Total polysaccharides from Camellia fraterna Hance and its preparation method and application

Abstract

The present invention relates to total polysaccharides from Camellia sinensis and a preparation method and application thereof. The preparation method comprises the following steps: (1) soaking Camellia sinensis medicinal materials in 95% ethanol overnight; (2) decocting the soaked Camellia sinensis medicinal materials with distilled water, filtering, concentrating the filtrate, centrifuging, and discarding the precipitate; (3) adding 95% ethanol to the supernatant until the alcohol content is 80%, sealing and standing for 24 hours, centrifuging, collecting the precipitate, and freeze-drying to obtain Camellia sinensis crude polysaccharides; (4) removing free protein from the Camellia sinensis crude polysaccharides by the Sevag method, and freeze-drying the aqueous phase to obtain the total polysaccharides from Camellia sinensis. The present invention proves through pharmacological experiments that the total polysaccharides from Camellia sinensis can be used in the preparation of drugs with weight loss function, in the preparation of drugs for lowering blood lipids, in the preparation of drugs for protecting the liver, in the preparation of drugs for treating or improving insulin resistance and glucose and lipid metabolism disorders, or in the preparation of drugs for treating or regulating intestinal flora disorders.

Description

Technical Field

[0001] The present invention relates to the field of natural product extraction and application, and in particular to Camellia sinensis total polysaccharide and a preparation method and application thereof. Background Art

[0002] Mountain green tea is a plant of the Aquifoliaceae family, Ilex hainanensis ( Ilex hainanensis Merr.) is processed and prepared from the dried leaves. It is a traditional Chinese medicinal herb used by ethnic minorities in Guangxi. It has the effects of clearing heat and detoxifying, reducing swelling and relieving pain, and promoting blood circulation and unblocking meridians. It is primarily used to lower blood pressure, blood lipids, and blood sugar, and protect the liver. Shanlucha and its preparations have been widely used clinically. Shanlucha Antihypertensive Tablets, a single-ingredient preparation made from Shanlucha, are included in Volume 1 of the Chinese Pharmacopoeia (2020 edition). Currently, research on the chemical composition and pharmacological effects of Shanlucha is relatively systematic. However, in addition to the already reported flavonoids, triterpenoid saponins, volatile oils, and organic acids, Shanlucha also contains a rich polysaccharide content, but there are currently no reports on the total polysaccharides in Shanlucha. Summary of the Invention

[0003] The purpose of the present invention is to provide total polysaccharides from Camellia oleifera and its preparation method and application, so as to promote the application of total polysaccharides from Camellia oleifera in pharmaceuticals and functional foods, thereby providing a new direction for the application of total polysaccharides from Camellia oleifera and a new idea for the treatment of some diseases.

[0004] The present invention extracts total green tea polysaccharides from mountain green tea. The total green tea polysaccharides have the effects of protecting the liver, lowering blood lipids, lowering blood sugar, regulating intestinal flora, and losing weight. The preparation method thereof comprises the following steps:

[0005] (1) Add 95% ethanol to the tea leaves and soak overnight;

[0006] (2) Decoction the soaked mountain green tea medicinal material with distilled water for 1-3 h, filter, concentrate the filtrate, centrifuge, and discard the precipitate;

[0007] (3) Add 95% ethanol to the supernatant obtained in step (2) until the alcohol content is 80%, seal and let stand for 12-36 hours, centrifuge, collect the precipitate, freeze-dry, and obtain the crude polysaccharide of Camellia sinensis.

[0008] (4) The Sevag method was used to remove free proteins from the crude polysaccharides of Camellia sinensis, and then the aqueous phase was freeze-dried to obtain the total polysaccharides of Camellia sinensis.

[0009] The present invention conducted pharmacological experiments on total Camellia sinensis polysaccharides, using an obese mouse model to investigate their ability to inhibit weight gain in obese mice and evaluate their weight-loss effects. Experiments with obese mice demonstrated that total Camellia sinensis polysaccharides had no significant toxic effects on normal mice and had no effect on their food intake. With the exception of the low-dose group, the body weights of mice in the medium and high-dose groups all decreased significantly. Compared with the model group, all dose groups showed significant improvements in liver weight, gonadal fat, and perirenal fat weight, with a dose-dependent effect. These experimental results demonstrate that the total Camellia sinensis polysaccharides provided by the present invention have a significant weight-loss effect and can be used to prepare drugs and / or foods with weight-loss properties. Specifically, total Camellia sinensis polysaccharides can be used in the preparation of drugs and / or foods with weight-loss properties.

