Crude polysaccharide of radix quatrainii and its preparation method and application in alleviating intestinal damage caused by plateau hypoxia

Abstract

The present invention provides crude polysaccharide of radix quatrain and a preparation method thereof and its role in alleviating intestinal damage caused by plateau hypoxia, and the present invention belongs to the field of medical technology. The preparation method of crude polysaccharide of radix quatrain provided by the present invention comprises the following steps: pulverizing radix quatrain after freeze-drying, removing pigments, lipids and monosaccharides, and then vacuum filtering to obtain insoluble residues; extracting crude polysaccharide of radix quatrain from the insoluble residues by ultrasonic-assisted hot water extraction. The obtained radix quatrain polysaccharide can reduce oxidative stress caused by plateau hypoxia by its antioxidant activity, thereby alleviating intestinal damage caused by hypoxia.

Description

Technical Field

[0001] The invention relates to the field of medical technology, and in particular to crude polysaccharide of genkwa root and a preparation method thereof, and the function of the polysaccharide in alleviating intestinal damage caused by plateau hypoxia. Background Art

[0002] As the third pole of the world, the Qinghai-Tibet Plateau is known as the "Roof of the World". It covers an area of ​​2.5 million square kilometers and has an average altitude of more than 4,000 meters. It has a special climate environment such as low oxygen, low temperature, low pressure and high ultraviolet radiation. Among them, hypoxia is the main factor affecting the human body. The changes in body function, metabolism and structure caused by hypoxia are the unique pathophysiological reactions of plateau-related diseases. Long-term exposure to high-altitude low-pressure hypoxia can cause damage to different tissues and organs, especially the gastrointestinal tract, which is the first to suffer from ischemia and hypoxia. The main site of intestinal damage caused by plateau hypoxia is the ileum. Hypoxia may induce severe primary intestinal barrier dysfunction, leading to bacterial and endotoxin translocation, causing systemic inflammatory response, and then causing plateau multiple organ dysfunction syndrome. Under hypoxic conditions, a large number of toxic reactive oxygen metabolites can cause cell energy depletion and acidosis, destroy the morphological structure of intestinal villi, increase the permeability of intestinal mucosal epithelial cells, and reduce the absorption function, affecting the digestion, absorption and transfer of nutrients. When the intestinal barrier function is damaged, harmful substances produced by the intestinal flora will enter the blood and cause damage to the body. Therefore, people are more likely to suffer from intestinal mucosal barrier function damage in the hypoxic environment of the plateau than in the plains. However, the existing literature focuses more on common intestinal damage, such as lesions and damage in the colon.

[0003] As the core of the intestinal microecological system, the intestinal microbiota has evolved with the host and is an integral part of the human body. One of the most notable characteristics of the intestinal flora is its stability. If it loses balance, various intestinal and extraintestinal diseases will occur. Therefore, maintaining the balance of the intestinal microecology is very important for humans to resist infectious diseases caused by intestinal pathogens.

[0004] The root of radix guaiac contains abundant macro- and trace elements and vitamins, and a full range of amino acids, with essential amino acids accounting for 48.46% of the total amino acids. It is a typical natural high-potassium, low-sodium, selenium-rich food. However, the content of ingredients in the roots of radix guaiac grown in different regions is different. For example, the fresh roots of radix guaiac in the Tibet Autonomous Region contain 0.18% crude total acid, 3.64% total sugar, and 1.35% crude protein. It has a sweet and sour taste and is a high-plant protein crop. The ingredients are different from those of the roots of radix guaiac grown in plains in many aspects. As a cost-effective medicinal and edible plant, the development of radix guaiac is still in the basic stage and has broad research space and application prospects. Summary of the invention

[0005] The purpose of the present invention is to extract crude polysaccharides from the root of genus Perilla, utilize the antioxidant activity thereof, reduce the oxidative stress caused by plateau hypoxia, and further alleviate the intestinal damage caused by plateau hypoxia.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing crude polysaccharide from Radix Gentianae, comprising the following steps:

[0008] (1) The genus of quail is freeze-dried and then crushed to remove pigments, lipids, and monosaccharides, and then vacuum filtered to obtain an insoluble residue;

[0009] (2) Extracting polysaccharide from the insoluble residue by ultrasonic-assisted hot water extraction.

[0010] Preferably, in step (1), pigments, lipids and monosaccharides are removed from the radix genus using petroleum ether.

