Plant polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine extracts, and discloses plant polysaccharide as well as a preparation method and application thereof. According to the application, a ginseng, poria cocos and rhizoma atractylodis macrocephalae powder traditional Chinese medicine composition is taken as an extraction raw material, plant polysaccharide consisting of 3-4% of arabinose, 7-8% of galactose, 39-40% of glucose, 1-2% of xylose, 29-30% of fructose and 19-20% of galacturonic acid is extracted from the ginseng, poria cocos and rhizoma atractylodis macrocephalae powder traditional Chinese medicine composition, and the molecular weight of the plant polysaccharide is 4-30kDa. Compared with total polysaccharides and other molecular weight polysaccharides, the lactase expression and activity can be improved more remarkably, and the problem of lactase insufficiency in vivo is solved; meanwhile, inflammatory response caused by diarrhea induced by high lactose can be effectively relieved, the damaged intestinal mucosal barrier can be repaired, the diarrhea state caused by high lactose is further relieved, and the prebiotics are expected to be developed into prebiotics capable of improving lactase expression and activity, promoting lactose digestion, relieving intestinal inflammation and repairing the damaged intestinal mucosal barrier. The method is applied to foods, medicines and health-care products.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine extracts, and in particular to a plant polysaccharide, a preparation method thereof, and an application thereof. Background Art

[0002] Lactose intolerance (LI) is a disease caused by lactase deficiency, which cannot completely digest lactose, resulting in symptoms such as nausea, borborygmus, abdominal pain, abdominal distension, and diarrhea. It is common in the Asian population. For the treatment of LI, lactose intake is usually restricted. However, long-term dietary restrictions can cause deficiencies in calcium and vitamins, exacerbate some underlying diseases such as osteoporosis, reduce the quality of life, and even cause depression.

[0003] It has been reported that plant crude polysaccharide extracts can play a good alleviating effect on diarrhea caused by high lactose by regulating the intestinal flora. Polysaccharides, as a carbon source for intestinal microbial fermentation, can be used as functional foods or prebiotics to prevent intestinal microbiota dysbiosis. However, there is currently no plant polysaccharide that can both increase the expression of lactase in the body and improve the activity of lactase. Summary of the Invention

[0004] In view of this, the purpose of the present application is to provide a plant polysaccharide and a preparation method thereof, so that the plant polysaccharide can not only significantly improve the activity of lactase, but also significantly increase the expression of lactase;

[0005] Another purpose of the present application is to provide a plant polysaccharide and a preparation method thereof, so that the plant polysaccharide can relieve intestinal inflammation caused by high lactose and repair the damaged intestinal mucosal barrier.

[0006] Another purpose of the present application is to provide the application of the above-mentioned plant polysaccharide in the preparation of products for improving lactose expression and enzyme activity or products for relieving intestinal inflammation and repairing the damaged intestinal mucosal barrier.

[0007] To solve the above technical problems / achieve the above purposes or at least partially solve the above technical problems / achieve the above purposes, as the first aspect of the present application, a plant polysaccharide is provided, calculated by weight percentage, including:

[0008] Arabinose 3-4%, galactose 7-8%, glucose 39-40%, xylose 1-2%, fructose 29-30%, and galacturonic acid 19-20%.

[0009] Optionally, the molecular weight of the plant polysaccharide is 4-30 kDa.

[0010] As the second aspect of the present application, a preparation method of the plant polysaccharide described in the present application is provided, including:

[0011] Extract the total polysaccharide from the traditional Chinese medicine composition composed of ginseng, poria cocos, atractylodes macrocephala, Chinese yam, white hyacinth bean, lotus seed, coix seed, amomum villosum, platycodon grandiflorum, and liquorice;

[0012] After redissolving the total polysaccharide in water, perform the first ethanol precipitation with the first ethanol solution, and centrifuge to obtain the supernatant;

[0013] Add the second ethanol solution to the supernatant for the second ethanol precipitation, and centrifuge to obtain the precipitate to obtain the plant polysaccharide;

[0014] Among them, the volume fraction of the first ethanol solution is 20 - 30%; the volume fraction of the second ethanol solution is 50 - 60%.

[0015] Optionally, the traditional Chinese medicine composition is composed of, by weight percentage: 12.9% of ginseng, 12.9% of poria cocos, 12.9% of atractylodes macrocephala, 12.9% of Chinese yam, 9.5% of white hyacinth bean, 6.5% of lotus seed, 6.5% of coix seed, 6.5% of amomum villosum, 6.5% of platycodon grandiflorum, and 12.9% of liquorice.

[0016] Further optionally, the total polysaccharide is separated from the traditional Chinese medicine extract by the water decoction method.

[0017] Optionally, the temperatures of the first ethanol precipitation and the second ethanol precipitation are independently selected from 0 - 4°C respectively.

[0018] As the third aspect of the present application, there is provided the use of the plant polysaccharide in the preparation of a product for improving lactase expression and activity or a product for alleviating intestinal inflammation and repairing damaged intestinal mucosal barrier.

[0019] Optionally, the product includes food, health products, and drugs.

[0020] As the fourth aspect of the present application, there is provided a product for improving lactase expression and activity or alleviating intestinal inflammation and repairing damaged intestinal mucosal barrier, which includes the plant polysaccharide of the present application.