[0010] Furthermore, the total polysaccharide of Camellia sinensis is used in the preparation of medicines and / or foods for treating or preventing obesity caused by a high-fat diet.

[0011] The present invention also investigated the ability of Camellia sinensis polysaccharides to reduce serum TC, TG, and LDL-C levels using an obese mouse model, evaluating the lipid-lowering effect of Camellia sinensis polysaccharides. The obese mouse experiments demonstrated that Camellia sinensis polysaccharides significantly improved serum LDL-C levels in a dose-dependent manner, while also reducing serum TC and TG levels. These experimental results demonstrate that the Camellia sinensis polysaccharides provided by the present invention have a significant lipid-lowering effect and can be used in the preparation of lipid-lowering drugs and / or foods.

[0012] Furthermore, lipid-lowering drugs and / or foods refer to drugs or foods that lower low-density lipoprotein cholesterol, or drugs or foods that simultaneously lower low-density lipoprotein cholesterol, total cholesterol, and triglycerides.

[0013] Furthermore, the total polysaccharide of Camellia sinensis is used in the preparation of medicines and / or foods for treating or preventing hyperlipidemia caused by a high-fat diet.

[0014] The present invention also used an obese mouse model to investigate the effects of Camellia sinensis total polysaccharides on liver weight, serum AST and ALT levels, liver tissue morphology, and inflammatory factors in the liver, evaluating the anti-liver damage and hepatoprotective effects of Camellia sinensis total polysaccharides. Experiments with obese mice showed that Camellia sinensis total polysaccharides significantly reduced liver weight and improved hepatocyte fatty degeneration. They also significantly reduced serum AST and ALT levels, and reduced liver levels of inflammatory factors such as IL-1β, IL-6, and TNF-α. These experimental results demonstrate that the Camellia sinensis total polysaccharides provided by the present invention have significant hepatoprotective effects and can be used in the preparation of liver-protective drugs and / or foods.

[0015] Furthermore, the invention relates to the use of total polysaccharides from Camellia sinensis in the preparation of medicines and / or foods for treating or preventing liver damage caused by a high-fat diet.

[0016] Furthermore, liver damage is caused by viruses, drugs, fatty degeneration, and tumors, including fatty liver and hepatitis.

[0017] Furthermore, drugs and / or foods for preventing liver damage and protecting the liver refer to drugs or foods that lower serum AST and ALT, or drugs or foods that lower inflammatory factors IL-1β, IL-6 and TNF-α in the liver, or drugs or foods that improve liver tissue morphology.

[0018] The present invention also investigated the effects of Camellia sinensis total polysaccharides on glucose tolerance and insulin levels using an obese mouse model, evaluating their efficacy in improving insulin resistance and glucolipid metabolism disorders. Glucose tolerance tests in obese mice showed significant decreases in fasting and peak blood glucose levels in the low, medium, and high dose groups, suggesting that Camellia sinensis total polysaccharides can improve glucose intolerance in mice to a certain extent. Furthermore, serum insulin level measurements showed that all doses of Camellia sinensis total polysaccharides significantly reduced the homeostasis model insulin resistance index (HOMA-IR) and improved elevated serum insulin levels. These experimental results demonstrate that the Camellia sinensis total polysaccharides provided by the present invention have significant effects on improving insulin resistance and glucolipid metabolism disorders, and can be used in the preparation of drugs and / or foods for treating or improving insulin resistance and glucolipid metabolism disorders.

[0019] Furthermore, insulin resistance and glucose and lipid metabolism disorders refer to insulin resistance and glucose and lipid metabolism disorders caused by a high-fat diet.

[0020] The present invention also used an obese mouse model to investigate the effect of total polysaccharides from mountain green tea on intestinal flora and evaluate the role of total polysaccharides from mountain green tea in regulating intestinal flora. The obese mouse experiment showed that total polysaccharides from mountain green tea can significantly increase the Alpha diversity index of intestinal flora and increase the number of beneficial bacteria. Alistipes 、 Flavonifractor 、 Clostridium_IV abundance, reducing harmful bacteria Desulfovibrio 、 Mucispirillum These experimental results prove that the total polysaccharide of Camellia sinensis provided by the present invention has the function of regulating the structure and abundance of intestinal flora, and can be used in the preparation of medicines and / or foods for treating or regulating intestinal flora disorders.