[0011] Preferably, the method of ultrasonic-assisted hot water extraction in step (2) is specifically as follows:

[0012] a. Add water to the insoluble residue so that the solid-liquid ratio is 1:40~45g / mL, the extraction time is 50~60min, the extraction temperature is 55~65°C, the ultrasonic power is 300~400W, and a mixture is obtained;

[0013] b. Take the liquid portion of the mixture, 55-65°C rotary evaporation concentration to 1 / 4-1 / 3 of the original volume, remove the protein component, to obtain a prefabricated crude polysaccharide;

[0014] c. Add 2.5 to 3.5 times the volume of ethanol to the pre-prepared crude polysaccharide to a final concentration of 92 to 98% (v / v), let it stand overnight, then centrifuge at 4500 to 5500 rpm for 8 to 12 min, collect the precipitate, and lyophilize to obtain the crude polysaccharide of Dahurian root.

[0015] The invention also provides crude polysaccharide of genkwa root obtained by the preparation method.

[0016] The present invention also provides an application of the crude polysaccharide of Radix Gentianae in preparing a medicine for alleviating intestinal damage caused by plateau hypoxia.

[0017] The present invention selects the radix guajava from high altitude areas as a raw material to extract plant polysaccharides. The radix guajava from high altitude areas has good antioxidant properties, can better alleviate plateau oxidative stress and intestinal oxidative damage and inflammation, and regulate intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The crude polysaccharide of Parsley root in Experimental Example 1 has an effect on counteracting oxidative stress in mice with acute high altitude hypoxia;

[0019] Figure 2 This is a cross-sectional image showing that crude polysaccharide from the root of Perilla frutescens alleviates intestinal barrier damage and inflammation caused by plateau hypoxia stress in Experimental Example 1;

[0020] Figure 3 The effect of crude polysaccharide of genkwa root in experimental example 1 on relieving intestinal inflammation, improving intestinal mucosal permeability and repairing intestinal barrier in mice;

[0021] Figure 4 This is the result of the effect of crude polysaccharide from Parsley root on the level of SCFAs in Experimental Example 2;

[0022] Figure 5 This is the result of the crude polysaccharide of genus Parviflora regulating the intestinal flora disorder induced by plateau hypoxia stress in Experimental Example 3;

[0023] Figure 6 This is the effect of crude polysaccharide of Parsley root on the intestinal microbial diversity of mice in different groups in Experimental Example 3;

[0024] Figure 7 This is the effect of crude polysaccharide from Parsley Root on the differences in microbial composition between different groups of mice in a plateau environment in Experimental Example 3. DETAILED DESCRIPTION

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Example 1

[0027] Preparation of crude polysaccharide from radix quatrainii

[0028] (1) Pretreatment of radix daphne: Fresh radix daphne collected from the Tibet Autonomous Region was washed, sliced, freeze-dried in a freeze dryer, and then crushed. The sieve was passed through a 60-mesh sieve, and the sieve was taken and stirred with petroleum ether (1:10 solid-liquid ratio) to remove pigments, lipids and monosaccharides. This step was carried out in a fume hood at room temperature. The treated mixture was vacuum filtered to obtain an insoluble residue, and the remaining petroleum ether was evaporated in a fume hood to remove the remaining petroleum ether. The pretreated sample was placed in a 40°C oven and dried to constant weight for subsequent polysaccharide extraction.

[0029] (2) The polysaccharide of the root of larkspur was extracted from the pretreated sample by ultrasonic-assisted hot water extraction. The parameters were as follows: solid-liquid ratio of 1:43 g / mL, extraction time of 55 min, extraction temperature of 60 °C, and ultrasonic power of 360 W. The extracted mixture was filtered, and the liquid part was concentrated to 1 / 4 of the original volume by rotary evaporation at 60 °C. The protein component in the solution was removed by the Sevag method, and 3 times the volume of ethanol was added to a final concentration of 95% (v / v). The solution was allowed to stand at 4 °C for precipitation overnight, and the precipitate was collected by centrifugation at 5000 rpm for 10 min. The precipitate was freeze-dried (pre-frozen to -50 °C and the pressure was reduced to 40 Pa) to obtain the crude polysaccharide of the root of larkspur.

[0030] Experimental Example 1

[0031] In order to evaluate the oxidative stress response of crude polysaccharides of radix quatica on the whole body of mice, as well as the damage to ileal tissue and changes in intestinal microorganisms, an acute hypoxia mouse model was established: the mice were adaptively fed for 7 days and then divided into two groups (10 mice in each group): normobaric and normoxic group (oral administration of PBS) (N); normobaric and normoxic radix group (oral administration of crude polysaccharides of radix quatica) (NB) were placed in a normobaric and normoxic environment for 14 days, hypobaric oxygen chamber control group (oral administration of PBS) (H); hypobaric oxygen chamber radix group (oral administration of crude polysaccharides of radix quatica) (HB); hypobaric oxygen chamber radix fecal microbiota transplantation group (oral administration of bacterial suspension made from feces of group HB) (HBF), which were placed in a hypobaric and hypoxic chamber for 14 consecutive days. The hypobaric and hypoxic chamber was opened at 8:00 a.m. every day to carry out necessary medication and housekeeping. After the medication was completed, the hypobaric and hypoxic chamber was closed and the pressure was quickly increased to a simulated altitude of 2500 m. After stabilization for 10 minutes, it was increased to 5000 m.