[0021] The present application uses the Shenling Baizhu Powder traditional Chinese medicine composition as the extraction raw material, and extracts a plant polysaccharide composed of arabinose, galactose, glucose, xylose, fructose, and galacturonic acid in a specific ratio from it. Its molecular weight is 4 - 30 kDa. Compared with the total polysaccharide and other molecular weight polysaccharides, it can have a more significant effect of improving lactase expression and activity, solve the problem of insufficient lactase in the body; at the same time, it can effectively alleviate the inflammatory reaction caused by high-lactose-induced diarrhea, and can also repair the damaged intestinal mucosal barrier, further reducing the degree of diarrhea caused by high lactose. It is expected to be developed into a prebiotic for improving lactase expression and activity, promoting lactose digestion, alleviating intestinal inflammation, and repairing damaged intestinal mucosal barrier, and applied to food, drugs, and health products. Description of the Drawings

[0022] The accompanying drawings of the specification, which form a part of this application, are used to provide a further understanding of this application. The schematic embodiments of this application and their descriptions are used to explain this application and do not constitute an improper limitation to this application;

[0023] Figure 1 The figure shows the schematic diagram of the detection results of the fecal water content of rats in the high-lactose diarrhea rat model prepared in Example 1, as well as the fecal water content at three time points of 24 h - 48 h - 72 h after the start of modeling; among them, A is the average fecal water content result obtained by randomly collecting feces three times a week for three weeks after modeling; B is the number of rats with diarrhea at 24 h; C is the fecal water content at three time points of 24 h - 48 h - 72 h after the start of modeling; D is the average fecal water content at three time points of 24 h - 48 h - 72 h after the start of modeling;

[0024] Con: Healthy control group, HLD: Model group, SLP: Total polysaccharide group, SLP-1: Control polysaccharide group, SLP-2: Plant polysaccharide group of this application (the same below);

[0025] Figure 2 The figure shows the schematic diagram of the detection results of the grasping force of rats in the high-lactose diarrhea rat model prepared in Example 1;

[0026] Figure 3 The figure shows the schematic diagram of the detection results of the fecal pH value of rats in the high-lactose diarrhea rat model prepared in Example 1;

[0027] Figure 4 The figure shows the schematic diagram of the detection results of lactase activity after in vitro lactase intervention with the polysaccharide prepared in Example 1; A: Summary result of lactase activity; B - D: Lactase activity results at different time points; Lactase: Lactase control group;

[0028] Figure 5 The figure shows the schematic diagram of the detection results of the lactose content in the cecal contents of rats in the high-lactose diarrhea rat model prepared in Example 1;

[0029] Figure 6 The figure shows the schematic diagram of the detection results of the lactase gene and protein expression in the small intestine (jejunum) of rats in the high-lactose diarrhea rat model prepared in Example 1; A: Protein expression band of lactase; B: Relative protein expression level of lactase;

[0030] Figure 7 The figure shows the schematic diagram of the detection results of the immunofluorescence expression of lactase in the small intestine of rats in the high-lactose diarrhea rat model prepared in Example 1;

[0031] Figure 8The figure shows the schematic diagram of the detection results of rat serum and tissue inflammatory factors in the polysaccharide prepared in Example 1 on the high-lactose diarrhea rat model;

[0032] Figure 9 The figure shows the schematic diagram of the detection results of the expression of mucosal barrier-related genes in the small intestine and colon of rats in the high-lactose diarrhea rat model by the polysaccharide prepared in Example 1;

[0033] Figure 10 The figure shows the schematic diagram of the detection results of the expression of mucosal barrier-related genes in the colon of rats in the high-lactose diarrhea rat model by the polysaccharide prepared in Example 1;

[0034] Figure 11 The figure shows the schematic diagram of the detection results of the immunofluorescence expression in the colon of rats in the high-lactose diarrhea rat model by the polysaccharide prepared in Example 1. Detailed implementation manners

[0035] This application discloses a plant polysaccharide, its preparation method and application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in this application. The products, processes and applications described in this application have been described through preferred embodiments. It is obvious that relevant personnel can make changes or appropriate changes and combinations to the methods described in this article without departing from the content, spirit and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of this application.

[0036] It should be noted that in this article, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, the element defined by the statement "including one..." does not exclude the existence of another identical element in the process, method, article or device including the said element. At the same time, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other.

[0037] In the prior art, plant polysaccharides mostly regulate the distribution of intestinal flora to cope with diarrhea caused by high lactose. However, this regulatory mechanism often takes a long time to make the intestinal flora environment reach an ideal state. Being able to directly improve the deficiencies such as insufficient intestinal lactase and lack of intestinal mucosal barrier can quickly improve diarrhea induced by high lactose, which has not been involved in the current prior art research.

[0038] In the first aspect of the present application, a plant polysaccharide is provided, which, by weight percentage, includes:

[0039] Arabinose 3 - 4%, galactose 7 - 8%, glucose 39 - 40%, xylose 1 - 2%, fructose 29 - 30% and galacturonic acid 19 - 20%.

[0040] The molecular weight of the plant polysaccharide described in the present application is 4 - 30 kDa, and it is isolated from the polysaccharide extracted from Shenling Baizhu Powder, which is a classic traditional Chinese medicine formula for tonifying qi and strengthening the spleen. In the research of the present application, it was unexpectedly found that the polysaccharide component in this molecular weight range in the total polysaccharide of Shenling Baizhu Powder has the effect of directly increasing the expression and activity of lactase, and can repair the damaged intestinal mucosal barrier and eliminate intestinal inflammation, and shows better effects compared with polysaccharides and total polysaccharides in other molecular weight ranges and compositions.