[0021] Furthermore, intestinal flora disturbance refers to intestinal flora disturbance caused by a high-fat diet.

[0022] Furthermore, drugs and / or foods for treating or regulating intestinal flora disorders refer to drugs and / or foods that regulate the structure of intestinal flora and increase beneficial bacteria. Alistipes 、 Flavonifractor 、 Clostridium_IV abundance, reducing harmful bacteria Desulfovibrio 、 Mucispirillum Abundance of medicine or food.

[0023] When the total polysaccharide of Camellia sinensis is added as an active ingredient in medicine or food, the dosage of the total polysaccharide of Camellia sinensis is 240 mg / (kg·d) to 960 mg / (kg·d), preferably 960 mg / (kg·d). The total polysaccharide of Camellia sinensis can be mixed with excipients according to pharmaceutical methods to prepare a plurality of clinical drug dosage forms, usually oral preparations, which are selected from any one of powders, oral liquids, granules, tablets, capsules or pills.

[0024] The food mentioned in the present invention refers to various finished products and raw materials for human consumption or drinking, as well as articles that are traditionally both food and medicine, but does not include articles for therapeutic purposes. Specifically, the food mentioned in the present invention refers to substances that can be eaten or drunk by humans, including processed foods, semi-finished products and unprocessed foods, including health foods, but does not include tobacco or substances that are only used as medicines. The food mentioned in the present invention includes but is not limited to health foods. All food forms that can be accepted by those skilled in the art are within the scope of protection of the present invention. It can be one or more of solid products, dairy products, solution products, powder products or suspension products, and the present invention is not limited thereto.

[0025] The present invention also provides an active polysaccharide having the effects of protecting the liver, lowering blood lipids, lowering blood sugar, regulating intestinal flora and losing weight, wherein the active polysaccharide comprises the total polysaccharide of Camellia sinensis.

[0026] Furthermore, the preparation method of the active polysaccharide comprises the following steps:

[0027] (1) Add 95% ethanol to the tea leaves and soak overnight;

[0028] (2) Decoction the soaked mountain green tea medicinal material with distilled water for 1-3 h, filter, concentrate the filtrate, centrifuge, and discard the precipitate;

[0029] (3) Add 95% ethanol to the supernatant obtained in step (2) until the alcohol content is 80%, seal and let stand for 12-24 hours, centrifuge, collect the precipitate, freeze-dry, and obtain the crude polysaccharide of Camellia sinensis.

[0030] (4) The Sevag method was used to remove free proteins from the crude polysaccharides of Camellia sinensis, and then the aqueous phase was freeze-dried to obtain the total polysaccharides of Camellia sinensis.

[0031] Advantages and beneficial effects of the present invention:

[0032] (1) The total polysaccharide of Camellia sinensis provided by the present invention can still reduce body weight during a normal high-fat diet, and has a significant weight-loss effect. It can be used to prepare drugs and / or foods with weight-loss function, and effectively prevent obesity caused by a high-fat diet.

[0033] (2) The total polysaccharide of Camellia sinensis provided by the present invention also has a significant lipid-lowering effect and can be used in the preparation of lipid-lowering drugs and / or foods, effectively preventing hyperlipidemia caused by a high-fat diet.

[0034] (3) The total polysaccharide of Camellia sinensis provided by the present invention also has significant anti-liver damage and liver protection effects, and can be used in the preparation of anti-liver damage and liver protection drugs and / or foods, effectively preventing liver damage (fatty liver or hepatitis) caused by a high-fat diet.

[0035] (4) The total polysaccharide of Camellia sinensis provided by the present invention also has the effect of improving insulin resistance and glucose and lipid metabolism disorders, and can be used in the preparation of drugs and / or foods for treating or improving insulin resistance and glucose and lipid metabolism disorders, and effectively prevent symptoms of abnormal blood sugar such as decreased glucose tolerance and increased insulin resistance index caused by a high-fat diet.

[0036] (5) The total polysaccharide of Camellia sinensis provided by the present invention also has the function of regulating the structure and abundance of intestinal flora, and can be used in the preparation of drugs and / or foods for treating or regulating intestinal flora disorders, and effectively prevent the symptoms of intestinal flora disorders caused by a high-fat diet.

