Stevia extract and its application

Abstract

This invention provides stevia extract and its applications. Specifically, the extract is obtained by extracting, concentrating, and fermenting stevia raw materials. Applications include the use of stevia extract in the preparation of formulations and / or foods that improve lipid metabolism, protect liver tissue, inhibit weight gain and / or blood glucose elevation caused by high-fat, high-sugar diets, reduce serum and / or liver TG and / or TC levels, inhibit HDL elevation and / or LDL reduction caused by high-fat, high-sugar diets, reduce FFA levels and / or AI index, improve oxidative stress caused by high-fat, high-sugar diets, inhibit colon shortening caused by high-fat, high-sugar diets, improve intestinal digestion and absorption capacity, and improve intestinal microbiota composition. This provides a new theoretical basis for using stevia extract to improve lipid metabolism disorders caused by high-fat diets, and also offers new ideas for the high-value utilization of natural agricultural waste, significantly expanding the application field of stevia and further exploring its physiological efficacy.

Description

Technical Field

[0001] This invention provides a stevia extract and its application, belonging to the field of pure natural plant preparation technology. Background Technology

[0002] Obesity has become the third leading epidemiological factor affecting human health after smoking and AIDS, and the fifth leading cause of death worldwide. Obesity is not only closely related to many chronic diseases such as diabetes, hypertension, coronary heart disease, hyperlipidemia, stroke, and arthritis, but it is also a risk factor for many chronic non-communicable diseases. The occurrence of obesity is closely related to lipid metabolism. When the body's energy intake and expenditure are imbalanced, adipose tissue stores excess nutrients in the form of triglycerides, leading to an excessive increase in the number and size of adipocytes, and even adipocyte apoptosis or necrosis, resulting in a pathological state of excessive fat accumulation and / or abnormal distribution. On the other hand, the imbalance between energy intake and expenditure causes a stress response in the body's energy system, leading to changes in the intestinal metabolic ecosystem. Recent studies have confirmed that gut microbiota dysbiosis can alter intestinal permeability, allowing large amounts of free fatty acids to enter the liver, leading to massive lipid deposition in the liver, inducing oxidative damage to hepatocytes, and promoting the development of chronic diseases.

[0003] The gut microbiota plays a crucial role in life processes, influencing the absorption of substances, energy metabolism, immune organ development, and resistance to pathogens. When gut microbes break down exogenous foods, excess lipids exceeding the body's needs are stored, directly or indirectly leading to obesity. This physiological process alters lipid metabolites in serum, adipose tissue, and liver, affecting the metabolism of triglycerides, phosphatidylcholine, and short-chain fatty acids, thus indirectly influencing energy and lipid metabolism. Therefore, regulating the gut microbiota may improve obesity-related diseases, such as regulating fat accumulation and lipid metabolism. Besides regulating gut microbiota calories through exercise and dietary control, several medications can be used. The most commonly used are Bifidobacteria, Lactobacillus, and Lactobacillus acidophilus complex, but most require dietary control. Recent studies have increasingly found that foods rich in polyphenols have antioxidant, anti-tumor, and gut microbiota-improving effects. Therefore, developing safe and functional foods from natural plants to regulate gut microbiota structure and lipid metabolism is of great significance.

[0004] Stevia is a perennial herbaceous plant belonging to the Asteraceae family, originating in eastern Paraguay, South America. Also known as "sweet grass," stevia is now successfully cultivated in China and Southeast Asia. Stevia extract contains stevia sugar, which is 300 times sweeter than sucrose, while its energy content is only one-ninth that of sucrose. Although only 60-70g of stevia sugar can be extracted from 1kg of leaves, it remains a highly regarded safe natural sugar source. Stevia sugar not only has the characteristics of high sweetness, low energy, non-toxicity, and no side effects, but it is also rich in flavonoids, alkaloids, water-soluble chlorophyll, lutein, as well as neutral water-soluble oligosaccharides, free sugars, amino acids, and lipids. It has been widely used in various industries such as food, beverages, and medicine. However, research on other physiological applications of stevia extract is rarely published.

[0005] The foregoing background information is intended to help those skilled in the art understand prior art that is similar to the present invention, and to facilitate the understanding of the inventive concept and technical solution of the present invention. It should be clearly stated that, in the absence of clear evidence that the above content was disclosed before the filing date of this patent application, the foregoing background information should not be used to evaluate the novelty of the technical solution of this application. Summary of the Invention

[0006] Technical issues

[0007] In order to solve the aforementioned technical problems and to expand the application scope of stevia, the present invention aims to provide a stevia extract and its application. Under the premise of existing antioxidant activity and the efficacy of promoting the growth of probiotic Lactobacillus casei, the stevia extract is applied to the preparation of related preparations or foods that improve lipid metabolism, protect the liver, reduce serum lipoprotein, improve oxidative stress, and improve intestinal digestion and absorption and microbial composition, which significantly expands the application field of stevia and further explores its physiological effects.

[0008] Technical solution

[0009] To achieve the above objectives, the inventors of this application conducted in-depth research and unexpectedly discovered a variety of physiological applications of stevia extract. By applying it to the preparation of related formulations or videos that improve lipid metabolism, protect the liver, reduce serum lipoproteins, improve oxidative stress, and improve intestinal digestion and absorption and microbial composition, the application fields of stevia have been significantly expanded.

