor functional product, and can also be used for preparing an anti-tumor functional product. The radix tetrastigme polysaccharide F-THDP2 prepared based on Phellinus linteus fermentation can also be directly used as a functional product for enhancing intestinal microbial peristalsis, such as a functional product for regulating intestinal flora structure, and can also be used for preparing a functional product for enhancing intestinal microbial peristalsis, such as a functional product for regulating intestinal flora structure. The functional products comprise foods, health products, medicines or daily chemical products and the like. For example, the radix tetrastigme polysaccharide F-THDP2 prepared based on Phellinus linteus fermentation can be used for preparing antitumor foods, antitumor health products, antitumor drugs, foods for enhancing intestinal microorganism peristalsis, health products for enhancing intestinal microorganism peristalsis, drugs for enhancing intestinal microorganism peristalsis and the like.
The raw materials, reagents, consumables, instruments and the like used in the invention can be commercially available products.
Compared with the prior art, the invention has the following advantages:
according to the characteristics of nutrient components and effective components of the radix tetrastigme, the invention aims at the defects of high content of starch inactive ingredients and the like in the current radix tetrastigme product, and the prepared polysaccharide after the radix tetrastigme fermentation has good biological activity in the aspects of resisting tumors and enhancing intestinal microorganism peristalsis by inoculating biological enzymes generated by Phellinus fermentation to carry out enzymolysis on the starch inactive materials in the radix tetrastigme, so that radix tetrastigme resources are fully utilized. The preparation method can effectively biodegrade and utilize the nutritional ingredients and the effective ingredients in the radix tetrastigme, and is beneficial to improving the activity of the active polysaccharide in the final product. The method has the advantages of natural sources of raw materials, controllable quality, no pollution to the environment and suitability for industrial production.
The high-order structure of the radix tetrastigme polysaccharide F-THDP2 fermented by the Phellinus linteus is completely different from that of the radix tetrastigme polysaccharide before being fermented, and the biological activity of the radix tetrastigme polysaccharide is also different. After Phellinus linteus fermentation, the newly produced radix tetrastigme polysaccharide has higher medicinal value. The radix tetrastigme polysaccharide F-THDP2 fermented by the Phellinus linteus has good biological activity, such as good anti-tumor activity and good intestinal microorganism peristaltic activity enhancement, and has wide practical application value. The method for obtaining the radix tetrastigme polysaccharide F-THDP2 by the Phellinus linteus fermentation is convenient to operate, can obtain macromolecules with higher order and definite structure, and can be industrially popularized and produced.
The invention researches the change of the structure and activity of the radix tetrastigme polysaccharide F-THDP2 fermented by the Phellinus linteus. The primary monosaccharide composition and molecular weight of the Phellinus linteus-fermented radix tetrastigme polysaccharide F-THDP2 were significantly changed compared to the unfermented radix tetrastigme polysaccharide THDP2. F-THDP2 has a molecular weight of 12.3kDa to 12.8kDa. In addition, there was a significant difference in the glycosidic chains of F-THDP2 and THDP2, 1,2-linked alpha-D-Galp and 1,6-linked alpha-D-Manp backbones were established in F-THDP2, which was significantly different from the 1,4-linked alpha-D-Glcp and 1,4-linked beta-D-Galp backbones in THDP2. Furthermore, F-THDP2 exhibits a more flexible chain conformation in aqueous solution than THDP2. Remarkably, F-THDP2 showed better inhibition of HeLa cells by Fas/fasl-mediated Caspase-3 signaling pathway compared to the original polysaccharide. The structural and biological activity changes show that the fermentation modification of Phellinus is a novel method with great prospect, can effectively convert starch and other polysaccharides in radix tetrastigme into biological macromolecules with high biological activity, and has potential application prospect in industry.
Drawings
FIG. 1 is a graph showing Absorbance at 490nm (Absorbance at 490nm, designated A490) of an eluate collected by DEAE-cellulose-52 ion exchange chromatography of an aqueous solution of crude polysaccharide F-THDP of Fleckedflesh polypore fermented by Phellinus in example 1, with Tube numbers.
FIG. 2 shows the Absorbance at 490nm (absorptance at 490 nm) curve of polysaccharide F-THDP 2-containing eluate purified by polyacrylamide sephadex (Sephacryl S-100) in example 1, tube numbers.
FIG. 3 is a GC-MS spectrum of polysaccharide acetylated products; a is a standard substance control spectrum, B is a radix tetrastigme polysaccharide THDP2 sample spectrum before fermentation; c is a sample map of the polysaccharide F-THDP2 of the radix tetrastigme after fermentation; wherein, the ordinate Relative Abundance is the response value, and the abscissa is the retention time: minutes (min), rham is rhamnose, rib is ribose, ara is arabinose, fuc is fucose, xyl is xylose, man is mannose, glc is glucose, gal is galactose.
FIG. 4a is F-THDP2 1 H-NMR spectrum, FIG. 4b shows F-THDP2 13 C-NMR spectrum, FIG. 4C HSQC spectrum of F-THDP2, FIG. 4d COSY spectrum of F-THDP2, FIG. 4e TCOSY spectrum of F-THDP2, FIG. 4F F-THDP2 1 H- 13 C HMBC pattern, FIG. 4g is NOESY pattern of F-THDP2.
FIG. 5a is a structural formula of F-THDP 2; fig. 5b is a structural formula of THDP2.