[0037] (6) The total polysaccharide of Camellia sinensis provided by the present invention has the effects of protecting the liver, lowering blood lipids, lowering blood sugar, regulating intestinal flora, and losing weight. Taking the total polysaccharide of Camellia sinensis during a high-fat diet can not only reduce serum AST and ALT levels, improve hepatocyte fatty degeneration, reduce the levels of inflammatory factors IL-1β, IL-6 and TNF-α in the liver, and protect the liver from damage; it can also significantly improve serum LDL-C, TC, TG levels and insulin levels, avoid causing hyperlipidemia and hyperglycemia, and reduce the incidence of cardiovascular and cerebrovascular diseases; at the same time, it can slow down weight gain and reduce weight, regulate intestinal flora, and comprehensively regulate the health of people on a high-fat diet and prolong life. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the average daily food intake change of mice during the treatment period;

[0039] Among them, normal group (Chow), model group (HFD), fenofibrate group (HFD+FNB), total polysaccharide group of mountain green tea (Chow+IMP-H), low-dose total polysaccharide group of mountain green tea (HFD+IMP-L), medium-dose total polysaccharide group of mountain green tea (HFD+IMP-M), and high-dose total polysaccharide group of mountain green tea (HFD+IMP-H);

[0040] Figure 2 The weekly body weight changes of mice during the treatment period;

[0041] Figure 3H&E staining of mouse liver tissue pathological sections;

[0042] Figure 4 The results of the glucose tolerance test;

[0043] Figure 5 The results of principal component analysis of mouse intestinal flora. DETAILED DESCRIPTION

[0044] In order to more clearly illustrate the technical solution of the present invention, the present invention is further described below by specific examples, but these examples are only for illustration, not limitation of the present invention. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0045] The present invention extracts total polysaccharides from Camellia sinensis and conducts pharmacological experiments on the total polysaccharides. The experimental results show that:

[0046] (1) The total polysaccharide of Camellia sinensis provided by the present invention can still reduce body weight during a normal high-fat diet, and has a significant weight-loss effect, and can be used to prepare drugs and / or foods with weight-loss function; in particular, it can be used in the preparation of drugs and / or foods for treating or preventing obesity caused by a high-fat diet.

[0047] (2) The total polysaccharide of Camellia sinensis provided by the present invention has a significant lipid-lowering effect and can be used in the preparation of lipid-lowering drugs and / or foods, especially in the preparation of drugs and / or foods for treating or preventing hyperlipidemia caused by a high-fat diet.

[0048] (3) The total polysaccharide of Camellia sinensis provided by the present invention has significant anti-liver damage and liver protection effects, and can be used in the preparation of liver protection drugs and / or foods, especially in the preparation of drugs and / or foods for treating or preventing liver damage caused by a high-fat diet.

[0049] (4) The total polysaccharide of Camellia sinensis provided by the present invention has a significant effect on improving insulin resistance and glucose and lipid metabolism disorders, and can be used in the preparation of drugs and / or foods for treating or improving insulin resistance and glucose and lipid metabolism disorders, especially insulin resistance and glucose and lipid metabolism disorders caused by a high-fat diet.

[0050] (5) The total polysaccharide of Camellia sinensis provided by the present invention has the function of regulating the structure and abundance of intestinal flora, and can be used in the preparation of drugs and / or foods for treating or regulating intestinal flora disorders, especially intestinal flora disorders caused by a high-fat diet.

[0051] The present invention will be further described below in conjunction with the embodiments:

[0052] Example 1

[0053] The total polysaccharide of Camellia sinensis of the present invention is prepared by the following preparation method:

[0054] Weigh 5 kg of Camellia sinensis (L. truncatum) and soak overnight in 95% ethanol. Decoction was then performed with 10-fold distilled water for 2 h. The filtrate was filtered and concentrated to 1 / 5 of its original volume in a 60°C water bath. The filtrate was centrifuged at 5000 rpm for 15 min, and the precipitate was discarded. 95% ethanol was added to the supernatant until the alcohol content was 80%. The mixture was sealed and incubated at 4°C for 24 h. The precipitate was then centrifuged at 5000 rpm for 15 min. The precipitate was collected and freeze-dried to obtain crude Camellia sinensis polysaccharides. Free protein was removed from the crude Camellia sinensis polysaccharides using the Sevag method, and the aqueous phase was freeze-dried to obtain total Camellia sinensis polysaccharides.