[0041] In the second aspect of the present application, a preparation method of the plant polysaccharide described in the present application is provided, including:

[0042] Extracting the total polysaccharide from a traditional Chinese medicine composition composed of ginseng, poria cocos, atractylodes macrocephala, Chinese yam, white hyacinth bean, lotus seed, coix seed, amomum villosum, platycodon grandiflorum and liquorice;

[0043] After redissolving the total polysaccharide in water, performing the first alcohol precipitation with a first ethanol solution, and centrifuging to obtain the supernatant;

[0044] Adding a second ethanol solution to the supernatant, performing the second alcohol precipitation, and centrifuging to obtain the precipitate, thereby obtaining the plant polysaccharide;

[0045] Wherein, the volume fraction of the first ethanol solution is 20 - 30%; the volume fraction of the second ethanol solution is 50 - 60%.

[0046] In some embodiments of the present application, the traditional Chinese medicine composition, by weight percentage, is composed of: ginseng 12.9%, poria cocos 12.9%, atractylodes macrocephala 12.9%, Chinese yam 12.9%, white hyacinth bean 9.5%, lotus seed 6.5%, coix seed 6.5%, amomum villosum 6.5%, platycodon grandiflorum 6.5% and liquorice 12.9%. Among them, each component uses processed traditional Chinese medicine decoction pieces. In some other embodiments of the present application, the processing methods of each component in the traditional Chinese medicine composition are as follows:

[0047] Ginseng: Moisten until thoroughly penetrated, cut into thin slices, dry, or powder it when in use;

[0048] Poria: Soak, wash, moisten, steam slightly, promptly peel off the outer skin, cut into pieces or thick slices, and dry in the sun;

[0049] Atractylodes macrocephala: Remove impurities, wash, moisten until thoroughly penetrated, cut into thick slices and dry; then stir-fry Atractylodes macrocephala with honey-fried bran until it turns yellowish-brown and emits a burnt aroma, and sift off the honey-fried bran;

[0050] Chinese yam: Remove impurities, soak and moisten until thoroughly penetrated, cut into thick slices, dry, and stir-fry until yellow according to the bran-frying method;

[0051] White hyacinth bean: Remove impurities, stir-fry with the clear-frying method until slightly yellow with burnt spots, and crush it when in use;

[0052] Lotus seed: Slightly soak, moisten until thoroughly penetrated, cut open, remove the core, and dry;

[0053] Coix seed: Remove impurities, stir-fry with the bran-frying method until slightly yellow;

[0054] Amomum villosum: Remove impurities, and crush it when in use;

[0055] Platycodon grandiflorum: Remove impurities, wash, moisten until thoroughly penetrated, cut into thick slices, and dry;

[0056] Licorice root: Remove impurities, wash, moisten until thoroughly penetrated, cut into thick slices, and dry; it can also be honey-fried until the surface turns golden yellow.

[0057] In some other embodiments, the ginseng can be replaced with codonopsis pilosula, cut into sections and dried, and the dosage of codonopsis pilosula can be appropriately increased.

[0058] In some embodiments of the present application, the total polysaccharide is separated from the traditional Chinese medicine extract by the water decoction method. In some other embodiments of the present application, the traditional Chinese medicine extract is obtained by the following method:

[0059] Weigh the traditional Chinese medicine composition, add water and boil. The ratio of material to liquid can be selected as 1:(10 - 15). Separate the water decoction liquid. The water decoction can be repeated multiple times, and the water decoction liquids each time are combined; the methods for separating the total crude polysaccharide include but are not limited to alcohol precipitation, column chromatography, and membrane separation. After the separation of the total crude polysaccharide, it is subjected to one or more purification processes such as protein removal, decolorization, and impurity removal. After purification, the purified total polysaccharide can still be separated by methods such as alcohol precipitation, column chromatography, and membrane separation.

[0060] In certain embodiments of the present application, the first ethanol precipitation uses a first ethanol solution with a low volume fraction to separate the non-target polysaccharide precipitate. If the volume fraction of the first ethanol solution is increased, the polysaccharide within the target range will also precipitate, resulting in losses in the yield and efficacy of the final plant polysaccharide. After separating the non-target polysaccharide precipitate, the supernatant containing the target polysaccharide is taken for the second ethanol precipitation. In this stage, a second ethanol solution with a higher volume fraction is used for precipitation, which can precipitate the target polysaccharide. However, a second ethanol solution with a lower or higher volume fraction will cause incomplete precipitation of the target polysaccharide or contamination by other polysaccharides, also resulting in losses in the yield and efficacy of the final plant polysaccharide. The standing temperatures for the first and second ethanol precipitations are independently selected from 0 - 4°C, the standing times are independently selected from 10 - 12 h, the centrifugation speeds are independently selected from 5000 - 6000 r / min, and the centrifugation times are independently selected from 10 - 20 min.

[0061] In certain embodiments of the present application, the volume of the first ethanol solution added is 0.25 - 0.43 times that of the total polysaccharide solution redissolved with water, and the concentration of the total polysaccharide solution redissolved with water is 2 - 5%; the volume of the second ethanol solution added is 1 - 1.5 times that of the supernatant.

[0062] In the third aspect of the present application, the lactase activities of the total polysaccharide (SLP), the polysaccharide after the first ethanol precipitation (SLP - 1), and the polysaccharide after the second ethanol precipitation (the plant polysaccharide SLP - 2 of the present application) are detected both in vivo and in vitro. The results show that in the in vitro lactase activity detection, the plant polysaccharide of the present application can significantly increase the in vitro lactase activity, with a better effect than SLP and SLP - 1. In the in vivo lactase activity detection, compared with the healthy control group (Con), the lactose content in the cecum of the model group (HLD) and the SLP - 1 group fed with a high - lactose diet increased significantly. However, the lactose content in the cecum of the SLP group and the SLP - 2 group fed with a high - lactose diet only increased slightly and was significantly lower than that of the HLD group and the SLP - 1 group. In addition, the detection of the lactase protein expression level in the small intestine tissue also proves that the expression level of the SLP - 2 group is higher. The above experimental results demonstrate that the plant polysaccharide isolated in the present application has an obvious effect of increasing lactase expression and activity, while polysaccharides with other molecular weights and compositions have limited effects on lactase expression and activity.