FIG. 6a is THDP2 1 The H-NMR spectrum, FIG. 6b is THDP2 13 C-NMR spectrum, FIG. 6C HSQC spectrum of THDP2, FIG. 6d COSY spectrum of THDP2, FIG. 6e TCOSY spectrum of THDP2, FIG. 6f THDP2 1 H- 13 HMBC profile, fig. 6g is NOESY profile of THDP2.
Fig. 7a is a laser light scattering diagram of THDP 2; FIG. 7b is a laser light scattering diagram of F-THDP 2; FIG. 7c is a plot of molecular weight Mw versus viscosity [ eta ] for F-THDP 2; wherein, the ordinate Relative Scale is the Relative proportion, and the abscissa time (min) is the time (min);
FIG. 8a is a two-dimensional Atomic Force Microscope (AFM) map of THDP 2; FIG. 8b is a graph of linear height measurements in the topography of FIG. 8 a; FIG. 8c is a graph of a linear energy spectrum of the topography of FIG. 8 a; FIG. 8d is a two-dimensional atomic force microscope map of F-THDP 2; FIG. 8e is a graph of linear height measurements in the profile of FIG. 8 d; FIG. 8f is a graph of a linear energy spectrum of the topography of FIG. 8 d.
FIG. 9 is a graph showing the Absorbance at 490nm (Absorbance at 490nm, designated A490) of an eluate collected by DEAE cellulose-52 ion exchange chromatography of an aqueous solution of crude polysaccharide THDP of radix tetrastigme without Phellinus linteus fermentation in comparative example 1, tube numbers.
FIG. 10 is a graph showing Absorbance at 490nm (Absorbance at 490 nm) of an eluate containing polysaccharide THDP2 purified by polyacrylamide sephadex (Sephacryl S-100) in comparative example 1, tube numbers.
Detailed Description
The following describes the invention in further detail with reference to specific embodiments and drawings, but is not intended to limit the scope of the technical scheme of the invention.
Phellinus linteus is Phellinus linteus (Sanghuangporus sanghuang).
Example 1
(1) Preparing fermented radix tetrastigme crude polysaccharide: taking 500g of radix tetrastigme tuber obtained by drying by adopting a vacuum freeze drying method, cutting into slices with the length of 3mm plus or minus 2mm, sterilizing at 121 ℃ for 25min, cooling to room temperature, and inoculating Phellinus; in a sealed Erlenmeyer flask, the humidity is kept at 58% +/-2%, fermentation culture is carried out for 20 days at 26 ℃, the obtained fermentation product is extracted for 2.5 hours at 95 ℃ +/-5 ℃ by using distilled water with the weight of 3 times of the weight of the fermentation product, the water extract is concentrated to obtain a concentrated solution, ethanol is added into the concentrated solution until the final concentration of the concentrated solution is 80% (v/v), alcohol precipitation is carried out, the obtained precipitate is subjected to removal of protein by adopting a Sevage reagent (the volume ratio of chloroform to n-butanol is 4:1), and the supernatant is freeze-dried to obtain the crude polysaccharide of radix tetrastigme (with the number of F-THDP) after fermentation of Phellinus.
(2) Purifying: and (3) separating and purifying the crude polysaccharide of radix tetrastigme (F-THDP) obtained in the step (1) after fermentation by Phellinus linteus. 10mL of crude polysaccharide aqueous solution (15 mg/mL) of the sample is injected into a column (6.5 cm. Times.40 cm) filled with DEAE cellulose-52 ion exchanger, and elution is carried out by increasing the gradient of NaCl aqueous solution (0-0.7 mol/L) at a flow rate of 2.5mL/min; 6mL of the eluate was collected in this order from each tube, and the absorbance at 490nm of each tube was measured by the phenol-sulfuric acid method, and the result was shown in FIG. 1, wherein the polysaccharide in the collected eluate of 0.05mol/L to 0.1mol/LNaCl aqueous solution was designated F-THDP1, the polysaccharide in the collected eluate of 0.15mol/L to 0.3mol/LNaCl aqueous solution was designated F-THDP2, and the polysaccharide in the collected eluate of 0.35mol/L to 0.5mol/LNaCl aqueous solution was designated F-THDP3. The eluent containing polysaccharide F-THDP1, the eluent containing polysaccharide F-THDP2 and the eluent containing polysaccharide F-THDP3 in FIG. 1 are dialyzed (dialysis bag with aperture of 5000Da-8000 Da) respectively, and each dialysate is chromatographed on a polyacrylamide sephadex (Sephacryl S-100) column (1.2 cm. Times.90 cm), and the loaded amount is 5mL, and eluted with 0.05mol/L phosphate buffer (pH 7.0) and 0.15mol/LNaCl aqueous solution (volume ratio of phosphate buffer to NaCl aqueous solution is 2:1) at a flow rate of 0.5 mL/min; collecting 3mL of eluent from each tube, measuring absorbance of each tube eluent at 490nm by phenol-sulfuric acid method, collecting eluent rich in polysaccharide F-THDP2 after chromatography, and the result is shown in figure 2; collecting the eluent rich in polysaccharide F-THDP1 after chromatography, and collecting the eluent rich in polysaccharide F-THDP3 after chromatography. Dialyzing the eluent rich in polysaccharide F-THDP2 (dialysis bag with aperture of 5000Da-8000 Da), and lyophilizing the dialysate to obtain radix Apioris Fortunei polysaccharide component F-THDP2 fermented by Phellinus. Dialyzing the eluent rich in polysaccharide F-THDP1 (dialysis bag with aperture of 5000Da-8000 Da), and lyophilizing the dialysate to obtain radix Apioris Fortunei polysaccharide component F-THDP1 fermented by Phellinus. Dialyzing the eluent rich in polysaccharide F-THDP3 (dialysis bag with aperture of 5000Da-8000 Da), and lyophilizing the dialysate to obtain radix Apioris Fortunei polysaccharide component F-THDP3 fermented by Phellinus.