[0055] Example 2

[0056] The present invention conducted a pharmacological experiment using the total polysaccharide of Camellia sinensis obtained in Example 1 above, and the specific results are as follows:

[0057] 1. Experimental animals and feed

[0058] Sixty-four SPF-grade C57BL / 6 male mice, weighing 18-22 g, were provided by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Regular diet (D12450J) and high-fat diet (D12492, 60 kcal%) were both produced by Research Diets, USA. The total polysaccharide of Camellia sinensis was mixed with ordinary feed at a content of 9.6 g / 1000 g to prepare the feed of Camellia sinensis total polysaccharide group (Chow+IMP-H) containing 9.6‰ of the drug; the total polysaccharide of Camellia sinensis was mixed with high-fat feed at a content of 2.4 g / 1000 g to prepare the feed of low-dose group (HFD+IMP-L) containing 2.4‰ of the drug; the total polysaccharide of Camellia sinensis was mixed with high-fat feed at a content of 4.8 g / 1000 g to prepare the feed of medium-dose group (HFD+IMP-M) containing 4.8‰ of the drug; the total polysaccharide of Camellia sinensis was mixed with high-fat feed at a content of 9.6 g / 1000 g to prepare the feed of high-dose group (HFD+IMP-H) containing 9.6‰ of the drug; the positive drug fenofibrate was mixed with 0.6 g / 1000 g g of the drug were mixed with high-fat feed to prepare the feed containing 0.6‰ fenofibrate group (HFD+FNB).

[0059] 2. Experimental methods

[0060] 2.1 Animal husbandry

[0061] The experimental animals were housed in the Capital Medical University Experimental Animal Center, four per cage, at 20-25°C, with a 12h:12h daylight cycle, and free access to food and water. After one week of adaptive feeding, the animals were randomly divided into a normal group (20 mice) and a model group (44 mice). The normal group received a normal diet, while the model group received a high-fat diet for 8 weeks to induce a NAFLD model. Four mice were randomly selected from each group, and the success of the model was assessed by weight and pathological examination. After successful model establishment, mice were treated with various doses of total polysaccharides from Camellia sinensis for 8 weeks (9-16 weeks), while continuing to maintain a high-fat diet. Animals were randomly divided into the following groups: normal control group (Chow), Camellia sinensis polysaccharide group (Chow + IMP-H), model group (HFD), low-dose Camellia sinensis polysaccharide group (HFD + IMP-L), medium-dose Camellia sinensis polysaccharide group (HFD + IMP-M), high-dose Camellia sinensis polysaccharide group (HFD + IMP-H), and fenofibrate group (HFD + FNB). They were fed the corresponding diets. Body weight and food intake were measured weekly. Glucose tolerance was assessed in each group at week 15 of treatment. At week 16, samples were collected (blood was drawn from the apex of the heart; liver, gonadal fat, and perirenal fat were collected and weighed; and cecal contents were obtained and fixed with 4% paraformaldehyde or snap-frozen in liquid nitrogen and stored at -80°C, as appropriate).

[0062] 2.2 Index determination

[0063] The levels of TC, TG, LDL-C, and transaminases in mouse serum were determined using a kit according to the instructions. The liver was embedded in paraffin and stained with H&E. The cecal contents of the mice were collected and the total bacterial DNA was extracted using a DNA extraction kit. The V3-V4 region sequence of the 16S rRNA gene was amplified. The PCR amplification obtained above was quantified and homogenized, and then the library was constructed using Illumina Miseq The optimized sequences were sequenced using the PCR platform. Based on a 97% similarity, OTU classification was performed on the obtained sequences, and the abundance of intestinal flora was analyzed at each taxonomic level.

[0064] 3. Statistical analysis: All data in this experiment were expressed as mean ± standard deviation (𝑥̅±SD). SPSS 19.0 software was used to perform one-way analysis of variance and t-test to analyze the differences between the groups. P <0.05 was used as the reference standard for statistical difference.