[0010] That is, the present invention includes the following technical solutions.

[0011] Option 1: Application of stevia extract in the preparation of formulations and / or foods that improve lipid metabolism.

[0012] Option 2: The use of stevia extract in the preparation of formulations and / or foods that protect liver tissue.

[0013] Option 3: Application of stevia extract in the preparation of formulations that inhibit weight gain and / or blood glucose elevation caused by a high-fat, high-sugar diet.

[0014] Option 4: The use of stevia extract in the preparation of formulations and / or foods that reduce the levels of TG and / or TC in serum and / or liver.

[0015] Option 5: The use of stevia extract in the preparation of formulations and / or foods that inhibit the increase in HDL and / or decrease in LDL caused by a high-fat, high-sugar diet.

[0016] Option 6: Application of stevia extract in the preparation of formulations and / or foods with reduced FFA content and / or AI index.

[0017] Option 7: Application of stevia extract in the preparation of formulations and / or foods that improve oxidative stress caused by high-fat, high-sugar diets.

[0018] Option 8: The use of stevia extract in the preparation of formulations and / or foods that inhibit colonic shortening caused by a high-fat, high-sugar diet.

[0019] Option 9: Application of stevia extract in the preparation of formulations and / or foods that improve intestinal digestion and absorption.

[0020] Option 10: The use of stevia extract in the preparation of formulations and / or foods that improve gut microbiota composition.

[0021] Furthermore, the applications include the use of stevia extract in the preparation of formulations and / or foods that increase the abundance of intestinal lactobacillus.

[0022] Furthermore, the applications include the use of stevia extract in the preparation of formulations and / or foods that reduce the abundance ratio of Firmicutes / Bacteroidetes.

[0023] Option 11: The use of stevia extract and stevia callus extract in the preparation of the formulations and / or foods described in the above options.

[0024] Furthermore, the formulations described in the aforementioned schemes include at least one of the following: liquid formulations, oil formulations, powder formulations, extract formulations, crystalline formulations, granular formulations, capsule formulations, tablets, soft capsule formulations, and gel formulations.

[0025] Furthermore, the preparations described in the aforementioned schemes also include at least one of feed additives and livestock breeding agents.

[0026] Furthermore, the foods mentioned in the aforementioned schemes include at least one of the following: food additives, functional foods, beverages, health products, condiments, baked goods, candies, dairy products, meat products, convenience foods, puffed foods, frozen foods, and instant foods.

[0027] Furthermore, the stevia extracts described in the aforementioned schemes are specifically prepared via the following steps:

[0028] S1. Extract stevia raw materials;

[0029] S2. Mix and concentrate the extract;

[0030] S3. Ferment the concentrated liquid to obtain the final product.

[0031] Furthermore, the stevia raw material includes stevia stems and / or stevia leaves.

[0032] Furthermore, the extraction is specifically carried out at a material-to-liquid ratio of 1:5 to 30 and a temperature above 90°C for 60 to 180 minutes; furthermore, ultrasonic-assisted extraction time can be at least 1 / 3, and the ultrasonic frequency of ultrasonic-assisted extraction is 20 kHz to 60 kHz and the ultrasonic intensity is 80 to 200 W.

[0033] Furthermore, the mixing and concentration is carried out by heated open-air concentration or heated vacuum concentration, and the concentration is increased to Brix 20 or higher, that is, the content of water-soluble solids is 20% or higher.

[0034] Furthermore, the fermentation involves fermenting the concentrate with lactic acid bacteria under light-protected conditions for at least 6 months. The lactic acid bacteria are either naturally occurring lactic acid bacteria found in the stems of stevia or added lactic acid bacteria.

[0035] Furthermore, in the aforementioned solutions, the stevia extract is used in combination with the stevia callus extract.

[0036] Furthermore, the preparation method of the stevia callus extract includes:

[0037] T1. Select healthy stevia leaves, stems, stem tips and / or root tips as explants, disinfect them and place them in an induction medium for culture, add plant growth regulators to induce callus formation, screen well-grown callus for expanded culture, and take well-grown callus to crush and use.

[0038] T2. Stevia callus extract is obtained by extracting, concentrating, and fermenting the pulverized callus tissue.

[0039] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined to obtain specific implementation methods.

[0040] The raw materials or reagents involved in this invention are all commercially available products, and the operations involved are all routine operations in the field unless otherwise specified.