Comparative example 1
(1) Preparation of radix tetrastigme crude polysaccharide: taking 500g of radix tetrastigme tuber obtained by drying by adopting a vacuum freeze drying method, extracting with distilled water at 95+/-5 ℃ for 2.5 hours, concentrating the obtained water extract to obtain a concentrated solution, adding ethanol into the concentrated solution until the final concentration of the concentrated solution is 80% (v/v), carrying out alcohol precipitation, removing protein from the obtained precipitate by adopting a Sevage reagent (the volume ratio of chloroform to n-butanol is 4:1), and freeze-drying supernatant to obtain crude polysaccharide (number is THDP) before fermentation of radix tetrastigme.
(2) Purifying: the crude polysaccharide of radix Apioris (THDP) obtained in the step (1) was isolated and purified according to the purification procedure in example 1, wherein the polysaccharide in the collected eluate of 0.05mol/L to 0.1mol/LNaCl aqueous solution was designated THDP1, the polysaccharide in the collected eluate of 0.15mol/L to 0.3mol/LNaCl aqueous solution was designated THDP2, and the polysaccharide in the collected eluate of 0.35mol/L to 0.5mol/LNaCl aqueous solution was designated THDP3. Finally, the radix tetrastigme polysaccharide component THDP2 before fermentation is obtained.
Example 2
(1) Preparing fermented radix tetrastigme crude polysaccharide: taking 500g of radix tetrastigme tuber obtained by drying by adopting a vacuum freeze drying method, cutting into slices with the length of 3mm plus or minus 2mm, sterilizing for 30min at 120 ℃, cooling to 24 ℃, and inoculating Phellinus; in a sealed Erlenmeyer flask, the humidity is kept at 55% +/-5%, the fermentation and culture are carried out for 25 days at 24 ℃, the obtained fermentation product is extracted for 4 hours at 95+/-5 ℃ by using distilled water with the weight of 6 times of the weight of the fermentation product, the water extract is concentrated to obtain a concentrated solution, an ethanol aqueous solution with the volume percentage concentration of 90% is added into the concentrated solution until the final concentration of the concentrated solution is 80% (v/v) for alcohol precipitation, the obtained precipitate is subjected to protein removal by adopting a Sevage reagent (the volume ratio of chloroform to n-butanol is 4:1), and the supernatant is freeze-dried to obtain the radix tetrastigme crude polysaccharide (with the number of F-THDP) after the fermentation of the Phellinus.
(2) Purifying: 10mL of an aqueous solution (20 mg/mL) of crude polysaccharide from Hemsleya stenoptera (F-THDP) was injected into a column (6.5 cm. Times.40 cm) packed with DEAE-cellulose-52 ion exchanger, wherein the column (1.2 cm. Times.90 cm) of Sephacryl S-100 was eluted with 0.05mol/L of phosphate buffer (pH 7.0) and 0.15mol/L of aqueous solution of LNaCl (the volume ratio of phosphate buffer to aqueous solution of NaCl: 3:1) at a flow rate of 0.5mL/min, and the remaining operations were the same as in example 1 to obtain a polysaccharide fraction F-THDP2 of Hemsleya stenoptera after fermentation.
Example 3
(1) Preparing fermented radix tetrastigme crude polysaccharide: taking 500g of radix tetrastigme tuber obtained by drying by adopting a vacuum freeze drying method, cutting into slices with the length of 3mm plus or minus 2mm, sterilizing for 20min at the temperature of 125 ℃, cooling to the temperature of 25 ℃, and inoculating Phellinus; in a sealed Erlenmeyer flask, the humidity is kept at 60% +/-5%, fermentation culture is carried out for 18 days at 28 ℃, the obtained fermentation product is extracted for 2 hours at 95+/-5 ℃ by using distilled water with the weight of 2 times of the weight of the fermentation product, the water extract is concentrated to obtain a concentrated solution, an ethanol aqueous solution with the volume percentage concentration of 95% is added into the concentrated solution until the final concentration of the concentrated solution is 80% (v/v) for alcohol precipitation, the obtained precipitate is subjected to protein removal by adopting a Sevage reagent (the volume ratio of chloroform to n-butanol is 4:1), and the supernatant is freeze-dried to obtain the radix tetrastigme crude polysaccharide (with the number of F-THDP) after fermentation by Phellinus.
(2) Purifying: 10mL of an aqueous solution (25 mg/mL) of crude radix tetrastigme polysaccharide (F-THDP) was injected into a column (6.5 cm. Times.40 cm) packed with DEAE-cellulose-52 ion exchanger, and the remaining operations were the same as in example 1 to obtain a radix tetrastigme polysaccharide component F-THDP2 after fermentation with Phellinus linteus.