[0063] The present application also detects the serum and tissue inflammatory factors of each group. The results show that the concentration of inflammatory factors in the SLP - 2 group is the lowest among all groups and is basically close to that of the Con group. At the same time, the detection results of the genes and proteins related to the intestinal mucosal barrier show that, compared with other groups, the SLP - 2 group can express the genes and proteins related to the intestinal mucosal barrier more significantly.

[0064] Based on the above excellent technical effects, the present application provides the use of the plant polysaccharide in the preparation of products for enhancing lactase expression and activity or products for alleviating intestinal inflammation and repairing damaged intestinal mucosal barriers. Among them, the present application does not limit the type of products, and any suitable product form can be within the scope of application, such as foods, health products, drugs, etc.

[0065] In the fourth aspect of the present application, a product for enhancing lactase expression and activity or alleviating intestinal inflammation and establishing an intestinal mucosal barrier is provided, which comprises the plant polysaccharide described in the present application. For example, the product can be a dairy product added with the plant polysaccharide described in the present application, or a health product for improving intestinal lactase expression and activity added with the plant polysaccharide and / or other functional ingredients and additives described in the present application, or a drug clinically used to eliminate lactose-induced diarrhea with the plant polysaccharide described in the present application as the main active ingredient, and corresponding pharmaceutical excipients can be selected according to the dosage form requirements.

[0066] In each group of comparative experiments provided in the present application, unless otherwise specified, except for the differences pointed out in each group, other experimental conditions, materials, etc. are kept consistent for comparability. In addition, the materials used in the present application can all be obtained through commercial channels.

[0067] The following further describes a plant polysaccharide, its preparation method and application provided by the present application.

[0068] Example 1:

[0069] Shenling Baizhu Powder is composed of the following raw materials by weight percentage: ginseng 12.9%, Poria cocos 12.9%, Atractylodes macrocephala (fried) 12.9%, Chinese yam (dry product) 12.9%, white hyacinth bean (fried) 9.5%, lotus seed (kernel removed) 6.5%, coix seed (fried) 6.5%, Amomum villosum 6.5%, Platycodon grandiflorum 6.5%, and licorice 12.9%.

[0070] (1) Weigh the raw materials of Shenling Baizhu Powder according to the above weight percentage, add distilled water according to the solid-liquid ratio of 1:10 - 15, boil and then pour out the liquid. Add the same volume of distilled water again, boil and take the liquid. Combine the two liquid portions;

[0071] (2) After concentrating and centrifuging the liquid portion, add absolute ethanol for alcohol precipitation, centrifuge and take the precipitate, and bake it dry by infrared to obtain the crude polysaccharide precipitate of Shenling Baizhu Powder, with a yield of 10 - 15%;

[0072] (3) Take the crude polysaccharide for redissolution, and the solution is deproteinized by the Sevag method. Specifically, add chloroform - n - butanol solution, shake well and then centrifuge, pour the chloroform layer and the turbid layer containing proteins into a separating funnel for liquid separation, discard the chloroform and proteins, mix the upper layer solution with the previous supernatant, and repeat 5 times;

[0073] (4) Finally, add ethanol with a volume fraction of 95% to the protein-free solution, and centrifuge after alcohol precipitation. The precipitate is dried by infrared baking to obtain the total polysaccharide of Shenling Baizhu Powder, denoted as SLP, with a yield of 40 - 50%;

[0074] Dissolve the total polysaccharide of Shenling Baizhu Powder and take 20 μl after treatment for determination on a high-performance gel permeation chromatography (HPGPC) TSK-gel G3000 PWXL chromatographic column. The molecular weights of the two main components of the polysaccharide in Shenling Baizhu Powder are 4 kDa and 80 kDa respectively. An Agilent Eclipse XDB-C18 chromatographic column is selected to determine that the polysaccharide in Shenling Baizhu Powder mainly includes mannose, rhamnose, glucose, galactose, xylose, etc.;

[0075] (5) Take an appropriate amount of the total polysaccharide of Shenling Baizhu, add ultrapure water and stir until completely dissolved to obtain solution A. Slowly add anhydrous ethanol with a volume fraction of 20 - 30% to solution A, then let it stand at 4 °C for 10 - 12 h, and then centrifuge at a speed of 5000 - 6000 r / min for 10 - 20 min. Take the supernatant B, and collect the precipitate denoted as SLP-1 (control polysaccharide);

[0076] (6) Continue to add anhydrous ethanol with a volume fraction of 50 - 60% to the supernatant B, then let it stand at 4 °C for 10 - 12 h, and then centrifuge at a speed of 5000 - 6000 r / min for 10 - 20 min. Collect the precipitate C denoted as SLP-2 (the plant polysaccharide of this application);

[0077] Control the temperature below 60 °C and the time within 30 min to dry SLP, SLP-1, and SLP-2 to a constant weight.