[0065] 4. Experimental results

[0066] 4.1 Effects on food intake, body weight, organ weight, and blood lipids in mice

[0067] Compared with the normal group, the body weight, liver weight, gonadal fat and perirenal fat weight of mice in the model group were significantly increased ( P<0.01). This indicates that the high-fat diet significantly increased the body weight and organ mass of mice. The above indicators of the normal-administered mountain green tea group showed no significant changes compared with the normal group, indicating that the total polysaccharide of mountain green tea had no obvious toxic reaction to normal mice and could be used in the future. During the treatment, the average daily food intake of mice ( Figure 1 ) had no significant changes; compared with the model group, the body weight of mice in the low- and medium-dose groups increased slowly, while the body weight of mice in the high-dose group decreased gradually (Table 1). At the end of the 16th week, except for the low-dose group, the body weight of mice in the medium- and high-dose groups decreased significantly ( P <0.01). This indicates that Camellia sinensis polysaccharides have a significant effect on improving the abnormal body weight and organ weight of mice induced by a high-fat diet, suggesting that Camellia sinensis polysaccharides may have a certain weight loss effect. Compared with the model group, the above indicators in the positive drug group were significantly improved ( P <0.01).

[0068] Compared with the normal group, the serum TC, TG and LDL-C levels of mice in the model group were significantly increased ( P <0.01 or P <0.05). This indicates that a high-fat diet induced dyslipidemia in mice. Compared with the model group, total polysaccharides from Camellia sinensis significantly improved serum LDL-C levels in a dose-dependent manner ( P <0.01); reduced serum TC levels, but only the high-dose group showed significant difference ( P <0.05); reduced serum TG levels. Compared with the model group, the fenofibrate group had significant improvements in serum TC, TG and LDL-C levels ( P <0.01).

[0069]

[0070] 4.2 Effects on liver function in mice

[0071] Compared with the normal group, the serum AST and ALT levels of mice in the model group were significantly increased ( P <0.01), indicating that high-fat diet caused liver damage in mice. Compared with the model group, the groups treated with various doses of Camellia sinensis polysaccharides significantly reduced serum AST and ALT levels ( P <0.01). Similarly, compared with the model group, the serum AST and ALT levels in the fenofibrate group were significantly higher ( P <0.01) decreased.

[0072]

[0073] pass Figure 3 Observation of H&E stained sections of mouse liver tissue showed that the liver cells of the normal group mice were uniform in size, arranged radially around the central vein, and the cell nuclei were clearly visible; the liver cells of the model group mice were swollen and disorderly arranged, with obvious fatty degeneration and ballooning of the liver, and the cell nuclei were marginalized; after intervention with total polysaccharides from Camellia dasyphylla, fat vacuoles were more obvious in the low-dose group, fatty liver was significantly improved in the medium-dose group, and there were almost no fat vacuoles and no ballooning in the high-dose group.

[0074] 4.3 Effects on insulin function in mice

[0075] Figure 4 The results of the glucose tolerance test showed that the blood glucose of the mice in the normal group reached its maximum value about 30 minutes after the injection of glucose, and the blood glucose returned to the initial level within 120 minutes; the fasting blood glucose value and maximum value of the mice in the model group were significantly higher than those in the normal group, and failed to return to the initial value 120 minutes after the injection of glucose, indicating that a high-fat diet would lead to a decrease in the mice's ability to regulate blood glucose. Compared with the model group, the fasting blood glucose and blood glucose peak values of the low, medium, and high-dose groups of mountain green tea were significantly reduced, indicating that the total polysaccharide of mountain green tea can improve the blood glucose intolerance of obese mice to a certain extent. The results of the serum insulin level measurement showed more intuitively that the insulin level in the mice in the high-fat diet group was significantly higher than that in the normal group ( P <0.01), and the administration of total polysaccharides from Camellia sinensis at all doses can significantly improve the elevated serum insulin level and bring it closer to normal levels. The insulin resistance index (HOMA-IR) is used to assess the state of insulin resistance. In this experiment, the calculation results of HOMA-IR showed that the HOMA-IR of the model group mice was significantly increased compared with the normal group ( P <0.01); Compared with the model group, the HOMA-IR was significantly reduced in the different dose-dependent groups of Camellia sinensis total polysaccharide administration, indicating that Camellia sinensis total polysaccharide has a good effect on improving the insulin resistance of mice induced by a high-fat diet.

[0076]

[0077] 4.4 Effects on Chronic Inflammation

[0078] Compared with the normal group, the levels of IL-1β, IL-6 and TNF-α in the liver of mice in the model group were significantly increased ( P <0.01), indicating that high-fat diet feeding triggered an inflammatory response in the liver or the whole body of mice. Compared with the model group, the levels of the three inflammatory factors in the liver of mice in each dose group of total polysaccharide from Camellia sinensis were significantly reduced ( P <0.01 or P <0.05), and improved the inflammatory response.