Regarding structural identification and performance analysis of polysaccharide before and after fermentation of radix tetrastigme:
1. monosaccharide composition
Taking 3mg of polysaccharide, placing the polysaccharide into a thin-wall long test tube, adding 4.0mL of 2.0mol/L trifluoroacetic acid, sealing the tube, and hydrolyzing at 110 ℃ for 2 hours. After hydrolysis, the solution in the test tube was evaporated to dryness under reduced pressure at a temperature lower than 40 ℃, then evaporated to dryness by adding methanol, and the step of "evaporating to dryness by adding methanol" was repeated 4 to 5 times to completely remove trifluoroacetic acid, thereby obtaining a completely acid-hydrolyzed sample.
Dissolving various monosaccharide standard substances and completely acid hydrolyzed samples in 3mL of distilled water respectively, adding 30mg of sodium borohydride, sealing, reducing for 3h at room temperature, neutralizing excessive sodium borohydride with glacial acetic acid, adding a small amount of methanol, concentrating under reduced pressure, evaporating to dryness, and repeating the steps of adding a small amount of methanol, concentrating under reduced pressure, evaporating to dryness for 4-5 times. 4mL of acetic anhydride (acetic anhydride) is added, the reaction is carried out for 1h at 100 ℃, 3mL of toluene is added, and the mixture is concentrated under reduced pressure and evaporated to dryness, thus obtaining an acetylated product. The acetylated products are respectively dissolved by 3mL of chloroform and transferred to a separating funnel, distilled water with equal amount is added for fully and uniformly mixing, after standing, the upper layer aqueous solution is removed, and the steps are repeated for 3-4 times. The chloroform layer is dried with a proper amount of anhydrous sodium sulfate, filtered, and the volume is fixed to 10mL by chloroform, thus obtaining the acetylated product, and the acetylated product is analyzed by GC-MS.
GC-MS conditions: DB-5 capillary column (30 m x 0.25mm x 0.25 μm) is adopted, the temperature is programmed (the initial column temperature is 120 ℃, the temperature is increased to 240 ℃ at 10 ℃/min and kept for 6.5 min), the interface temperature is 250 ℃, the ion source temperature is 250 ℃, the sample injection amount is 2.0 mu L, helium is used as carrier gas, and the flow rate is 1.0mL/min.
The GC-MS spectrum of the acetylated product of the polysaccharide F-THDP2 hydrolysate after fermentation is shown in FIG. 3C, the GC-MS spectrum of the acetylated product of the polysaccharide THDP2 hydrolysate before fermentation is shown in FIG. 3B, and the corresponding monosaccharide standard spectrum (FIG. 3A) is shown:
the monosaccharide composition of polysaccharide F-THDP2 in example 1 consisted of D-galactose, D-mannose and L-fucose, wherein the molar ratio of D-galactose, D-mannose and L-fucose was 12.6:2.3:1.0, glucose was not contained in F-THDP2, and the content of galactose was drastically increased, galactose was the main monosaccharide of F-THDP2, and the mole percentage of galactose was 79.25%.
The monosaccharide composition of polysaccharide F-THDP2 in example 2 consisted of D-galactose, D-mannose and L-fucose, wherein the molar ratio of D-galactose, D-mannose and L-fucose was 11.8:2.6:1.0, glucose was not contained in F-THDP2, and the content of galactose was drastically increased, galactose was the main monosaccharide of F-THDP2, and the molar percentage of galactose was 76.62%.
The monosaccharide composition of polysaccharide F-THDP2 in example 3 consisted of D-galactose, D-mannose and L-fucose, wherein the molar ratio of D-galactose, D-mannose and L-fucose was 12.0:2.0:1.0, glucose was not contained in F-THDP2, and the content of galactose was drastically increased, galactose was the main monosaccharide of F-THDP2, and the mole percentage of galactose was 80.0%.
The monosaccharide composition of polysaccharide THDP2 in comparative example 1 consisted of D-glucose, D-galactose and D-mannose, with a molar ratio of D-glucose, D-galactose and D-mannose of 9.5:4.1:1.1, indicating that THDP2 is free of fucose, glucose is the main monosaccharide of THDP2, and the molar percentage of glucose is 64.63%.