[0041] Beneficial effects

[0042] According to this invention, stevia extract is obtained by extracting, concentrating, and fermenting stevia raw materials. This extract not only possesses excellent antioxidant activity, but also effectively inhibits weight gain and blood glucose elevation caused by a high-fat, high-sugar diet. Simultaneously, it regulates lipid metabolism, protects liver tissue, maintains balance in various parts of the liver, and inhibits lipid accumulation. It also regulates oxidative stress, particularly alleviating changes in serum oxidative stress indicators induced by a high-fat, high-sugar diet. Furthermore, it has a certain ameliorative effect on changes in intestinal morphology and structure in mice induced by a high-fat diet, and stevia extract can improve the intestinal digestive and absorptive capacity of mice fed a high-fat diet. Stevia extract intervention can gradually shift the intestinal microbial composition of mice subjected to high-sugar, high-fat intervention towards a normal state. On the other hand, stevia extract can improve colonic morphology, promote food digestion and absorption, and regulate the abundance of Firmicutes and Bacteroidetes. Correlation analysis shows that HDL, SOD, GSH-Px are significantly positively correlated with the relative abundance of Prevotellaceae and Akkermansia. This study provides a new theoretical basis for using stevia extract to improve lipid metabolism disorders caused by a high-fat diet, and also offers new ideas for the high-value utilization of natural agricultural waste.

[0043] The present invention adopts the above-mentioned technical solution to achieve the above objectives, which makes up for the shortcomings of the prior art, is reasonably designed, and is easy to operate. Attached Figure Description

[0044] To make the above and / or other objects, features, advantages and examples of the present invention more apparent and understandable, the accompanying drawings used in the specific embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0045] Figure 1This indicates the effects of stevia extract on growth, organs, and blood glucose in mice; where A) represents weight gain; B) represents final body weight; C) represents body length; D) represents tail length; E) represents abdominal fat weight; F) represents a photograph of the mouse; G) represents blood glucose level; H) represents kidney weight; I) represents liver weight; and J) represents testicular weight. Different letters indicate significant differences at p < 0.05 as determined by analysis of variance (Duncan's test), *p < 0.05, **p < 0.01, ***p < 0.001 indicating significant differences compared to groups determined by Student's t-test; NS does not represent any significance.

[0046] Figure 2 This indicates the effects of stevia extract on serum and liver lipids; where A) represents serum triglyceride (TG) content; B) represents serum total cholesterol (TC) content; C) represents serum high-density lipoprotein (HDL) content; D) represents serum low-density lipoprotein (HDL) content; E) represents serum free fatty acid (FFA) content; F) represents the AI ​​index; G) represents liver total cholesterol (TC) content; H) represents liver triglyceride (TG) content; different letters indicate significant differences at p < 0.05 as determined by analysis of variance (Duncan test), *p < 0.05, **p < 0.01, ***p < 0.001 indicate significant differences compared to the groups determined by Student's t-test; NS does not represent any significance.

[0047] Figure 3 The values ​​represent the effects of stevia extract on liver injury; where A) represents serum alanine aminotransferase (ALT) activity; B) represents serum aspartate aminotransferase (AST) activity; and C) represents liver H&E stained sections. Different letters indicate significant differences at p < 0.05 as determined by analysis of variance (Duncan test), where *p < 0.05, **p < 0.01, and ***p < 0.001 indicate significant differences compared to groups determined by Student's t-test. NS does not represent any significance.

[0048] Figure 4 The results indicate the effect of stevia extract on oxidative stress; where A) represents serum superoxide dismutase (SOD) activity; B) represents serum malondialdehyde (MDA) content; and C) represents serum glutathione peroxidase (GSH-Px) activity. Different letters indicate significant differences at p < 0.05 as determined by analysis of variance (Duncan test), *p < 0.05, **p < 0.01, ***p < 0.001 indicating significant differences compared to groups determined by Student's t-test; NS does not represent any significance.

[0049] Figure 5The text indicates the effect of stevia extract on intestinal damage; where A) represents a colon photograph; B) represents colon length; C) represents a colon H&E stained photograph; and D) represents the water content of intestinal contents. Different letters indicate significant differences at p < 0.05 as determined by analysis of variance (Duncan test), *p < 0.05, **p < 0.01, ***p < 0.001 indicating significant differences compared to groups determined by Student's t-test; NS does not represent any significance.

[0050] Figure 6 The diagram represents the effects of stevia extract on gut microbiota; where A) represents the ACE index; B) represents the Chao1 index; C) represents the Simpson index; D) represents the relative abundance at the phylum level; E) represents the relative abundance at the genus level; F) represents the Pearson relationship heatmap; NS do not represent any meaning.

[0051] Figure 7 This indicates the effects of the combined use of stevia extract and stevia callus extract on blood glucose, serum lipids, and liver lipids; where A represents blood glucose levels; B represents total cholesterol content in the liver; and C) represents triglyceride content in the liver. Detailed Implementation

[0052] Those skilled in the art can refer to the content of this document and appropriately replace and / or modify the process parameters to achieve the desired results. However, it should be particularly noted that all similar replacements and / or modifications are obvious to those skilled in the art and are considered to be included in this invention. The products and preparation methods described in this invention have been described through preferred examples, and those skilled in the art can obviously modify or appropriately change and combine the products and preparation methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0053] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. This invention uses the methods and materials described herein; however, other suitable methods and materials known in the art may also be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patent cases, provisional applications, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, the definitions included in this specification shall prevail.

[0054] Unless otherwise stated, all percentages, parts, proportions, etc. are by weight; other statements include, but are not limited to, “%”, “wt%”, “mass%” meaning weight percentage, “mol%” meaning mole percentage, and “vol%” meaning volume percentage.

