CN116284464A - Polygonatum cyrtonema polysaccharide and preparation method and application thereof - Google Patents
Polygonatum cyrtonema polysaccharide and preparation method and application thereof Download PDFInfo
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
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Abstract
The invention relates to a polygonatum cyrtonema polysaccharide, a preparation method and application thereof, which specifically comprises the following steps: (1) Pulverizing dried rhizome of Polygonatum cyrtonema Fabricius, adding water, performing ultrasonic extraction, collecting extractive solution, and concentrating to obtain crude extract; (2) Adding proper amount of water into the crude extract, carrying out ultrasonic dissolution, then fixing the volume, adding absolute ethyl alcohol with the volume of 8-10 times, standing at 0-4 ℃, centrifuging, discarding supernatant, collecting precipitate, washing the precipitate with ethyl alcohol, and freeze-drying to obtain crude polysaccharide; (3) Preparing crude polysaccharide into crude polysaccharide solution with mass concentration of 1.0g/L, adding chloroform-n-butanol mixed solution, shaking, standing, centrifuging, discarding intermediate protein layer and lower organic layer, repeating the operation until no obvious white precipitate appears, adding 8-10 times of absolute ethanol into water layer, standing at 0-4deg.C, centrifuging, discarding supernatant, collecting precipitate, washing precipitate with ethanol, and lyophilizing to obtain rhizoma Polygonati polysaccharide.
Description
Technical Field
The invention belongs to the field of natural plant extracts, and particularly relates to a polygonatum cyrtonema polysaccharide and a preparation method and application thereof.
Background
The Polygonatum cyrtonema Sieb, also known as rhizoma polygonati, rhizoma zingiberis, is a dried rhizome of Polygonatum cyrtonema Sieb Polygonatum cyrtonema hua of Polygonatum genus of Liliaceae (Liliaceae), which is received in China pharmacopoeia (one part) of 2015 year edition together with dried rhizome of Polygonatum cyrtonema Sieb Polygonatum Kingianum Coll et hemsl. Is a plant with homology of medicine and food. China is mainly distributed in Yangtze river basin, guangdong, guangxi and other places. The polygonatum polysaccharide is a main pharmacological active ingredient of polygonatum cyrtonema, has the effects of reducing blood sugar and blood fat, resisting tumors, bacteria, inflammation, viruses, immunoregulation, oxidization and protecting liver and kidney, and the related research report of the polygonatum cyrtonema polysaccharide on intestinal flora is not found in literature search. The variation of the composition structure of intestinal flora has a certain relation with the immune function of the organism, such as obesity, insulin resistance, autoimmune diseases, inflammatory enteritis, autism, cardiovascular and cerebrovascular diseases, tumor and the like. Therefore, the method has great significance for researching the intestinal flora regulating activity of the polygonatum polysaccharide.
Disclosure of Invention
The invention provides a polygonatum cyrtonema polysaccharide with an effect of regulating intestinal flora, which is characterized in that the preparation method of the polygonatum cyrtonema polysaccharide comprises the following steps:
(1) Pulverizing dried rhizome of Polygonatum cyrtonema Fabricius, adding water, performing ultrasonic extraction, collecting extractive solution, and concentrating to obtain crude extract;
(2) Adding proper amount of water into the crude extract, carrying out ultrasonic dissolution, then fixing the volume, adding absolute ethyl alcohol with the volume of 8-10 times, standing at 0-4 ℃, centrifuging, discarding supernatant, collecting precipitate, washing the precipitate with ethyl alcohol, and freeze-drying to obtain crude polysaccharide;
(3) Preparing crude polysaccharide into crude polysaccharide solution with mass concentration of 1.0g/L, adding mixed solution of chloroform-n-butanol (volume ratio of 4:1), shaking, standing, centrifuging, discarding intermediate protein layer and lower organic layer, repeating the operation for 1-3 times until no obvious white precipitate appears, adding 8-10 times volume of absolute ethanol into water layer, standing at 0-4deg.C, centrifuging, discarding supernatant, collecting precipitate, washing precipitate with ethanol, and lyophilizing to obtain rhizoma Polygonati polysaccharide.
The water consumption in the step (1) is 6-10 times of the rhizome medicinal material quality of Polygonatum cyrtonema Fabricius, the ultrasonic extraction temperature is preferably 80-90 ℃, the extraction times are preferably 2-5 times, the ultrasonic frequency is 30-40kHz, the ultrasonic extraction time is 0.5-1.0h each time, and the water is selected from distilled water or deionized water. The pulverization is preferably carried out to 20-100 mesh.