The above results indicate that glucose in radix tetrastigme is metabolized and converted primarily to galactose during fermentation. Furthermore, F-THDP2 contains a new monosaccharide compared to THDP 2: fucose, indicating that F-THDP2 is a new polysaccharide biosynthetically produced during fermentation, not the degradation product of the original tetrastigme polysaccharide. 2. Physicochemical property component and molecular weight detection
The weight percentage of the total polysaccharide detected by the phenol-sulfuric acid method of the radix tetrastigme polysaccharide F-THDP2 fermented by Phellinus in the example 1 is 99.76%. The laser light scattering graph (fig. 7 b) shows that the detected 90 ° light scattering LS signal, the difference detector RI signal and the signal peak of the viscosity detector VIS have similar peak shapes, almost completely overlapping, which indicates that the delay between the two detectors has been accurately corrected. The RI signal of sample F-THDP2 showed a single symmetrical peak shape for the polysaccharide, indicating that F-THDP2 is a polysaccharide with a uniform molecular weight distribution, with a molecular weight Mw=1.23×10 4 Da。
In the embodiment 2, the weight percentage of the total polysaccharide is 99.13% detected by a phenol-sulfuric acid method of the radix tetrastigme polysaccharide F-THDP2 fermented by Phellinus linteus; the RI signal shows a single symmetrical peak pattern for the polysaccharide, indicating that F-THDP2 is a polysaccharide with a uniform molecular weight distribution, with a molecular weight Mw=1.26X10 4 Da。
In example 3, the weight percentage of the total polysaccharide of the radix tetrastigme polysaccharide F-THDP2 fermented by Phellinus is 99.37% by using a phenol-sulfuric acid method; the RI signal shows a single symmetrical peak pattern for the polysaccharide, indicating that F-THDP2 is a polysaccharide with a uniform molecular weight distribution, with a molecular weight Mw=1.28X10 4 Da。
The weight percentage of the total polysaccharide of the radix tetrastigme polysaccharide component THDP2 before fermentation (unfermented) in comparative example 1 is 99.71 percent by using a phenol-sulfuric acid method. The laser light scattering graph (fig. 7 a) shows that the detected 90 ° light scattering LS signal, the difference detector RI signal and the signal peak of the viscosity detector VIS have similar peak shapes, almost completely overlapping, which indicates that the delay between the two detectors has been accurately corrected. The RI signal of sample THDP2 showed a single symmetrical peak shape for polysaccharide, indicating that THDP2 is a polysaccharide with uniform molecular weight distribution, with molecular weight mw=3.58×10 4 Da。
THDP2 and F-THDP2 had molecular weights of 3.58X10, respectively 4 g/mol and 1.23X10 4 g/mol-1.28×10 4 g/mol, showing that the molecular weight of polysaccharide F-THDP2 after the fermentation of the radix tetrastigme is obviously lower than that of the unfermented polysaccharide of the radix tetrastigmeTHDP2。
3. Methylation analysis
A2 mg polysaccharide sample was dissolved in 1mL dimethyl sulfoxide (DMSO), sealed with nitrogen, sonicated to aid dissolution for a short time, and then methylated according to the method of Ciucan, et al (Ciucan, L., & Kerek, F.. A simple and repid method for permethylation ofcarbohydrates. Carbohydrate Research,131, 209-217).
F-THDP2 is subjected to three times of methylation, acid hydrolysis, reduction and acetylation to prepare a partially methylated Aldi alcohol acetate derivative, and GC-MS analysis is carried out (see table 1). As can be seen from table 1: polysaccharide F-THDP2 after fermentation of radix tetrastigme contains 6 residues, 2-Galp-1 (26.72 mol%), 6-Galp-1 (6.43 mol%), 2,6-Galp-1 (21.15 mol%), 2, 6-mann-1 (13.35 mol%), T-Fucp (6.32 mol%) and T-Galp (26.03 mol%), respectively.
THDP2 was subjected to three methylation steps, acid hydrolysis, reduction and acetylation to prepare a partially methylated Aldi alcohol acetate derivative, and GC-MS analysis was performed (see Table 2). As can be seen from table 2: the glycosidic linkage connection mode of the non-fermented polysaccharide THDP2 of radix tetrastigme mainly comprises T-Manp (7.11 mol percent), T-Glcp (21.44 mol percent), 4-Galp-1 (28.49 mol percent), 4,6-Glcp-1 (28.67 mol percent) and 4-Glcp-1 (14.29 mol percent). The glycosidic bond connection mode of polysaccharide before and after the fermentation of the radix tetrastigme is obviously changed.
TABLE 1F-THDP 2 methylation analysis
| Methylated monosaccharide residues | Connection mode | Molar ratio of | Major fragment ions (m/z) |
| 2,3,4,6-Me 4 -Fucp | 1-linked Fucp | 6.32 | 59,71,89,101,117,129,144,161 |
| 2,3,4,6-Me 4 -Galp | 1-linked Galp | 26.03 | 87,99,102,117,129,205 |
| 3,4,6-Me 3 -Galp | 1,2-linked Galp | 26.72 | 59,71,129,145,161,189 |
| 2,3,4-Me 3 -Galp | 1,6-linked Galp | 6.43 | 71,87,99,117,129,189,233 |
| 3,4-Me 2 -Galp | 1,2,6-linked Galp | 21.15 | 87,99,129,189 |
| 3,4-Me 2 -Manp | 1,2,6-linked Manp | 13.35 | 87,99,129,189 |
TABLE 2 THDP2 methylation analysis
| Methylated monosaccharide residues | Connection mode | Molar ratio of | Major fragment ions (m/z) |
| 2,3-Me 2 -Glcp | 1,4,6-linked Glcp | 28.67 | 43,85,102,117,127,159,201,261 |
| 2,3,6-Me 3 -Glcp | 1,4-linked Glcp | 14.29 | 43,45,71,87,102,113,117,129,233 |
| 2,3,4,6-Me 4 -Manp | 1-linked Manp | 7.11 | 43,59,71,87,101,117,129,145,161,205 |
| 2,3,4,6-Me 4 -Glcp | 1-linked Glcp | 21.44 | 43,45,59,71,87,101,117,129,145,205 |
| 2,3,6-Me 3 -Galp | 1,4-linked Galp | 28.49 | 43,59,71,87,99,102,117,129,159,233 |
4. Nuclear magnetic resonance
60mg of polysaccharide was dissolved in 0.5mL of deuterium water, respectively, and 600MHz NMR scan was performed on Bruker-AVIII500M, switzerland.
According to F-THDP2 1 H-NMR (see FIG. 4 a), 13 C-NMR (see FIG. 4 b) combined with HSQC spectra (see FIG. 4C) etc., 6 peaks were detected more significantly for analysis. The main chain of polysaccharide F-THDP2 after the radix tetrastigme fermentation consists of a → 2) -alpha-D-Galp- (1 → sum → 2, 6) -alpha-D-Manp- (1 → main chain, and the branched chain consists of end groups beta-D-Galp, → 6) -alpha-3-O-Me-D-Galp- (1 → and end groups alpha-L-Fucp residues.