[0078] Example 2:

[0079] Use an ion chromatograph to determine the monosaccharide composition of SLP-1 and SLP-2. Both SLP-1 and SLP-2 are composed of arabinose, galactose, glucose, xylose, fructose, and galacturonic acid, but their composition ratios and molecular weights are different, as shown in Table 1 below:

[0080] Table 1

[0081]

[0082] Determine the molecular weights of SLP-1 and SLP-2 by high-performance gel permeation chromatography (HPGPC). Among them, the molecular weight of SLP-1 is mainly 50 - 60 kDa, and the molecular weight of SLP-2 is mainly 4 - 30 kDa. Analyze the functional groups of the polysaccharide by Fourier transform infrared spectroscopy FT-IR. The results of SLP-1 show that the absorption band is at 3600 - 3200 cm -1is the stretching vibration absorption peak of -OH. The absorption peaks in this region are characteristic peaks of saccharides. Specifically as follows: 3567 cm -1 at, 3191 cm -1 at are the stretching vibration absorption peaks of O-H and are characteristic peaks of saccharides. 2927 cm -1 at is the stretching vibration absorption peak of C-H and is a characteristic peak of saccharides. At 1656 cm -1 at, 1604 cm -1 at there are absorption peaks, which may be attributed to the stretching vibration of C=O. At 1415 cm -1 at, 1151 cm -1 at, 1081 cm -1 at there are absorption peaks, which may be attributed to the stretching vibration of C-O. At 1020 cm -1 at there is an absorption peak, which may be attributed to the stretching vibration of O-H. At 836 cm -1 at there is an absorption peak, which may be attributed to the angular vibration of C-H of α-anomeric epimerization. At 761 cm -1 at there is an absorption peak, which may be attributed to the symmetric ring stretching vibration of the pyranose ring. The results of SLP-2 show that the absorption band is at 3600 - 3200 cm -1 is the stretching vibration absorption peak of -OH. The absorption peaks in this region are characteristic peaks of saccharides. Specifically as follows: 3288 cm -1 at is the stretching vibration absorption peak of O-H and is a characteristic peak of saccharides. 2933 cm -1 at is the stretching vibration absorption peak of C-H and is a characteristic peak of saccharides. At 1745 cm -1 at, 1639 cm -1 at there are absorption peaks, which may be attributed to the stretching vibration of C=O. At 1419 cm -1 at, 1151 cm -1 at, 1079 cm -1 at there are absorption peaks, which may be attributed to the stretching vibration of C-O. At 1240 cm -1 at, 1020 cm -1 at there are absorption peaks, which may be attributed to the stretching vibration of O-H. At 831 cm -1 at there is an absorption peak, which may be attributed to the angular vibration of C-H of α-anomeric epimerization. At 935 cm -1 at there is an absorption peak, which may be attributed to the asymmetric ring stretching vibration of the pyranose ring.

[0083] Example 3:

[0084] Experimental animals: SPF-grade male SD rats with a body weight of 260 ± 20 g were selected. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the license number is SCXK(Beijing) 2021-0006. The feed was purchased from K'ao Hsiang Hsieh Li (Tianjin) Feed Co., Ltd.

[0085] After 7 days of adaptive feeding with normal feed, the experiment was started. The rats were randomly divided into a healthy control group (hereinafter referred to as Con), a model group HLD group (hereinafter referred to as HLD), an SLP group, an SLP-1 group, and an SLP-2 group, with 8 rats in each group. The Con group was fed with normal feed, and the other groups were fed with gradient high-lactose feed to prepare a high-lactose diarrhea rat model. The gradient high-lactose feed feeding means that after one week of feeding with 30% high-lactose feed, it was continued to be fed with 40% high-lactose feed for one week, and then with 50% high-lactose feed for one week.

[0086] During the feeding of the gradient high-lactose feed, drugs were administered. The dosage of the drug was calculated by comprehensively considering the dosage of Shenling Baizhu San, the extraction rate of polysaccharides, the content of polysaccharides, and the body surface area of the rats. Specifically, the dosage of the SLP group was 330 mg / kg, the dosage of the SLP-1 group was 170 mg / kg, and the dosage of the SLP-2 group was 87 mg / kg. The administration method was intragastric administration. The Con group and the HLD group were given ultrapure water at 1 ml / 100 g, and intragastric administration was carried out at the same time point every day.

[0087] 1. Detection of physiological indicators

[0088] After the intervention, the small intestine tissue, colon tissue, cecal contents, abdominal aortic blood, and hepatic portal vein blood were retained under pentobarbital sodium anesthesia and detected after treatment.

[0089] (1) Detection of fecal water content

[0090] Feces were collected at 24 h, 48 h, and 72 h after modeling to observe the fecal characteristics and the state around the anus of the rats. Three times a week, the fecal samples were immediately weighed (wet weight) after collection, and then placed in an oven at 60 °C for 24 h of drying. After drying, the samples were weighed again to obtain the dry weight. The fecal water content was calculated using the following formula:

[0091] Fecal water content = (fecal wet weight – fecal dry weight) / fecal wet weight × 100%.

[0092] From Figure 1As can be seen from Figure A, compared with the Con group (64.16±1.11%), the fecal water content in the HLD group (82.77±2.50%, ***p<0.001) was significantly increased, and the fecal water content in the SLP-1 group (80.48±2.67%, ***p<0.001) was significantly increased after administration, indicating that the improvement effect of the polysaccharide in the SLP-1 group was limited; compared with the HLD group, the fecal water content in the SLP group (71.52±1.81%, **p<0.01) was significantly decreased after administration, and the fecal water content in the SLP-2 group (70.24±2.93%, **p<0.01) was also significantly decreased after administration. The fecal water content in the SLP-2 group was comparable to that in the SLP group after administration, and both were significantly less than that in the SLP-1 group (*p<0.05).

[0093] Figure B shows that at 24 h, diarrhea occurred in 75% of the rats in the SLP group, 50% in the SLP-2 group, while in the HLD group and the SLP-1 group, 100% had started diarrhea.