The water consumption in the step (2) is preferably 0.6-1.0 times of the rhizome medicinal material mass of Polygonatum cyrtonema, the ultrasonic frequency is 30-40kHz, the standing time is preferably 8-10 h, and the centrifugal rotating speed is preferably 3000 r.min -1 The time is preferably 3-5min.
The dosage of the chloroform-n-butanol mixed solution in the step (3) is preferably 20% -25% of the volume of the crude polysaccharide solution; the standing time is preferably 3-5 h, and the centrifugal rotating speed is preferably 3000 r.min -1 The time is preferably 3-5min.
The ethanol which is washed and precipitated in the steps (2) and (3) is preferably ethanol solution with the volume fraction of 90-95 percent.
Another embodiment of the present invention provides an application of the polygonatum cyrtonema polysaccharide in adjusting colony richness of intestinal flora and correcting disturbance of immunosuppressive intestinal flora.
Another embodiment of the present invention provides an application of the polygonatum cyrtonema polysaccharide in adjusting the diversity of the intestinal flora of mice under immunosuppression.
Another embodiment of the present invention provides the use of the above-described polygonatum cyrtonema polysaccharide for reducing the abundance of the paralacteroides colonies in the intestinal flora.
Another embodiment of the present invention provides the use of the above-described polygonatum cyrtonema polysaccharide for increasing the abundance of Lactobacillus; and/or increasing the abundance of f_lachnospiraceae_unclassified, lachnospiraceae NK4a136 (lachnospiraceae_nk 4a 136_group) colonies in the intestinal flora.
Another embodiment of the present invention provides an application of the polygonatum cyrtonema polysaccharide in preparing an intestinal flora regulator.
An intestinal flora regulator is characterized by taking the polygonatum cyrtonema polysaccharide as an active ingredient. Other intestinal flora modulators may also be included. Pharmaceutically acceptable auxiliary materials can be also included, and the dosage form is selected from solid preparations, liquid preparations and the like. The administration amount is preferably 200 to 600mg/kg, more preferably 400 to 600mg/kg, still more preferably 400 and 600mg/kg.
Compared with the prior art, the invention has the advantages that: according to the invention, pure active Polygonatum cyrtonema polysaccharide is obtained by methods such as ultrasonic water extraction, alcohol precipitation and impurity removal, an immunosuppression mouse model is established by adopting injection cyclophosphamide, polygonatum cyrtonema polysaccharide is administered in a stomach-filling administration mode, and the Polygonatum cyrtonema polysaccharide is found to be capable of adjusting colony abundance of intestinal flora of mice, correcting disturbance of intestinal flora of immunosuppression mice, and has a regulating effect on intestinal flora of immunosuppression mice, so that the Polygonatum cyrtonema polysaccharide is hopeful to be developed as an intestinal flora regulator.
Drawings
FIG. 1 is an OTU abundance cluster heat map.
Figure 2 is an OTU wien plot or petal plot.
Fig. 3 is a histogram of species distribution.
Fig. 4 is a PCA plot.
FIG. 5 is a graph showing the comparison of strain abundance differences.
Detailed Description
1. Experimental materials
1.1 Experimental raw materials and animals
Polygonatum cyrtonema 2019 was purchased in Baixi Sixilin county, guangxi, identified as Polygonatum cyrtonema Polygonatum cyrtonema Hua by the university of Guangxi traditional Chinese medicine Tan Xiaoming researchers, samples were stored in the university of Guangxi traditional Chinese medicine science laboratory center, and male mice (weight 20+ -2 g) were purchased from Hunan Srilk land laboratory animals Limited.
1.2 major laboratory instruments and reagents
Electronic balance (PL 203, metrele Torisuo instruments (Shanghai; T500, change Co., ltd.), high-speed multifunctional pulverizer (WJX-100, shanghai Yuan Wako industry Co., ltd.), digital constant temperature water bath (HH-6, changzhou national electric apparatus Co., ltd.), desk Centrifuge (Centrifuge 5810R, ai Bende Chinese Co., ltd.), ultrasonic cleaner (SB-800 DTD, ningbo New Zhi Biotech Co., ltd.), freeze dryer (Alpha 2-4LSCbasic, CHRIST Co., ltd.), 0.9% sodium chloride injection (Hut Bi Tongsu pharmaceutical Co., ltd.), PBS Buffer (NA, fuzhou Miss), lying Buffer (8304565, BD).