According to THDP2 1 H-NMR (see FIG. 6 a), 13 C-NMR (see FIG. 6 b) in combination with HSQC spectrum (see FIG. 6C) etc., the main chain of the non-fermented polysaccharide THDP2 of radix tetrastigme consists of → 4) - α -D-Glcp- (1 → and → 4) - β -D-Galp- (1 → and the branches consist of α -D-Manp and α -D-Glcp.
TABLE 3 chemical shift of F-THDP2 sugar residues
TABLE 4 chemical shift holohd 2 sugar residues
In conclusion, it is confirmed that F-THDP2 is composed of polysaccharide with a weight percentage of more than 99%; the monosaccharide composition is composed of D-galactose, D-mannose, L-fucose and 3-O-methyl-D-galactose, wherein the molar ratio of D-galactose, D-mannose, L-fucose and 3-O-methyl-D-galactose is 8.5-11.0:1.5-3:0.8-1.5:1.0. the laser light scattering method proves that the polymer is a single component and has a weight average molecular weight of 12.3KDa to 12.8KDa. The nuclear magnetic resonance spectrum determines the connection mode of the glycosidic bond, and the main chain structure of the structural unit of the polysaccharide is (1-2) connected alpha-D-galactose residue and (1-6) connected alpha-D-mannose residue; the C-6 position of an alpha-D-galactose residue with a (1-2) linkage is replaced by a beta-D-galactose end group, the C-2 position of two alpha-D-mannose residues with a (1-6) linkage on the main chain is replaced by a first branched chain consisting of an alpha-3-O-methyl-D-galactose residue with a beta-D-galactose end group with a (1-6) linkage, and an alpha-L-fucose end group. The specific structural unit may be one structural unit shown in fig. 5a, or may be structural units in which three branches are arranged in other arrangement sequences.
5. Higher conformation of polysaccharide chains
THDP2 and F-THDP2 had molecular weights of 3.58X10, respectively, using a SEC-MALLS-Vis laser light scattering system 4 g/mol and 1.23X10 4 g/mol, the molecular weight of polysaccharide F-THDP2 after the fermentation of the radix tetrastigme is lower than that of polysaccharide THDP2 after the fermentation of the radix tetrastigme.
The calculation of the Mark-Houwink-Sakurada equation shows that the alpha value of the polysaccharide THDP2 before fermentation is 0.32, and the alpha value of the polysaccharide F-THDP2 after fermentation is 0.76, which shows that the polysaccharide THDP2 before fermentation is in a compact chain structure in solution, and the polysaccharide F-THDP2 after fermentation is converted from the compact chain structure to a free extended chain conformation in solution.
The two-dimensional atomic force morphology of the polysaccharide is obtained by respectively smearing the F-THDP2 aqueous solution in the embodiment 1, the embodiment 2 and the embodiment 3 with the mass percentage concentration of 5-10% on a mica sheet, and the result shows that the polysaccharide chains of the THDP2 are mutually wound to form an aggregate to form a compact coil with the height of 1.18nm plus or minus 0.09nm (the embodiment 1 is shown in figure 8 a). In contrast, from F-THDP2 a number of extended molecular chains were observed (example 1, FIG. 8 d), with chain heights of 0.51 nm.+ -. 0.07nm, and F-THDP2 exhibited a more extended flexible conformation. 6. Comparison of biological Activity of radix Apioris Fortunei polysaccharide before and after fermentation
1. In vitro anti-tumor (MTT method)
Taking A549 cells, heLa cells and MCF-7 cells in logarithmic growth phase, digesting the adherent cells with 0.25% (mass percent) trypsin,adjusting cell concentration to 1-10X10 with RPMI1640 complete culture solution 4 The cell suspension of each mL is inoculated into a 96-well culture plate according to 500 cells/well after accurate cell count, 100 mu L of each well is added, and the mixture is placed at 37 ℃ and 5% (volume percent) of CO 2 Culturing in saturated humidity incubator for 24 hr, adding complete culture medium to dilute the sample into 10 μl of sample solution with different concentrations, adding equal volume of RPMll640 complete culture medium as blank control group, arranging 5 multiple holes each, and transferring cell culture plate into CO 2 In an incubator at 37℃5% CO 2 Culturing for 24h and 48h under saturated humidity condition. After the completion of the culture, 50. Mu.L of a freshly prepared solution of tetramethylazo-salt (MTT) was added to each well, incubated for 4 hours, MTT was reduced, and when the appearance of a filamentous purple crystal around the cells in the well plate was seen under an inverted microscope, 200. Mu.L of dimethyl sulfoxide (DMSO) was added to each well, and after shaking with a plate shaker, the Optical Density (OD) was measured at 570nm using an enzyme-labeled instrument, and a control group was set, and the inhibition ratio of the cells was calculated according to the formula. Inhibition (%) = (control OD value-dosing OD value)/control OD value x 100%.