[0094] Figure C shows the fecal water content of feces collected at three time points of 24 - 48 - 72 h, from high to low were the HLD group, the SLP-1 group, the SLP group, the SLP-2 group, and the Con group.

[0095] Figure D shows the average fecal water content at the three fecal collection time points of 24 - 48 - 72 h. Compared with the Con group (61.32±3.45), the fecal water content in the HLD group (86.07±4.00, ***p<0.001), the SLP group (77.51±3.30, **p<0.01), the SLP-1 group (83.80±4.93, **p<0.01), and the SLP-2 group (73.99±4.06, *p<0.05) was significantly increased; compared with the HLD group (86.07±4.00), the fecal water content in the SLP-2 group (73.99±4.06, *p<0.05) was significantly decreased.

[0096] The above results of fecal water content indicate that the plant polysaccharide of the present application with a molecular weight of 4 - 30 kDa as the active ingredient can reduce the water content in the feces of animals in the lactose-induced diarrhea model, and the effect is more significant compared with other groups, which is directly related to its special effects of significantly increasing lactase expression and activity, as well as reducing inflammation and repairing the intestinal mucosal barrier.

[0097] (2) Grip strength detection

[0098] Use a rat grip strength meter, adjust the crossbar device on the grip strength meter, place the rat on the crossbar of the grip strength meter, ensure that its limbs naturally grasp the crossbar, gently pull the rat, and record the maximum value of each grip strength. Each rat is subjected to 3 independent grip strength tests, and appropriate rest can be taken between each test to avoid fatigue.

[0099] From Figure 2 It can be seen that compared with the Con group (1817.38 ± 45.31 N), the grasping force of the HLD group (1547.84 ± 20.19, ***p < 0.001) decreased significantly, the grasping force of the SLP group (1686.41 ± 24.60 N, *p < 0.05) decreased after administration, and the grasping force of the SLP-1 group (1620.05 ± 63.28 N, **p < 0.01) also decreased significantly after administration; compared with the HLD group, the grasping force of the SLP group (1686.41 ± 24.60 N, *p < 0.05) increased after administration, and the grasping force of the SLP-2 group (1744.29 ± 29.08 N, **p < 0.01) increased significantly after administration. The grasping force of the SLP-2 group after administration was greater than that of the SLP-1 group (*p < 0.05).

[0100] The results of the above grasping force detection show that the plant polysaccharide of the present application with a molecular weight of 4 - 30 kDa as the active ingredient can increase the grasping force of animals in the lactose-induced diarrhea model, and the effect is more significant compared with other groups, which is directly related to its special effects of significantly increasing lactase expression and activity, and reducing inflammation and repairing the intestinal mucosal barrier.

[0101] (3) Fecal pH value detection

[0102] Collect fresh fecal samples and mix them with distilled water at a ratio of 1:10. Filter the slurry to remove solid particles, and measure the pH value using a pH meter (Mettler Toledo, Zurich, Switzerland), which has been calibrated with a standard buffer solution. Record the pH value immediately after mixing.

[0103] From Figure 3 It can be seen that compared with the Con group (7.28 ± 0.10), the fecal pH value of the HLD group (5.18 ± 0.25, ***p < 0.001) decreased significantly, the fecal pH value of the SLP group (5.83 ± 0.19, ***p < 0.001) decreased significantly after administration, the fecal pH value of the SLP-1 group (5.21 ± 0.19, ***p < 0.001) decreased significantly after administration, and the fecal pH value of the SLP-2 group (6.04 ± 0.12, ***p < 0.001) decreased significantly after administration;

[0104] The fecal pH value of the SLP-1 group after administration was equivalent to that of the HLD group, both less than that of the SLP group (*p < 0.05). Compared with the HLD group, the fecal pH value of the SLP-2 group increased significantly after administration (**p < 0.01), and compared with the SLP-1 group, the fecal pH value of the SLP-2 group increased significantly after administration (**p < 0.01).

[0105] The above fecal pH value detection results show that although the pH values of each group are significantly lower than those of the Con group, in terms of the improvement effect, compared with the HLD group, the plant polysaccharide of the present application with a molecular weight of 4-30 kDa as the active ingredient can increase the fecal pH value of animals in the lactose-induced diarrhea model, and the effect is more significant than that of other groups. This is directly related to its special effects of significantly increasing lactase expression and activity, as well as reducing inflammation and repairing the intestinal mucosal barrier.

[0106] 2. Lactase activity detection

[0107] (1) In vitro lactase activity detection

[0108] Dissolve lactase (1.5 mg / mL) and SLP, SLP-2 (2.0 mg / mL) in 0.2 M PBS (pH 7.0) respectively. Subsequently, completely mix 1.0 mL of each sample solution at 37 °C. Measure the OD at 600 nm at 0, 30, 60, 120, and 180 minutes respectively. 600 And measure the absorbance value according to the operation instructions of the lactase activity detection kit (Beijing Solarbio Science & Technology Co., Ltd.).

[0109] It can be directly obtained from Table 2 and Figure 4 that: compared with the Lactase group, SLP group, and SLP-1 group, the SLP-2 group increased significantly at 30, 60, 120, and 180 min, proving that the plant polysaccharide provided by the present application has a very significant promoting effect on improving lactase activity.

[0110] Table 2

[0111]

[0112]

[0113] (2) Detection of lactose content in cecal contents

[0114] Take 300 mg of cecal contents, add 600 μL of pure water, mix well, centrifuge at 12000 rpm for 10 min, take the supernatant, filter the supernatant through a 0.22 μm filter, wash the filter with an appropriate amount of pure water, and then dry it in an oven. Re-dissolve it with 300 μL of pure water, centrifuge at 12000 rpm for 10 min, and take the supernatant for testing. Detect the lactose content according to the operation instructions of the lactose microplate detection kit (cohesion).