[0032] 1.1 In the study of the effect of crude polysaccharide of radix quatrain on the anti-oxidative stress of mice with acute high altitude hypoxia, the antioxidant activity SOD, GSH-Px, MDA and total antioxidant capacity T-AOC in the serum of the above five groups of mice were determined. Figure 1 As shown in the results, compared with the N group, the H group significantly reduced the serum SOD, GSH-Px and total antioxidant capacity T-AOC in the simulated plateau environment. Compared with the H group, the HB group had enhanced GSH-Px and SOD activities after intervention with radix quatrain crude polysaccharides, while the activity of total antioxidant capacity T-AOC increased significantly. The SOD, GSH-Px and T-AOC activities of the HBF group were all enhanced. There was no significant change in the NB group compared with the N group. MDA, as a lipid oxidation product, can reflect the degree of oxidative reaction in the body. In the serum of mice after hypoxia, the content of MDA was significantly higher than that of the N group, but after intervention with radix quatrain crude polysaccharides, its content decreased. This shows that radix quatrain crude polysaccharides can alleviate plateau oxidative stress.

[0033] 1.2. Polysaccharides from radix quatrains alleviate intestinal barrier damage and inflammation caused by plateau hypoxia stress

[0034] The ileum tissues of the five groups of mice were cut into pieces of about 1 cm each, rinsed with physiological saline and fixed in 4% paraformaldehyde, embedded in paraffin, and then the sections were stained with hematoxylin-eosin (H&E). The section images were collected and analyzed.

[0035] A certain amount of ileum tissue was taken, and the supernatant was collected after tissue homogenization. ELISA kits were used to detect IL-6, IL-1β, and TNF-α. IBM SPSS Statistics was used to perform statistical analysis of IL-6, IL-1β, and TNF-α levels by one-way analysis of variance (ANOVA) to detect the normality and statistical significance of the data.

[0036] Physical and chemical kits were used to detect ileal tissue diamine oxidase (DAO) and myeloperoxidase (MPO). IBM SPSS Statistics was used to perform statistical analysis of DAO and MPO levels by one-way analysis of variance (ANOVA) to detect the normality and statistical significance of the data.

[0037] like Figure 2 As shown in the figure, the ileum of the experimental mice in group N was intact and orderly, and the intestinal villi were long and dense. Compared with group N, there was no significant change in group NB, and the ileum of mice in group H showed obvious histopathological damage, with sparse, shortened, and loosely arranged small intestinal villi, and the intestinal mucosa fell off and showed signs of atrophy. Compared with group H, the intestinal villi of mice in group HB were relatively long, and the intestinal villi of mice in group HBF were relatively long and arranged relatively tightly, indicating that the low-pressure hypoxic environment can cause intestinal mucosal damage, increased intestinal mucosal permeability, and destruction of the intestinal mucosal barrier.

[0038] like Figure 3 As shown in the results, acute hypobaric and hypoxic stress significantly increased the activity of DAO and MPO in the ileum of mice. Compared with the N group, the activity of DAO and MPO in the H group was significantly increased. Compared with the H group, the HB group significantly reduced the activity of DAO and MPO in the ileum, and the activity of DAO and MPO in the ileum of HBF was reduced. In addition, compared with the N group, the expression levels of IL-6, IL-1β and TNF-α inflammatory factors in the ileum tissue of the H group mice were significantly increased. Compared with the H group, the HB group significantly reduced the expression level of inflammatory factors in the ileum, and the expression level of inflammatory factors in HBF was also reduced. These results indicate that the hypoxic environment in high altitude areas may cause intestinal inflammation in mice, increase intestinal mucosal permeability or damage the intestinal barrier, and intervention with crude polysaccharides from the root of genus quatrain can alleviate these effects to a certain extent.

[0039] Experimental Example 2

[0040] Evaluation of the effects of crude polysaccharides from radix quatrains on SCFAs levels

[0041] Mouse fecal sample pretreatment

[0042] (1) After the sample is thawed, weigh 20 mg of the sample into a 2 mL centrifuge tube and add a small steel ball;

[0043] (2) Add 1000 μL of phosphoric acid solution (0.5%, v / v), grind with a 30 Hz ball mill for 1 min, vortex at 2500 r / min for 10 min, sonicate at 4°C for 5 min, and centrifuge at 4°C and 12000 r / min for 10 min;

[0044] (3) Pipette 100 μL of supernatant into the corresponding 1.5 mL centrifuge tube, add 500 μL of MTBE extractant containing internal standard, vortex at 2500 r / min for 3 min, sonicate at 4°C for 5 min, and centrifuge at 4°C and 12000 r / min for 10 min;

[0045] (4) Pipette 200 μL of supernatant into the liner of the injection bottle for GC-MS / MS analysis.