TABLE 5 comparison of antitumor Activity of radix Apioris Fortunei polysaccharide before and after fermentation
| Sugar component | Concentration (mg/mL) | HeLa cell inhibition Rate (%) |
| F-THDP2 in example 1 | 0.6 | 57.51 **## |
| F-THDP2 in example 2 | 0.6 | 56.34 **## |
| F-THDP2 in example 3 | 0.6 | 56.03 **## |
| F-THDP1 in example 1 | 0.6 | 13.72 |
| F-THDP3 in example 1 | 0.6 | 18.54 |
| THDP2 | 0.6 | 41.67 |
| Blank control group | 0 | 2.31 |
Note that: F-THDP2 was compared with F-THDP1, F-THDP3, and the blank, respectively, and P <0.05 and P <0.01 respectively. F-THDP2 is compared to comparative example 1THDP2, # represents P <0.05, # represents P <0.01.
TABLE 6 comparison of antitumor Activity of radix Apioris Fortunei polysaccharide before and after fermentation
| Sugar component | Concentration (mg-mL) | Inhibition of A549 cells (%) |
| F-THDP2 in example 1 | 0.6 | 52.85 **## |
| F-THDP2 in example 2 | 0.6 | 52.21 **## |
| F-THDP2 in example 3 | 0.6 | 51.75 **## |
| F-THDP1 in example 1 | 0.6 | 15.23 |
| F-THDP3 in example 1 | 0.6 | 12.66 |
| THDP2 | 0.6 | 40.12 |
| Blank control group | 0 | 1.98 |
Note that: F-THDP2 was compared with F-THDP1, F-THDP3, and the blank, respectively, and P <0.05 and P <0.01 respectively. F-THDP2 is compared to comparative example 1THDP2, # represents P <0.05, # represents P <0.01.
TABLE 7 comparison of antitumor Activity of Aphyllophorum polysaccharide before and after fermentation
Note that: F-THDP2 was compared with F-THDP1, F-THDP3, and the blank, respectively, and P <0.05 and P <0.01 respectively. F-THDP2 is compared to comparative example 1THDP2, # represents P <0.05, # represents P <0.01.
Comparing the in vitro anti-tumor capability of 3 components F-THDP2, F-THDP1 and F-THDP3 obtained by separating the fermented radix tetrastigme crude polysaccharide by DEAE cellulose-52, as can be seen from tables 5-7, compared with a blank control group, the F-THDP1 and F-THDP3 have no obvious difference in inhibition rate of 3 cells A549 cells, heLa cells and MCF-7 cells, indicating that the F-THDP1 and F-THDP3 components have no capability of inhibiting tumor cells; compared with THDP2, the anticancer activity of F-THDP2 on 3 cell lines is obviously better than that of THDP2 (P < 0.01) at the same concentration, which indicates that the anticancer activity of polysaccharide F-THDP2 after radix tetrastigme fermentation is obviously enhanced.
2. Action on intestinal microorganisms
100 healthy mice are randomly grouped, 10 mice in each group are administrated by gastric lavage 1 time a day, the concentration of polysaccharide aqueous solution is 0.1mg/mL, the dosage is 50mg polysaccharide/kg body weight of the mice each time, the mice are sacrificed after continuous administration for 14d, and tissues are collected. The mice in the blank group were perfused with an equal volume of physiological saline to the dose of the dosing group, 1 time per day, sacrificed after 14 days, and tissues were collected. The cecal content of the mice was placed in a centrifuge tube containing 4.5mL of sterile physiological saline and shaken well in a micro-shaker, which was 10 -1 Multiple dilution, 10 -1 Centrifuging the diluted solution, collecting supernatant 0.5mL, adding into a centrifuge tube containing 4.5mL sterile physiological saline, shaking in a micro-oscillator to obtain 10 -2 Multiple dilution, according to the above steps until dilution to 10 -7 Multiple times. Will be10 -7 The bacteria were cultured in the double dilution, lactobacillus was anaerobically cultured at 37℃for 48 hours using LBS agar medium, E.coli was aerobically cultured at 37℃for 24 hours using EMB agar medium, bifidobacterium was anaerobically cultured at 37℃for 48 hours using BS agar medium, and 3 replicates of each bacteria culture test were set. After the bacterial culture test, the bacteria were subjected to plate colony counting, and the number of bacteria contained in lactobacillus, bifidobacterium and escherichia coli was counted.
Table 8 effect units on microbial flora: lg CFU/g (10) 1 CFU/g)
| Sugar component | Lactobacillus (lactic acid bacterium) | Bifidobacterium strain | Coli bacterium |
| F-THDP2 in example 1 | 11.03±0.14 **## | 9.87±0.17 **## | 4.56±0.14 **## |
| F-THDP2 in example 2 | 11.09±0.15 **## | 9.74±0.15 **## | 4.62±0.11 **## |
| F-THDP2 in example 3 | 11.27±0.13 **## | 9.92±0.14 **## | 4.67±0.13 **## |
| F-THDP1 in example 1 | 7.53±0.12 | 7.09±0.14 | 6.25±0.12 |
| F-THDP3 in example 1 | 7.41±0.13 | 7.02±0.12 | 6.27±0.14 |
| THDP2 | 7.98±0.14 | 6.87±0.11 | 6.31±0.11 |
| Blank group | 7.13±0.15 | 6.34±0.12 | 6.36±0.14 |
Note that: F-THDP2 was compared with F-THDP1, F-THDP3, blank, respectively, and P <0.05 and P <0.01.F-THDP2 is compared to comparative example 1THDP2, # represents P <0.05, # represents P <0.01.