2. Experimental method
2.1 preparation of Polygonatum cyrtonema polysaccharide
(1) Accurately weighing 500g of dried rhizome of Polygonatum cyrtonema Fabricius, pulverizing, sieving with 20 mesh sieve, adding 4000mL of distilled water, extracting with 90 deg.C ultrasonic wave (40 kHz) for 3 times each for 1.0 hr, collecting extractive solution, and concentrating to obtain crude extract.
(2) Adding proper amount of water (300 mL) into the crude extract obtained in the step (1), dissolving by ultrasonic (40 kHz), fixing volume (to 500 mL), adding 4000mL of absolute ethanol, standing at 0-4 ℃ for 8h, centrifuging (3000 r.min) -1 3 min), the supernatant was discarded, the precipitate was collected, washed with 95% ethanol by volume fraction, and freeze-dried to give 80.5g of crude polysaccharide.
(3) Preparing crude polysaccharide solution (1.0L) with mass concentration of 1.0g/L from appropriate amount of crude polysaccharide, adding mixed solution (200 mL) of chloroform-n-butanol (volume ratio of 4:1), shaking vigorously, standing for 4 hr, and centrifuging (3000 r.min) -1 3 min), discarding the intermediate protein layer and the lower organic layer, repeating the operation for 1-3 times until no obvious white precipitate appears, adding 8-10 times volume of absolute ethanol into the water layer, standing at 0-4deg.C for 8 hr, and centrifuging (3000 r.min) -1 3 min), removing the supernatant, collecting the precipitate, washing the precipitate with 90% ethanol by volume fraction, and freeze-drying to obtain 69.0g of rhizoma Polygonati polysaccharide.
2.2 pharmaceutical preparation
60mg/kg cyclophosphamide configuration: 0.2g cyclophosphamide is taken and added with 30mL distilled water for dissolution.
200mg/kg Polygonatum cyrtonema polysaccharide configuration: 200mg of polygonatum cyrtonema polysaccharide is dissolved in 10mL of distilled water.
400mg/kg Polygonatum cyrtonema polysaccharide: 400mg of polygonatum cyrtonema polysaccharide is taken and dissolved in 10mL of distilled water.
600mg/kg Polygonatum cyrtonema polysaccharide configuration: 600mg of Polygonatum cyrtonema polysaccharide is dissolved in 10mL of distilled water.
2.3 grouping, modeling and administration of mice
Male mice of 18-22g were selected. After 6 days of adaptive feeding, the strain is randomly divided into 5 groups, wherein 5 groups are a normal group (CK), a Model Group (MG), a low-dose group of polygonatum cyrtonema polysaccharide (200 MG/kg, GH 200), a medium-dose group of polygonatum cyrtonema polysaccharide (400 MG/kg, GH 400) and a high-dose group of polygonatum cyrtonema polysaccharide (600 MG/kg, GH 600). The drug group was administered once daily by gavage (1 g:0.01mL of the weight of the mice), and the control group was administered with the same amount of physiological saline (1 g:0.01mL of the weight of the mice) as the model group for 7 days. In addition to the normal group, 0.2mL (1 g:0.1mL of the weight of the mice) of cyclophosphamide was injected intraperitoneally at 60mg/kg on the second, fourth and sixth days of feeding to induce immunosuppression in the mice. Each group was normally fed and drunk.
2.4 biological sample collection and index determination
2.4.1 fecal collection
On the seventh day after continuous administration, the mouse feces were collected and stored in a-80 ℃ refrigerator for biological sample treatment.
2.4.2 determination of intestinal flora (16S MetaVx TM Method)
(1) DNA extraction/QC
Extracting DNA from a sample using a DNA extraction kit, using
dsDNA HS Assay Kit the DNA concentration was measured.
(2) PCR amplification and library construction
A series of PCR primers designed for Jin Weizhi were used to amplify 2 hypervariable regions, including V3 and V4, on the 16S rDNA of prokaryotes using 20-30ng of DNA as template. The V3 and V4 regions were amplified using an upstream primer comprising the "cctacggrrbgcascagkvrgaat" sequence and a downstream primer comprising the "GGACTACNVGGGTWTCTAATCC" sequence. In addition, the adaptors with Index were added by PCR to the ends of the PCR products of 16S rDNA for NGS sequencing under the amplification conditions as shown in Table 1:
TABLE 1 amplification conditions
PCR reaction parameters: the pre-denaturation parameters 94℃for 3min X1, denaturation parameters 94℃for 5sec, annealing parameters 57℃for 90sec, extension for 10s at 72℃and final extension parameters for 5min were performed for 24 cycles.