[0115] According to the detection results of the lactose content in cecal contents, the influence degree of each group of polysaccharides on lactase in vivo can be indirectly compared. From Figure 5It was concluded that: compared with the Con group (53.90±17.34), the lactose content in the HLD group (114.19±16.85, **p<0.01) and the SLP-1 group (131.37±17.95, ***p<0.001) increased significantly, indicating that the SLP-1 group could not effectively improve the activity of lactase in vivo; compared with the HLD group (114.19±16.85), the content in the SLP group (69.06±8.50, *p<0.05) decreased significantly; compared with the SLP-1 group (131.37±17.95), the content in the SLP group (69.06±8.50, **p<0.01) and the SLP-2 group (83.81±7.07, *p<0.05) decreased significantly.

[0116] (3) Detection of lactase protein expression level in small intestine tissue

[0117] Detection of lactase protein expression level in small intestine tissue: The protein samples of the tissue were lysed with RIPA lysis buffer containing protease, phosphatase inhibitor and PMSF. The protein concentration was quantified by BCA protein assay. 25 μg of protein was loaded onto an SDS PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder, incubated with the primary antibody overnight at 4°C, and incubated with the secondary antibody for 1 hour. The protein bands were determined using a GelViewb chemiluminescence imaging system (Guangzhou, China). The optical density was quantified using Image Lab1.41 software.

[0118] It was concluded from Figure 6 that: Figure A shows the protein expression bands of lactase, and Figure B shows the relative protein expression level of lactase. Compared with the Con group (1.00±0.00), the HLD group (1.13±0.23), the SLP group (1.28±0.32), and the SLP-1 group (0.95±0.28), the protein expression level in the SLP-2 group (2.15±0.20, *p<0.05 compared with the SLP group, **p<0.01 for the other groups) increased significantly, indicating that the plant polysaccharide of the present application can increase the expression of lactase in vivo.

[0119] (4) Immunofluorescence detection of lactase

[0120] After dewaxing and rehydrating the paraffin sections, antigen retrieval was performed using EDTA. The sections were rinsed 3 times with PBS buffer for 3 minutes each time, blocked with 1% BSA, and incubated at room temperature for 30 minutes. The blocking solution was gently blotted dry, and the primary antibody was added dropwise to the sections, followed by incubation overnight at 4°C in a humidified chamber. After rewarming for 30 minutes, the sections were rinsed 3 times with PBS buffer for 5 minutes each time. After gently blotting dry, the diluted fluorescent secondary antibody was added dropwise to cover the tissue, and incubated at room temperature in the dark for 120 minutes. The sections were washed 3 times with PBS buffer for 5 minutes each time, and after gently blotting dry, DAPI staining solution was added and incubated at room temperature in the dark for 10 minutes. Anti-tissue autofluorescence: The sections were washed 3 times with PBS buffer, and anti-tissue autofluorescence agent was gently added and incubated at room temperature for 10 minutes. Mounting: The sections were washed 3 times with PBS buffer for 5 minutes each time, blotted dry, and then mounted with anti-fluorescence quenching mounting medium. Observation and image acquisition were performed using a fluorescence microscope.

[0121] From Figure 7 It was obtained that compared with the Con group (3.19 ± 0.44), HLD group (5.76 ± 2.43), SLP group (10.54 ± 3.94), and SLP-1 group (5.39 ± 1.00), the fluorescence intensity of the SLP-2 group (47.26 ± 4.14, ***p < 0.001) was significantly increased, and the fluorescence detection results further confirmed that the plant polysaccharide of this application could significantly increase lactase expression.

[0122] 3. Detection of serum and tissue inflammatory factors

[0123] The whole blood specimens were left standing at room temperature for 2 hours, centrifuged at 3000 rpm at 4°C for 15 minutes, and the upper-layer serum was collected. After aliquoting, it was transferred to a -80°C refrigerator for storage. Before detection, the serum was taken out and thawed at room temperature. The colon tissue was added with 10 times the volume of normal saline according to the ratio of weight (mg): volume (uL) = 1:10, mechanically homogenized under low-temperature conditions at 2500 - 3000 revolutions per minute, centrifuged for 10 minutes, and the upper-layer supernatant was taken for determination. The experimental operations were carried out according to the instructions of the kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.), and IL-6 and IL-1β were detected respectively.

[0124] From Figure 8 It was obtained from Figure A that compared with the HLD group (0.22 ± 0.02 ng / mL), the content of the inflammatory factor IL-6 in the serum of the SLP-2 group (0.17 ± 0.02 ng / mL, *p < 0.05) was significantly decreased after administration, and the content of IL-6 in the other groups was higher than that of the SLP-2 group.

[0125] From Figure 8As shown in Figure B, compared with the Con group (3.21 ± 0.31 pg / mg), the content of inflammatory factor IL-6 in the colon tissue of the HLD group (4.49 ± 0.51 pg / mg, *p < 0.05) increased significantly; compared with the HLD group, the content of inflammatory factor IL-6 in the colon tissue after administration of the SLP-2 group (3.36 ± 0.35 pg / mg, *p < 0.05) decreased significantly, and the effect was better than that of the SLP group and the SLP-1 group, and was close to the IL-6 content of the Con group.