Comparing the effect of 3 components F-THDP2, F-THDP1 and F-THDP3 obtained by separating the fermented radix tetrastigme crude polysaccharide by DEAE cellulose-52 on the intestinal microbiota of mice, it can be seen from Table 8 that, compared with a blank group, F-THDP1 and F-THDP3 have no obvious difference on lactobacillus, bifidobacterium and escherichia coli, indicating that the F-THDP1 and F-THDP3 components have no effect of regulating intestinal strains; F-THDP2 significantly increased the number of lactobacilli and bifidobacteria compared to THDP2 (p < 0.01), significantly decreased the number of E.coli (p < 0.01). Lactobacillus and bifidobacterium are common probiotics, and the proliferation of a large amount of lactobacillus and bifidobacterium can inhibit the proliferation of pathogenic microorganisms, so that the lactobacillus and bifidobacterium have important significance for guaranteeing intestinal health, and the F-THDP2 can regulate the intestinal flora structure.
The radix tetrastigme polysaccharide based on Phellinus linteus fermentation in the invention consists of more than 99% of polysaccharide by weight percent; the polysaccharide consists of galactose, mannose, fucose and 3-O-methyl-galactose, wherein the molar ratio of galactose, mannose, fucose and 3-O-methyl-galactose is 8.5-11.0:1.5-3:0.8-1.5:1.0. the weight average molecular weight of the radix tetrastigme polysaccharide after Phellinus fermentation is 10kDa-15kDa. The polysaccharide has strong biological activity, such as enhanced antitumor activity and enhanced intestinal microorganism peristalsis activity. The change of parameters in the preparation method does not affect the preparation of the polysaccharide, so that the preparation of the polysaccharide can be realized by any combination of parameters in the preparation method. And will not be described in detail herein.
Claims (10)
1. A radix tetrastigme polysaccharide prepared based on Phellinus fermentation is characterized by comprising more than 99% of polysaccharide by weight percent; the polysaccharide consists of galactose, mannose, fucose and 3-O-methyl-galactose, wherein the molar ratio of galactose, mannose, fucose and 3-O-methyl-galactose is 8.5-11.0:1.5-3:0.8-1.5:1.0.
2. the tetrastigme polysaccharide of claim 1, wherein the galactose is alpha-galactose and beta-galactose, the mannose is alpha-mannose, the fucose is alpha-fucose, and the 3-O-methyl-galactose is alpha-3-O-methyl-galactose.
3. The tetrastigme polysaccharide according to claim 1 or 2, wherein the galactose is α -D-galactose and β -D-galactose, the mannose is α -D-mannose, the fucose is α -L-fucose, and the 3-O-methyl-galactose is α -3-O-methyl-D-galactose.
4. A tetrastigme polysaccharide according to claim 3, wherein the backbone structure in the structural unit of the polysaccharide is a (1→2) linked α -D-galactose residue and a (1→6) linked α -D-mannose residue; the C-6 position of an alpha-D-galactose residue with a (1-2) linkage is replaced by a beta-D-galactose end group, the C-2 position of two alpha-D-mannose residues with a (1-6) linkage on the main chain is replaced by a first branched chain consisting of an alpha-3-O-methyl-D-galactose residue with a beta-D-galactose end group with a (1-6) linkage, and an alpha-L-fucose end group.
5. The tetrastigme polysaccharide of claim 4, wherein the beta-D-galactose terminal group is substituted at the C-6 position of any one (1→2) linked alpha-D-galactose residue on the backbone, the first branch and one alpha-L-fucose terminal group being arranged in any order at the C-2 position of two consecutive (1→6) linked alpha-D-mannose residues.
6. The tetrastigme polysaccharide of any one of claims 1-5, wherein the tetrastigme polysaccharide has a weight average molecular weight of from 10KDa to 15KDa.
7. The method for preparing a polysaccharide from radix tetrastigme according to any one of claims 1 to 6, comprising the steps of:
(1) Preparing fermented radix tetrastigme crude polysaccharide: fermenting radix tetrastigme tubers by Phellinus, extracting a fermentation product with water, precipitating with ethanol, removing protein and freeze-drying supernatant to obtain radix tetrastigme crude polysaccharide after Phellinus fermentation;
(2) Purifying: and (3) carrying out column chromatography filled with DEAE cellulose ion exchanger on the aqueous solution of the crude radix tetrastigme polysaccharide obtained in the step (1) after the fermentation of the Phellinus, collecting the eluent eluted by 0.15mol/L-0.3mol/LNaCl aqueous solution, carrying out gel filtration chromatography, collecting the eluent containing the polysaccharide, and carrying out dialysis and freeze-drying to obtain the radix tetrastigme polysaccharide after the fermentation of the Phellinus.
8. The method of claim 7, wherein step (1) comprises: slicing radix tetrastigme tubers, sterilizing, cooling, inoculating Phellinus, fermenting and culturing, extracting a fermentation product with water at 90-100 ℃ to obtain an aqueous extract, concentrating to obtain a concentrated solution, removing proteins from a precipitate obtained by alcohol precipitation of the concentrated solution by using a Sevage reagent, and freeze-drying a supernatant to obtain crude radix tetrastigme polysaccharide fermented by Phellinus.
9. The method according to claim 7 or 8, wherein the conditions of the fermentation culture are: the temperature is 24-28 ℃, the humidity is 50-65%, and the culture time is 18-25 days.
10. Use of a radix tetrastigme polysaccharide according to any one of claims 1-6 for the preparation of a functional product comprising a food, a health product, a pharmaceutical product or a daily chemical product for anti-tumour and/or enhancing intestinal microbial peristalsis.
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