[0079]

[0080] 4.5 Effects on the intestinal flora of mice

[0081] Cecal contents were collected from each group of mice and sequenced using the V3-V4 region of the 16S rRNA gene as the target. The abundance and structure of the cecal microbiota in the normal, normal dosing, model, and high-dose total polysaccharide groups were determined, yielding a total of 508 OTUs.

[0082] The principal component analysis of intestinal flora intuitively shows the differences between the intestinal flora of each group of mice, reflecting the diversity of microbial colonies between the groups. Figure 5 As shown, the normal group and the model group were completely separated in terms of spatial distance, proving that a high-fat diet can alter the intestinal flora structure of mice. The flora structure of the group receiving total polysaccharides from Camellia sinensis (HFD+IMP) showed varying degrees of change and was completely separated from the model group, and closer to the normal group.

[0083] Alpha diversity is an analysis of species diversity in a single sample, including the Chao index and the Ace index. Both are used to assess the richness of the community in the sample. The larger the index, the richer the species in the sample. Compared with the normal group, the Chao and Ace indices of the model group were significantly reduced ( P <0.01), indicating that high-fat diet feeding significantly reduced the species richness of intestinal flora in mice; compared with the model group, the Chao and Ace indices of intestinal flora in the high-dose group of total polysaccharides from Camellia sinensis were significantly increased ( P <0.01), and showed a good effect in regulating the overall structure and abundance of intestinal flora in obese mice induced by high-fat diet.

[0084] After species annotation, at the genus level, the mice in the model group Desulfovibrio, Mucispirillum The abundance of genera increased, Alistipes, Clostridium_IV The abundance of other genera decreased. Mucispirillum It is a genus of bacteria in the phylum Ferrobacterium. Desulfovibrio Genera such as and are usually inflammation-related pathogens, and their abundance increased significantly in the model group mice ( P <0.01), the abundance of both in the total polysaccharide group of Camellia sinensis was significantly reduced ( P <0.01), suggesting that the total polysaccharide of mountain green tea may improve the intestinal pathological state of the model group mice by regulating the changes in the structure of intestinal flora. Alistipes, Flavonifractor, Clostridium_IV The abundance of PThese bacterial genera can break down indigestible carbohydrates and produce short-chain fatty acids. These small-molecule short-chain fatty acids are often involved in energy production and have anti-inflammatory activity, which can protect the intestine from pathogens and diseases.

[0085]

[0086]

[0087] Finally, it should be noted that the above examples are merely specific embodiments of the invention. Clearly, the invention is not limited to the above embodiments and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosed content of the invention should be considered within the scope of protection of the invention.

Claims

1. The total polysaccharide of mountain green tea is used in the preparation of drugs with weight loss function, or in the preparation of drugs for treating or regulating intestinal flora disorders caused by a high-fat diet, for regulating the structure of intestinal flora, increasing the abundance of beneficial bacteria Alistipes, Flavonifractor, and Clostridium_IV, and reducing the abundance of harmful bacteria Desulfovibrio and Mucispirillum; The total polysaccharide of mountain green tea has the effects of protecting the liver, lowering blood lipids, lowering blood sugar, regulating intestinal flora, and losing weight. The preparation method thereof comprises the following steps: (1) Add 95% ethanol to the tea leaves and soak overnight; (2) Decoction the soaked mountain green tea medicinal material with distilled water for 1-3 h, filter, concentrate the filtrate, centrifuge, and discard the precipitate; (3) Add 95% ethanol to the supernatant obtained in step (2) until the alcohol content is 80%, seal and let stand for 12-36 hours, centrifuge, collect the precipitate, freeze-dry, and obtain the crude polysaccharide of Camellia sinensis; (4) The Sevag method was used to remove free proteins from the crude polysaccharides of Camellia sinensis, and then the aqueous phase was freeze-dried to obtain the total polysaccharides of Camellia sinensis.

2. The use according to claim 1, characterized in that When the total polysaccharide of Camellia oleifera is added into medicine as an active ingredient, the dosage of the total polysaccharide of Camellia oleifera is 240 mg / kg•d-960 mg / kg•d.

3. The use according to claim 1, characterized in that The medicine is an oral preparation, including powder, oral liquid, granules, tablets, capsules and pills.

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