[0126] As shown in Figure 8 As shown in Figure C, compared with the Con group (0.57 ± 0.05 pg / mg), the content of inflammatory factor IL-1β in the colon tissue of the HLD group (0.85 ± 0.08 pg / mg, **p < 0.01) increased significantly, and the content of inflammatory factor IL-1β in the colon tissue after administration of the SLP group (0.76 ± 0.08 pg / mg, *p < 0.05) increased significantly; compared with the HLD group, the content of inflammatory factor IL-1β in the colon tissue after administration of the SLP-2 group (0.63 ± 0.09 pg / mg, *p < 0.05) decreased significantly, and the effect was better than that of the SLP group and the SLP-1 group.

[0127] 4. Detection of the expression levels of genes related to the mucosal barrier in the small intestine and colon tissues

[0128] Genes related to the mucosal barrier: Occludin gene, ZO-1 gene, Claudin-1 gene; Detection was carried out using a commercially available kit.

[0129] As shown in Figure 9 It was shown that compared with the HLD group, the expressions of genes of ZO-1 (1.66 ± 0.30, *p < 0.05), Claudin-1 (1.29 ± 0.31, ***p < 0.001), Occludin (0.81 ± 0.25, *p < 0.05) in the small intestine, and Occludin (1.73 ± 0.44, **p < 0.01), ZO-1 (2.61 ± 0.67, **p < 0.01), Claudin-1 (5.78 ± 0.93, ***p < 0.001) in the colon of the SLP-2 group increased significantly;

[0130] Compared with the SLP group, the expressions of genes of Occludin (0.81 ± 0.25, *p < 0.05) and Claudin-1 (1.29 ± 0.31, **p < 0.01) in the small intestine of the SLP-2 group increased significantly.

[0131] Compared with the SLP-1 group, the gene expressions of intestinal ZO-1 (1.66±0.30, *p<0.05) and Claudin-1 (1.29±0.31, **p<0.01) in the SLP-2 group, and Claudin-1 (5.78±0.93, ***p<0.001) in the colon were significantly increased.

[0132] The above results indicate that after the intervention of the plant polysaccharide of the present application, the expression levels of genes related to the intestinal mucosal barrier can be promoted, and the damaged intestinal mucosal barrier can be repaired, thereby further providing protection for the intestine.

[0133] 5. Detection of the expression levels of proteins related to the mucosal barrier in colon tissues

[0134] Proteins related to the mucosal barrier: Occludin protein, ZO-1 protein, Claudin-1 protein; Detection was carried out with reference to the detection method of lactase protein in intestinal tissues;

[0135] It was obtained from Figure 10 that compared with the HLD group, the SLP-2 group was significantly improved in the expression levels of the three proteins; at the same time, compared with the SLP group and the SLP-1 group, the expression levels of Occludin protein and ZO-1 protein in the SLP-2 group were significantly increased after intervention.

[0136] 6. Barrier immunofluorescence detection

[0137] Barrier protein: ZO-1; Detection was carried out with reference to the immunofluorescence detection method of lactase;

[0138] It was obtained from Figure 11 that compared with the Con group (22.43±2.00), there was no significant difference in the SLP-2 group (21.32±1.07), while in the HLD group (18.56±0.18, **p<0.01), the SLP group (19.98±0.52, *p<0.05), and the SLP-1 group (19.95±0.24, *p<0.05), the average fluorescence intensity was significantly decreased; compared with the HLD group (18.56±0.18), the average fluorescence intensity in the SLP-2 group (21.32±1.07, **p<0.01) was significantly increased.

[0139] The above are only the specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A plant polysaccharide, characterized in that, Comprising, by weight percentage: Arabinose 3 - 4%, galactose 7 - 8%, glucose 39 - 40%, xylose 1 - 2%, fructose 29 - 30% and galacturonic acid 19 - 20%.

2. The plant polysaccharide according to claim 1, wherein The molecular weight is 4 - 30 kDa.

3. A method for preparing a plant polysaccharide as described in claim 1, characterized in that, Comprising: Extracting the total polysaccharide from a traditional Chinese medicine composition consisting of ginseng, poria cocos, atractylodes macrocephala, Chinese yam, white hyacinth bean, lotus seed, coix seed, amomum villosum, platycodon grandiflorum and liquorice; After redissolving the total polysaccharide in water, performing the first alcohol precipitation with a first ethanol solution, and centrifuging to obtain the supernatant; Adding a second ethanol solution to the supernatant, performing the second alcohol precipitation, and centrifuging to obtain the precipitate, thereby obtaining the plant polysaccharide; Wherein, the volume fraction of the first ethanol solution is 20 - 30%; the volume fraction of the second ethanol solution is 50 - 60%.

4. The preparation method according to claim 3, characterized in that, The traditional Chinese medicine composition, by weight percentage, is composed of: ginseng 12.9%, poria cocos 12.9%, atractylodes macrocephala 12.9%, Chinese yam 12.9%, white hyacinth bean 9.5%, lotus seed 6.5%, coix seed 6.5%, amomum villosum 6.5%, platycodon grandiflorum 6.5% and liquorice 12.9%.

5. The preparation method according to claim 3 or 4, characterized in that The total polysaccharide is separated from the traditional Chinese medicine extract by water decoction method.

6. The preparation method according to claim 3, characterized in that, The temperatures of the first alcohol precipitation and the second alcohol precipitation are independently selected from 0 - 4°C respectively.

7. Use of the plant polysaccharide according to claim 1 or 2 in the preparation of a product for increasing lactase expression and activity or a product for alleviating intestinal inflammation and repairing damaged intestinal mucosal barrier.

8. The application according to claim 7, wherein The product includes food, health products, and drugs.

9. A product for enhancing lactase expression and activity, or alleviating intestinal inflammation and repairing damaged intestinal mucosal barrier, characterized in that, Comprising the plant polysaccharide according to claim 1 or 2.

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