[0046] SCFAs are the main metabolites produced by intestinal flora and play a vital role in maintaining intestinal function. Figure 4 As shown in the results, under the simulated plateau environment, compared with the N group, the concentration of short-chain fatty acids in the NB group increased, and the concentrations of acetate, propionate and butyrate in the H group decreased. Compared with the H group, the concentration of short-chain fatty acids in the HB group increased after intervention with genus arborvitae crude polysaccharide. There was no significant increase in short-chain fatty acids in the HBF group, indicating that genus arborvitae crude polysaccharide promoted the proliferation of some bacteria that produce short-chain fatty acids.

[0047] Experimental Example 3

[0048] Polysaccharides from Radix Euphorbiae radix regulate intestinal flora disturbance induced by plateau hypoxia stress

[0049] Metagenomic sequencing was performed on the five groups of mouse feces in Example 1, and the sequencing results were analyzed.

[0050] The stress of the plateau hypoxic environment can damage the intestinal mucosa, increase anaerobic metabolic byproducts, lead to an imbalance in intestinal flora, a decrease in beneficial bacteria, and the growth of harmful bacteria. Figure 5The abundance of bacteria at the species level is shown in the figure. Compared with the H group, after BRP (Crude Polysaccharide from Radix Corydalis) intervention, the relative abundance of beneficial bacteria in the HB group of mice increased, such as Akkermansia muciniphila, Lactobacillus intestinalis, Bacteroides thetaiotaomicron, Ileibacterium valens, Muribac lumintestinale, Lactobacillus reuteri, etc. The high-altitude hypoxic environment significantly increased the abundance of Clostridium and Alistipes shahii. These conditional pathogens may trigger the intestinal inflammatory process and accelerate the progression of altitude sickness.

[0051] like Figure 6 As shown in the figure, the Shannon diversity of the H and HB groups was higher than that of the N and NB groups, and there was no statistical difference between the other groups. The slightly higher Shannon diversity of the H and HB groups may be a response of the mice to the plateau environment when they are in a specific plateau environment.

[0052] Figure 7 As shown in the figure, principal component analysis showed that the distance between group N and group NB was close, indicating that the structure of intestinal flora was similar. However, group H was far away from group N and group NB, and the intestinal flora was less similar. This indicates that the intestinal flora structure of group H changed under the plateau environment. After intervention with crude polysaccharides from radix quatrainii, the intestinal flora structure of group HB was close to that of group N and group NB, indicating that crude polysaccharides from radix quatrainii can change the intestinal flora under the plateau environment and make it closer to the normal pressure group.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The use of fecal bacteria after intragastric administration of crude polysaccharide of Radix Corydalis in the preparation of a drug for alleviating intestinal damage caused by plateau hypoxia, characterized in that: The method for preparing the crude polysaccharide from Radix Gentianae comprises the following steps: (1) The genus of quail is freeze-dried and then crushed to remove pigments, lipids, and monosaccharides, and then vacuum filtered to obtain an insoluble residue; (2) extracting crude polysaccharides from the insoluble residue using an ultrasound-assisted hot water extraction method; The fecal bacteria is a bacterial suspension made from the feces of mice after intragastric administration of crude polysaccharide of Radix Paridis.

2. The use according to claim 1, characterized in that: In step (1), pigments, lipids and monosaccharides are removed from the radix quinata using petroleum ether.

3. The use according to claim 1, characterized in that: The method of ultrasonic-assisted hot water extraction in step (2) is specifically as follows: a. Add water to the insoluble residue so that the solid-liquid ratio is 1:40-45 g / mL, the extraction time is 50-60 min, the extraction temperature is 55-65 ° C, the ultrasonic power is 300-400 W, and a mixture is obtained; b. Take the liquid portion of the mixture, condense it by rotary evaporation at 55-65°C to 1 / 4-1 / 3 of the original volume, remove the protein component, and obtain a prefabricated crude polysaccharide; c. Add 2.5 to 3.5 times the volume of ethanol to the pre-prepared crude polysaccharide to a final concentration of 92 to 98% (v / v), let it stand for precipitation overnight, then centrifuge at 4500 to 5500 rpm for 8 to 12 minutes, collect the precipitate, and freeze-dry to obtain crude polysaccharide of the root of genus Georgi.

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