[0055] When quantities, concentrations, or other numerical values ​​or parameters are given as ranges, preferred ranges, or a series of upper and lower preferred values, it should be understood that they specifically disclose all ranges formed by any pair of values ​​of any larger or preferred range limit and any smaller or preferred range limit, regardless of whether the ranges are disclosed separately. For example, when describing a range of “1 to 5 (1-5)”, the described range should be understood to include ranges such as “1 to 4 (1-4)”, “1 to 3 (1-3)”, “1 to 2 (1-2)”, “1 to 2 (1-2) and 4 to 5 (4-5)”, “1 to 3 (1-3) and 5”, etc. Unless otherwise stated, wherever numerical ranges are described herein, the ranges include the range endpoints as well as all integers and fractions within that range.

[0056] Unless otherwise specified, the materials, methods, and examples described herein are exemplary and not limiting. While similar or equivalent methods and materials can be used to implement or test the invention, suitable methods and materials are described herein.

[0057] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Furthermore, in the absence of conflict, the embodiments and features in the embodiments of this application can be combined with each other.

[0058] To facilitate understanding of the embodiments of the present invention, the abbreviations and key terms that may be involved in the embodiments of the present invention will first be explained or defined. For undefined abbreviations or key terms, they are all conventionally understood by those skilled in the art.

[0059] TG: Triglycerides;

[0060] TC: Total cholesterol;

[0061] HDL: High-density lipoprotein;

[0062] LDL: Low-density lipoprotein;

[0063] SOD: Superoxide dismutase;

[0064] MDA: Malondialdehyde;

[0065] GSH-Px: Glutathione peroxidase;

[0066] FFA content: Free fatty acid content;

[0067] ALT: Alanine aminotransferase;

[0068] AST: Aspartate aminotransferase;

[0069] AI index: cardiovascular index;

[0070] ACE index: Angiotensin-converting enzyme content or activity;

[0071] Chao1 index: an indicator for measuring species richness.

[0072] The present invention is described in detail below.

[0073] Example 1:

[0074] A stevia extract, the preparation steps of which include:

[0075] S1. Take 20g of stevia stems, crush them through a 20-mesh sieve, add 400mL of deionized water, and extract at 90℃ for 120min. Then add 100mL of deionized water and extract at 90℃ for 10min (repeat twice). After filtration, concentrate under vacuum at 45℃ for 5h. After sealing, sterilize at 95℃ for 4h. Finally, inoculate with active lactic acid bacteria for fermentation for 12 months. Filter the fermentation broth to obtain stevia extract, with the following basic composition: water 84.13%, total sugar 7.86%, ash 4.36%, crude protein 3.59%, and crude fat 0.06%. The soluble solids content is 23.20%, and the total phenol content is 15.29mg / mL.

[0076] Example 2:

[0077] Animal experiments were conducted using the stevia extract obtained in Example 1 to verify its application effects in regulating lipid metabolism, protecting the liver, reducing serum lipoproteins, improving oxidative stress, improving intestinal digestion and absorption, and related physiological functions of microbial composition.

[0078] Part 1: Animal Experiment Design and Grouping

[0079] Fifty male Kunming mice, weighing 20–25 g, were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. They were housed in an SPF-protected environment at a temperature of (23±2)℃ and a relative humidity of 60%–70%. They had free access to food and water, and the day-night cycle was 12 hours. All animal experimental procedures were carried out in accordance with relevant animal management regulations.

[0080] The experimental design and procedure are shown in Table 1. After one week of acclimatization, mice were randomly divided into a control group, a model group, a low-dose group (0.05 g / kg, dry basis / mouse body weight), a medium-dose group (0.10 g / kg, dry basis / mouse body weight), and a high-dose group (0.20 g / kg, dry basis / mouse body weight), with 10 mice in each group. The control group was fed a routine diet, while the other groups were fed a high-fat diet for 20 days to establish the MAFLD mouse model. Low, medium, and high doses of stevia extract were prepared, and the corresponding concentrations were administered to the three formulation groups by gavage. The control and model groups were given the same volume of sterile water. The drugs were administered once daily for 30 consecutive days. After 20 days of high-fat diet, the model group showed more severe steatosis in the liver tissue than the control group, with a large number of fat vacuoles and elevated blood lipid levels, proving the model was successful. After the last gavage, the mice were fasted for 12 hours, but had free access to water. Blood was collected from the canthus of the eye, and serum was separated by centrifugation at 3000 rpm for 15 min; liver tissue was collected; and fecal samples were collected from each group of mice. All samples were immediately stored at -80℃ for subsequent analysis.

[0081] Table 1 - Experimental Design and Procedure Design

[0082]

[0083] Part Two: Experimental Verification Methods

[0084] 2.1 Pathological observation of liver and intestinal tissues

[0085] Mouse liver and intestinal tissues were fixed in fixative, embedded, sectioned, and stained with hematoxylin and eosin (HE). The tissues were then observed under a microscope to examine the fatty pathology of the liver and intestinal tissues.

[0086] 2.2 Detection of liver lipids, blood lipids and blood glucose levels

[0087] Blood glucose levels in collected plasma were measured using a rapid blood glucose meter. Serum AST, ALT, TG, TC, and FFA levels in each group of mice were measured using kits. Serum HDL and LDL levels were measured using ELISA. Liver TG and TC levels were measured using kits. (Kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd.)