And (3) identifying PCR amplification results: the PCR products were detected by electrophoresis on a 1.5% agarose gel.
(3) Sequencing on machine
Library concentrations were detected by an enzyme-labeled instrument. The library was quantified to 10nM and PE250/PE300 double-ended sequencing was performed according to the instructions of the Illumina Miseq (Illumina, san Diego, calif., USA) instrument, with sequence information read from Miseq self-contained MiSeq Control Software (MCS).
2.5 data processing
Experimental data were analyzed using Graphpad Prism 6.05 software, all in mean+ -SEM
Mean ± standard deviation, and simultaneously performing single-factor analysis of variance, P by t-test<0.05 is statistically significant.
Species taxonomic analysis was performed on the representative sequences of OTUs using RDP classifer (Ribosomal Database Program) bayesian algorithm, and the community composition of each sample was counted at different species classification levels. The samples were compared for significant microbial community differences by (un) weighted unifrac analysis.
3 detection result of intestinal flora of mice
3.1 analysis of intestinal flora composition of mice
According to the result of OTU (Operational Taxonomic Units, OTU) clustering analysis, a petal graph which can intuitively show the number composition similarity and overlapping condition of sample OTUs is drawn as shown in fig. 1, and the result shows that the number of the sample OTUs is 180, the number of the samples CK, MG, GH200, GH400 and GH600 is 2, the number of the samples CK is 16, the number of the samples GH200 is 1, the number of the samples GH400 is 1, and the number of the samples GH600 is 6, as shown in fig. 2.
At the genus level, the population fall composition of the intestinal flora of each group of mice is less different, but the relative abundance of different populations is more different. Wherein the dominant bacterial population of CK is mainly f __ Lachnospiraceae_Unclassified, trichosporon NK4A136 (Lachnospiraceae_N4A136_group); the dominant bacterial groups of MG are mainly Prevotella, parabaacteroides and Equisqualis (Alistipes); the dominant bacterial group of GH200 is mainly of the genus Prevotella (Alloprvotella), f __ Lachnospiraceae_Unclassified, biluomycetaceae NK4A136 group (Lachnospiraceae_N4A136_group); the dominant bacterial flora of GH400 is mainly Prevotella, f __ Lachnospiraceae_Unclassified, lactobacillus, and Prevotella ae_UCG-001; the dominant bacterial flora of GH600 is mainly Prevotella (Allopretum), f __ Lachnospirace_ Unclassified, parabacteroides.
The relative abundance of pseudoprasugrel (allocprevotella) flora in GH400 and GH600 was significantly increased compared to CK, MG and GH200, and the relative abundance of Lactobacillus (Lactobacillus) flora in GH400 was significantly increased compared to the other groups, as shown in fig. 3.
3.2PCA analysis
As can be seen from PCA analysis (Principal Component Analysis) of the test results, all samples of GH400 and GH600 significantly aggregated in both the PC1 and PC2 dimensions, with CK, MG being relatively closer to GH200, and relatively farther from GH400, GH600, but not significantly different; samples of CK, MG, GH200, GH400, and GH600 were substantially aggregated together in both dimensions PC1 and PC3, with CK and GH200 individual samples being relatively far apart, and with all samples of each experimental group being significantly aggregated in both dimensions PC2 and PC3, as shown in fig. 4.
3.3 analysis of significance of differences in the colony Structure between groups
And selecting a representative sequence for each OTU, and obtaining species classification information corresponding to the OTU. And labeling the representative sequences of species classification by using an RDP classifier to obtain community composition of each sample. Figure 5 shows the percentage of six major bacterial communities at the genus level for each sample. Compared with the normal group, CTX increases the abundance of the model group F_muticacueae_unclassified, bacteroides and Prevotella alloprevotella, and decreases the abundance of F_lachnospiraceae_unclassified, the group NK4A136 (Lachnospiraceae_N4A136_group) and Lactobacillus. After polysaccharide administration, the abundance of F_muebacueae_unclassified, bacteroides and Prevotella alloprevotella was decreased. The richness of the F_lachnospiraceae_unclassified, the Lachnospiraceae_N4A136 group of the Biluomycetaceae, and the Lactobacillus is significantly improved.