[0088] 2.3 Oxidative stress state

[0089] The levels of SOD, MDA, and GSH-PX in serum were detected using a kit purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0090] 2.4 Determination of water content and pH of intestinal contents

[0091] The determination of water content in intestinal contents was performed according to the international standard AOAC (1995). 0.5 g of the crushed intestinal contents was weighed into a centrifuge tube, 4.5 mL of distilled water was added, and after homogenization, the mixture was centrifuged at 10,000 rpm for 15 min in a high-speed centrifuge. The pH of the supernatant was then measured using a pH meter.

[0092] 2.5 Determination of intestinal flora structure

[0093] Total bacterial DNA was extracted from feces according to the instructions of the DP328 Fecal DNA Extraction Universal Kit (Tiangen Biotech (Beijing) Co., Ltd.). DNA purity and concentration were determined by agarose gel electrophoresis. A certain amount of sample DNA was placed in a centrifuge tube, and the sample was further diluted to 1 ng / μL with sterile water. Using the diluted genomic DNA as a template, the target 16S rDNA-V4 region fragment was amplified by PCR using the universal 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') PCR methods. Electrophoresis was performed on a 2% agarose gel. Equal amounts of the PCR products were mixed thoroughly, and then the PCR products were detected by 2% agarose gel electrophoresis. The target band was recovered using a gel extraction kit. Next, a library was constructed and quantified. After quantification, qualified libraries were selected for the next step of sequencing.

[0094] After sequencing, the raw data (Raw tags) were spliced ​​and filtered to obtain high-quality data (Clean tags). Chimeric sequences were then removed from the Clean tags to obtain the final valid data for the samples. A 97% similarity was defined as the clustering standard. Uparse software was used to cluster the effective tags of all samples, grouping the valid data into OTUs. The Mothur method and the SILVA132 SSU rRNA database were then used for species annotation. Species annotation analysis yielded community composition information for all samples at each taxonomic level. All sample data were then normalized, and R software was used for Alpha diversity analysis, principal coordinate analysis, OUTs abundance analysis, and functional prediction analysis. This yielded information on sample species richness and microbial diversity, and further comparative analysis of differences in fecal microbial structure and function among different groups was conducted.

[0095] Part Three: Data Analysis

[0096] Experimental data were analyzed using SPSS 23.0, with ( xMean squared (±s) was used. ANVOVA analysis was used for comparisons between groups, and independent samples t-tests were used for comparisons between two groups. p < 0.05 was considered statistically significant; graphs were created using GraphPad Prism 8.0 software.

[0097] Part Four: Results Analysis

[0098] 4.1 Effects of Stevia extract on food intake, blood glucose, and organ function in Kunming mice.

[0099] After 20 consecutive days on a high-fat diet, the levels of TG and TC in the blood lipids of Kunming rats were significantly higher than those in a standard diet, indicating that the hyperlipidemia model was successful.

[0100] Figure 1 This study demonstrated the effects of stevia extract on growth, organs, and blood glucose levels in mice. During a 30-day dietary intervention with stevia extract, [the following effects were observed]. Figure 1 As shown in A and B, the model group had a higher body weight than the control group. The stevia extract group inhibited the rapid weight gain caused by a high-fat diet, resulting in a lower body weight. The high-dose group showed a better effect (P = 0.094). Figure 1 The physical parameters of Kunming mice shown in C and D indicate that, compared with the control group, a high-fat diet resulted in shorter body and tail lengths in the mice (P < 0.05); intervention with stevia extract inhibited this change (P > 0.05), and there was no dose-dependent effect.

[0101] Excessive fat intake can cause body fat to accumulate in various parts of the body, so we will examine the weight of several major organs here. Figure 1 As shown in E-J, the liver, epididymal fat, and testicular weight in the model group were significantly higher than those in the control group (P < 0.05), but had almost no effect on the kidneys (P > 0.05). Abdominal fat in different doses of stevia extract groups was significantly lower than that in the model group (P < 0.05), indicating that stevia extract can promote lipid metabolism in Kunming rats and inhibit excessive fat storage. The liver is the main organ for lipid metabolism. When the body ingests excessive fat, the rate of lipid catabolism is lower than the rate of fat synthesis, leading to lipid accumulation in the liver and hepatomegaly. Stevia extract can inhibit lipid accumulation in the liver, with the medium-dose group showing a significant inhibitory effect (P < 0.05). Blood glucose is glucose in the blood and an important source of energy. High blood glucose levels can cause insulin resistance. Due to the high-fat, high-sugar diet, the blood glucose concentration in the model group was significantly higher than that in the control group (P < 0.001). Stevia extract intervention significantly reduced blood glucose concentration (P < 0.05) in a dose-dependent manner. These results demonstrate that stevia extract can effectively inhibit weight gain and blood sugar spikes caused by a high-fat, high-sugar diet.