4. Conclusion(s)
Experimental results show that the polygonatum cyrtonema polysaccharide can adjust the diversity and structural composition of the colony of the intestinal flora of the mice under immunosuppression, improve the richness of Lactobacillus, improve the richness of f __ Lachnospiraceae_Unclassified colony, reduce the richness of the colony of Parabacterides, adjust the richness of the colony of the intestinal flora of the mice, and correct the disorder of the intestinal flora of the immunosuppressed mice. The experimental result shows that the polygonatum cyrtonema polysaccharide has a certain regulation effect on the disturbance of intestinal flora of an immunosuppressed mouse, and can be used as an intestinal flora regulator or a related functional product or additive such as an immunomodulator and the like for development and application.
Claims (10)
1. The preparation method of the polygonatum cyrtonema polysaccharide with the effect of regulating intestinal flora is characterized by comprising the following steps of:
(1) Pulverizing dried rhizome of Polygonatum cyrtonema Fabricius, adding water, performing ultrasonic extraction, collecting extractive solution, and concentrating to obtain crude extract;
(2) Adding proper amount of water into the crude extract, carrying out ultrasonic dissolution, then fixing the volume, adding absolute ethyl alcohol with the volume of 8-10 times, standing at 0-4 ℃, centrifuging, discarding supernatant, collecting precipitate, washing the precipitate with ethyl alcohol, and freeze-drying to obtain crude polysaccharide;
(3) Preparing crude polysaccharide into crude polysaccharide solution with mass concentration of 1.0g/L, adding mixed solution of chloroform-n-butanol (volume ratio of 4:1), shaking, standing, centrifuging, discarding intermediate protein layer and lower organic layer, repeating the operation for 1-3 times until no obvious white precipitate appears, adding 8-10 times volume of absolute ethanol into water layer, standing at 0-4deg.C, centrifuging, discarding supernatant, collecting precipitate, washing precipitate with ethanol, and lyophilizing to obtain rhizoma Polygonati polysaccharide.
2. The Polygonatum cyrtonema Falcatum polysaccharide of claim 1, characterized in that the water consumption in the step (1) is 6-10 times of the rhizome medicinal material quality of Polygonatum cyrtonema Falcatum, the ultrasonic extraction temperature is preferably 80-90 ℃, the extraction times are preferably 2-5 times, the ultrasonic frequency is 30-40kHz, and the ultrasonic extraction time is 0.5-1.0h each time. The pulverization is preferably carried out to 20-100 mesh.
3. The polysaccharide of Polygonatum cyrtonema of any one of claims 1-2, characterized in that the water dosage in step (2) is preferably 0.6-1.0 times the rhizome of Polygonatum cyrtonema, the ultrasonic frequency is 30-40kHz, the standing time is preferably 8-10 h, and the centrifugal rotational speed is preferably 3000 r.min -1 The time is preferably 3-5min.
4. The polygonatum cyrtonema polysaccharide according to any one of claims 1-3, characterized in that the amount of the mixed solution of chloroform and n-butanol in the step (3) is preferably 20% -25% of the volume of the crude polysaccharide solution; the standing time is preferably 3-5 h, and the centrifugal rotating speed is preferably 3000 r.min -1 The time is preferably 3-5min.
5. The method for preparing a polysaccharide from Polygonatum cyrtonema of any one of claims 1 to 4, characterized by comprising the steps of any one of claims 1 to 4.
6. Use of the polysaccharide of Polygonatum cyrtonema of any one of claims 1-4 for modulating colony richness of intestinal flora, correcting disorders of immunosuppressive intestinal flora.
7. Use of the polygonatum cyrtonema polysaccharide according to any one of claims 1-4 for modulating the diversity of the intestinal flora of mice under immunosuppression.
8. Use of a polygonatum cyrtonema polysaccharide according to any one of claims 1-4 for reducing the abundance of paralacteroides colonies in the intestinal flora.
9. Use of the polygonatum cyrtonema polysaccharide according to any one of claims 1-4 for increasing the abundance of Lactobacillus; and/or increasing the abundance of f_lachnospiraceae_unclassified, lachnospiraceae NK4a136 (lachnospiraceae_nk 4a 136_group) colonies in the intestinal flora.
10. Use of the polysaccharide of Polygonatum cyrtonema of any one of claims 1-4 for the preparation of an intestinal flora modulator; an intestinal flora regulator is characterized by taking the polygonatum cyrtonema polysaccharide as an active ingredient. Other intestinal flora modulators may also be included. Pharmaceutically acceptable auxiliary materials can be also included, and the dosage form is selected from solid preparations, liquid preparations and the like. The administration amount is preferably 200 to 600mg/kg, more preferably 400 to 600mg/kg, still more preferably 400 and 600mg/kg.
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