[0102] 4.2 Effects of Stevia extract on serum and liver lipids in Kunming mice

[0103] The levels of four lipid markers (TG, TC, HDL-C, and LDL-C) reflect whether the body's lipid metabolism is normal. Figure 2 The effects of stevia extract on serum and liver lipids were demonstrated. Figure 2 As shown in A, B, G, and H, a high-fat, high-sugar diet significantly increased the levels of TG and TC in serum and liver (P < 0.001), while stevia extract significantly reduced the levels of TG and TC in serum and liver (P < 0.05), and this reduction was dose-dependent. Furthermore, from... Figure 2 C and D in the results indicate that stevia extract can significantly inhibit the decrease in HDL and increase in LDL caused by a high-fat, high-sugar diet (P < 0.05). Furthermore, from... Figure 2 As shown in E and F, serum free fatty acid content and AI index can also reflect lipid metabolism. High-fat and high-sugar diet significantly increased serum FFA content and AI index (P < 0.001), while stevia extract significantly reduced FFA content and AI index (P < 0.001). Figure 2 The results indicate that different doses of stevia extract have different regulatory patterns on the four lipid parameters in mice fed a high-fat diet, suggesting that stevia extract can affect the lipid metabolism level of mice fed a high-fat, high-sugar diet, and that this effect is dose-dependent.

[0104] 4.3 Observation of the effects of stevia extract on liver function and pathological sections in Kunming mice

[0105] A high-fat, high-sugar diet can induce lipid metabolism disorders, and it is necessary to confirm whether liver metabolic function is normal. Figure 3 The effects of stevia extract on liver injury were demonstrated by measuring serum ALT and AST levels. Figure 3 As shown in A and B, the serum ALT and AST levels in the high-fat, high-sugar diet group were significantly higher than those in the standard diet group (P < 0.001), while stevia extract significantly inhibited these levels (P < 0.01), and this inhibitory effect was dose-dependent. Figure 3 As shown in Figure C, H&E staining of liver tissue revealed a significant increase in liver lipid droplets and lipid accumulation in all high-fat diet groups compared to the control group. However, with increasing stevia extract concentration, the size and amount of liver lipids decreased, a result consistent with blood lipid levels and transaminase levels. This demonstrates that stevia extract can regulate lipid metabolism and protect liver tissue.

[0106] 4.4 Effects of Stevia extract on oxidative stress in Kunming mice

[0107] Recent studies have shown that high-fat diets increase oxidative stress and lead to lipid accumulation in the liver, resulting in other liver diseases. Therefore, the lipid metabolism-regulating and metabolic syndrome-alleviating activities of many anti-obesity and anti-diabetic drugs or natural products are closely related to their antioxidant and free radical scavenging activities. Among these, serum SOD and GSH-Px antioxidant enzymes can effectively scavenge excess free radicals, regulate reactive oxygen species (ROS) levels, and play an important role in controlling lipid peroxidation. Stevia extract has been confirmed to have good antioxidant activity; therefore, researching whether stevia extract can effectively prevent oxidative stress induced by high-fat diets is of great significance. Figure 4 This study demonstrates the effect of stevia extract on oxidative stress, by... Figure 4 As shown in A and C, the SOD and GSH-Px enzyme activities in the model group were significantly lower than those in the control group (P < 0.001). Stevia extract at different concentrations significantly increased SOD and GSH-Px enzyme activities (P < 0.05), exhibiting a clear dose-dependent effect. Notably, the SOD enzyme activity level in the high-dose group almost returned to that of a standard diet. MDA is an intermediate peroxidation product, indirectly reflecting the body's oxidative state. Figure 4 As shown in Figure B, the MDA level in the control group was significantly lower than that in the model group (P < 0.001). When the model group was treated with stevia extract, the MDA level significantly decreased (P < 0.05), similar to the above results, exhibiting a dose-dependent effect. Here we can conclude that stevia extract, with its excellent in vitro antioxidant activity, can regulate oxidative stress after ingestion, especially significantly alleviating changes in serum oxidative stress indicators induced by a high-fat, high-sugar diet.

[0108] 4.5 Effects of Stevia extract on intestinal morphology and barrier function in Kunming mice

[0109] The colon is an important part of the intestines for digesting food. Figure 5 The effects of stevia extract on intestinal damage were shown, from Figure 5 Figures A and B show that the colon length in the model group was shorter than that in the control group (P > 0.05). Different doses of stevia extract all inhibited colon shortening induced by a high-fat, high-sugar diet, with the low-dose group showing better results. Villus length and crypt depth are intuitive indicators for evaluating intestinal digestive performance. Longer villi and shallower crypts, along with a higher villus length-to-crypt depth ratio, indicate better digestion and absorption; conversely, lower villus length and crypt depth indicate poorer digestion and absorption. Colonic H&E stained sections are shown below. Figure 5As shown in Figure C, prolonged high-fat diet feeding significantly improved the morphology and structure of the mouse intestine. In the model group, the intestinal villi were significantly shorter and the crypts significantly deeper, indicating that a high-fat diet affects the digestive and absorptive capacity of the mouse intestine. Compared with the model group, the jejunal villi length in the low, medium, and high stevia extract addition groups showed a certain increasing trend with increasing stevia extract addition, while the crypt depth showed a decreasing trend. Furthermore, the intestinal morphology of the medium and high dose groups was almost similar to that of the control group. The water content of intestinal contents can indirectly reflect the digestive capacity of the intestine. Higher water content promotes peristalsis and digestion, as shown in the test results. Figure 5 As shown in Figure D, the water content of all three stevia extract groups was high, with no significant difference from the control group (P > 0.05), and higher than the model group. Furthermore, the intestinal water content of the high-dose group was significantly higher than that of the model group (P < 0.05). These results indicate that the addition of stevia extract has a certain ameliorative effect on the changes in intestinal morphology and structure induced by a high-fat diet in mice, suggesting that stevia extract has the ability to improve the intestinal digestive and absorptive capacity of mice fed a high-fat diet.

[0110] 4.6 Effects of Stevia extract on intestinal morphology and barrier function in Kunming mice

[0111] A growing body of research indicates that gut microbiota is related to host nutrient and energy uptake, glucose and cholesterol metabolism, insulin sensitivity, non-alcoholic fatty liver disease, and chronic inflammation. In this invention, we used the Illumina Miseq high-throughput sequencing platform to determine the V4 hypervariable region of the 16S rDNA of mouse gut microbiota to investigate the effects of a high-sugar, high-fat diet and stevia extract on mouse gut microbiota. The results are presented in [Table / Insert Results Here]. Figure 6 The effects of stevia extract on gut microbiota. The diversity of gut microbiota determines the gut microecology, which can be measured using the Chao1 index, ACE index, and Simpson index. The results are as follows: Figure 6 From A to C, it can be seen that a high-sugar, high-fat diet and stevia extract have virtually no impact on gut microbiota diversity.

[0112] Depend on Figure 6At the phylum level, the gut microbiota of mice was mainly composed of Firmicutes and Bacteroidetes. Firmicutes are sensitive to calories in food and promote calorie absorption; excessive abundance can lead to obesity. High-fat diets significantly increased Firmicutes abundance (P < 0.05), and intervention with stevia extract significantly improved this change induced by a high-fat diet. Compared with the model group, the abundance of Bacteroidetes was significantly increased in the medium-to-high dose stevia extract group, resulting in a decrease in the Firmicutes / Bacteroidetes (F / B) ratio. These results indicate that stevia extract intervention can gradually shift the gut microbiota composition of mice subjected to high-sugar, high-fat diets towards a normal state. The study also found that the addition of stevia extract could increase the abundance of Lactobacillus, but this was not dose-dependent.

[0113] To explore the relationship between gut microbiota and glucose and lipid metabolism as well as oxidative stress, we conducted a correlation analysis between the abundance of gut microbiota and related indicators, such as... Figure 6 As shown in F, the AI ​​index showed a significant positive correlation with the levels of Stenotrophomonas, Ruminococaceae, Odoribacter, and Acetatifactor. Notably, HDL levels, SOD, and GSH-Px enzyme activity were significantly positively correlated with the abundance of Prevotellaceae (which influences short-chain fatty acid synthesis) and Akkermansia (which inhibits LPS production). Short-chain fatty acids (SCFAs) are intestinal energy sources; higher SCFA levels promote intestinal microbial activity, while SCFA deficiency weakens their protective effect on the intestinal mucosal barrier. LPS (lipopolysaccharide) is an intestinal endotoxin; excessive levels can damage the intestinal mucosal barrier. TG, TC, LDL, AST, and ALT showed significant positive correlations with Rothia bacteria. Based on these results, we hypothesize that exogenous intervention with stevia extract enhances the protective effect of the intestinal mucosal barrier by increasing the abundance of Prevotellaceae and Akkermansia, promoting the production of SCFA, reducing the production of enterogenic endotoxin LPS, maintaining intestinal homeostasis, and thus regulating lipid metabolism through the gut-liver axis, maintaining lipid stability in various parts of the body, and protecting the liver.

[0114] Example 3:

[0115] A stevia callus extract is provided, the preparation steps of which include:

[0116] T1. Select healthy stevia shoot tips as explants, sterilize them, and culture them in the following induction medium: MS medium as the base, with the addition of 2.5% sucrose, 1.0 mg / L 6-BA, and 0.1 mg / L NAA, and culture at 25±2℃ and 1800 lx / 12h for 1 week; select well-grown callus tissue and expand it in the following expansion medium: B5 medium as the base, with the addition of 3.5% sucrose, 1.5 mg / L 6-BA, and 0.2 mg / L NAA, and culture at 25±2℃ and 1800 lx / 12h for 1 week; take well-grown callus tissue, pulverize it, and pass it through a 20-mesh sieve for later use;

[0117] T2. Mix the raw materials at a weight ratio of 1:1, add 20 times the amount of water, and extract at 95℃ for 120 minutes. During the 30th to 90th minute, use ultrasonic-assisted extraction with an ultrasonic frequency of 45kHz and an ultrasonic intensity of 100W to obtain the extract. Heat the extract and concentrate it under vacuum to Brix 25. Ferment the concentrate with naturally occurring lactic acid bacteria under light-protected conditions for 9 months to obtain the final product.

[0118] Example 4:

[0119] Stevia extract obtained in Example 1 and stevia callus extract obtained in Example 3 were mixed at a weight ratio of 1:1. Animal experiments were then conducted according to the first part of Example 2, using the same dosage as the low, medium and high dose groups of stevia extract.

[0120] Figure 7 The effects of the combined use of stevia extract and stevia callus extract on blood glucose, serum lipids, and liver lipids were demonstrated. Figure 7 As shown in A, when stevia extract and stevia callus extract are used in combination, the low-dose combination group has a better hypoglycemic effect than the medium-dose group using stevia extract alone, while the high-dose combination group has a very significant hypoglycemic effect, reducing blood glucose to levels close to the control group. This suggests that adding stevia callus extract to stevia extract may significantly enhance the latter's hypoglycemic effect, or that the combination of stevia extract and stevia callus extract exhibits an unexpected synergistic hypoglycemic effect. Therefore, the combination can effectively inhibit the rise in blood glucose caused by a high-fat, high-sugar diet. Figure 7As shown in B and C, compared with the use of stevia extract alone, its combination with stevia callus extract helps to further inhibit the increase of liver TC and TG caused by a high-fat, high-sugar diet. The inhibitory effect of the low-dose combination group on the increase of liver TC and TG is comparable to that of the high-dose stevia extract group. The inhibitory effect of the medium-dose combination group is significantly better than that of the high-dose stevia extract group. The high-dose combination group can inhibit the liver TC and TG content caused by a high-fat, high-sugar diet to a level close to that of the control group. The possible reason is that the addition of stevia callus extract enhances the inhibitory effect of stevia extract on the increase of liver TC and TG, or the combination of the two has a synergistic inhibitory effect. This indicates that the combination of stevia extract and stevia callus extract can affect the lipid metabolism level of mice fed a high-fat, high-sugar diet, and this effect is correlated with the dose of the combination mixture.

[0121] Further research confirmed that a mixture of stevia extract and stevia callus extract at a weight ratio of 1:3 to 5:1 significantly outperformed stevia extract alone at the same dosage. These effects included: improved lipid metabolism, liver protection, reduction of serum and liver total cholesterol (TC) levels, inhibition of HDL elevation and / or LDL reduction induced by a high-fat, high-sugar diet, reduction of FFA levels and / or AI index, improvement of oxidative stress induced by a high-fat, high-sugar diet, inhibition of colonic shortening induced by a high-fat, high-sugar diet, improved intestinal digestive and absorptive capacity, and improved gut microbiota composition. These findings provide a further optimized approach for using stevia extract and stevia callus extract to improve lipid metabolism disorders caused by a high-fat diet, and also offer new insights for the high-value utilization of natural agricultural waste.

[0122] The conventional techniques described in the above embodiments are existing technologies known to those skilled in the art, and therefore will not be described in detail here.

[0123] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

[0124] Although the present invention has been described in detail and specific embodiments have been cited, it will be apparent to those skilled in the art that various changes or modifications can be made without departing from the spirit and scope of the invention.

[0125] While the foregoing detailed descriptions have shown, described, and pointed out novel features applicable to various embodiments, it should be understood that various omissions, substitutions, and changes may be made to the form and details of the described apparatus or methods without departing from the spirit of this disclosure. Furthermore, the various features and methods described above may be used independently of each other or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of this disclosure. Many of the foregoing embodiments include similar components, and therefore, these similar components are interchangeable in different embodiments. Although the invention has been disclosed in the context of certain embodiments and examples, those skilled in the art will understand that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or applications, as well as their obvious modifications and equivalents. Therefore, the invention is not intended to be limited to the specific disclosure of the preferred embodiments herein.

[0126] All matters not covered in this invention are common knowledge.

Claims

1. Use of a Stevia rebaudiana extract in combination with a Stevia rebaudiana callus extract for the preparation of a formulation for improving hyperglycemia and hyperlipidemia, characterized in that, The improvement of high-fat and high-sugar performance is manifested as reducing blood sugar and / or inhibiting the increase of liver TC and TG; The preparation method of the stevia extract comprises: S1, taking stevia raw materials for extraction; S2, mixing and concentrating the extract; S3, fermenting the concentrated liquid to obtain the stevia extract; The preparation method of the stevia extract comprises: T1, selecting healthy stevia leaves, stems, stem tips and / or root tips as explants, disinfecting and culturing in an induction medium, adding plant growth regulators to induce callus formation, screening and expanding the culture of well-grown callus, and crushing the well-grown callus for use; T2, crushing the callus, extracting, concentrating and fermenting to obtain the stevia callus extract; The weight ratio of the stevia extract to the stevia callus extract is 1:

1.

2. The application of claim 1, wherein: In the preparation method of the stevia extract, the extraction is specifically carried out at a solid-liquid ratio of 1:5-30 and a temperature of 90°C or higher for 60-180 min.

3. The application of claim 1 or 2, wherein: In the preparation method of the stevia extract, the mixing and concentration are heating open concentration or heating vacuum concentration, and the concentration is carried out to a Brix of 20 or higher, i.e., a water-soluble solid content of 20% or higher.

4. The application of claim 1, wherein: The fermentation is the fermentation of the concentrated liquid with lactic acid bacteria under light-proof conditions for at least 6 months, and the lactic acid bacteria are natural lactic acid bacteria attached to the stevia stems or added lactic acid bacteria.

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