Pseudo-ginseng leaf fermentation product as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a panax notoginseng leaf fermentation product and a preparation method and application thereof.Panax notoginseng leaves are fermented by mixing lactobacillus delbrueckii subsp. Bulgaricus and streptococcus thermophilus, the content of rare ginsenosides F2, Rg3 and Rk1 in the fermented panax notoginseng leaf fermentation liquid is increased by 2 times, 9 times and 4 times or above respectively, and the content of the rare ginsenosides Rg3 and Rk1 is increased by 2 times or above. Two kinds of rare ginsenosides C-K and Rh2 are newly added; the total amount of rare ginsenosides F2, Rg3, Rk1, C-K and Rh2 is increased by 9 times or above, and mouse model experiments find that the panax notoginseng leaf fermented total saponin has the effects of remarkably reducing blood fat and liver fat of hyperlipidemia mice, relieving symptoms of the hyperlipidemia mice and relieving symptoms of non-alcoholic fatty liver diseases and has excellent anti-oxidative stress performance. The panax notoginseng leaf fermented product is simple to prepare and high in fermentation efficiency, and has wide edible and medicinal prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a notoginseng leaf fermentation product and a preparation method and application thereof. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Hyperlipidemia, also known as lipid metabolism disorder or abnormality, refers to a systemic lipid metabolism abnormality caused by various reasons, including high plasma total cholesterol, triglycerides and (or) low-density lipoprotein cholesterol and (or) low high-density lipoprotein cholesterol. Hyperlipidemia is an important factor in stroke, myocardial infarction, coronary heart disease, heart failure, hypertension, diabetes, fatty liver and obesity, and can induce cardiovascular and cerebrovascular diseases such as atherosclerosis, coronary heart disease, and myocardial infarction, which seriously threaten human health. In recent years, the incidence of hyperlipidemia in the Chinese population has increased significantly, which is one of the main reasons for the rapid increase in the number of sub-healthy people. The prevention and treatment of hyperlipidemia has become an urgent need of our society. The main drugs for the clinical treatment of hyperlipidemia are statins, fibrates, bile acid sequestrants and cholesterol absorption inhibitors, but long-term use will produce strong dependence, intolerance, abdominal discomfort, bloating, diarrhea, muscle toxicity and liver damage and other side effects. There is an urgent need to use natural plants to develop safe and efficient lipid-lowering drugs, foods and health products.

[0004] Non-alcoholic fatty liver disease refers to a clinical pathological syndrome characterized by excessive fat deposition in hepatocytes caused by factors other than alcohol and other clear liver damage. It is the most common metabolic stress liver damage and chronic liver disease. The global prevalence of non-alcoholic fatty liver disease in adults is about 32%, and the prevalence in my country is about 29%, and it continues to rise. Non-alcoholic fatty liver disease increases the risk of liver damage, cirrhosis and liver cancer. It is also a common comorbidity of type 2 diabetes, obesity and hypertension. About 55% of type 2 diabetes patients and 80% of obese people also suffer from non-alcoholic fatty liver disease. In the past 15 years, the mortality rate of non-alcoholic fatty liver disease has increased from 0.18% to 1.08%. Long-term use of traditional drugs such as lipid-lowering drugs and oral hypoglycemic drugs can cause side effects such as abdominal discomfort and liver damage. There is an urgent need to use natural plants to develop safe and efficient drugs, foods and health products to relieve non-alcoholic fatty liver disease.

[0005] Panax notoginseng Panax notoginseng(Burk) FHChen) is a plant of the genus Panax in the Araliaceae family. It is a traditional precious medicinal material in my country and a representative medicine for promoting blood circulation and removing blood stasis. It is mainly distributed in Yunnan, Guangxi, Jiangxi, Sichuan and other places. Traditionally, the dried roots and rhizomes of Panax notoginseng are used as medicine. Panax notoginseng has diverse biological activities. On the one hand, the use of Panax notoginseng is increasing. On the other hand, the problem of continuous cropping obstacles in long-term cultivation of Panax notoginseng has become increasingly prominent, resulting in insufficient resources of Panax notoginseng roots. It is urgent to develop and utilize Panax notoginseng leaf resources. The main active ingredient of Panax notoginseng is saponin compounds. Different parts of Panax notoginseng are rich in different types of saponins. At present, more than 200 saponins have been isolated and identified from different parts of Panax notoginseng, such as stems, leaves, flowers, and fruits. Different types of saponins have different biological activities. Ordinary ginsenosides contain sugar groups and have high molecular polarity. They are not easily absorbed through the intestinal mucosa. Therefore, they are not the molecular structure with the best pharmacological activity. They need to be metabolized and transformed by intestinal flora to reduce the molecular polarity so that they can better penetrate the intestinal wall and enter the blood circulation to exert their efficacy. Compared with common ginsenosides, rare saponins have higher biological activity and medicinal value. For example, some rare saponins such as Rh 2 , Rg 3 、F 2 It has anti-cancer, anti-thrombotic, anti-skin aging and therapeutic activities for nervous system diseases.

[0006] Rare saponins are prepared by microbial fermentation technology, which has a simple process, low energy consumption, strong selectivity, high content of rare saponins, and is not easy to cause environmental pollution. Summary of the invention

[0007] The purpose of the present invention is to provide a Panax notoginseng leaf fermentation product and its preparation method and application in view of the current urgent need to develop and utilize ginsenoside natural medicines, convert common ginsenosides in Panax notoginseng leaves into rare ginsenosides with more medicinal value through biological fermentation, and then prepare Panax notoginseng leaf fermentation product rich in rare ginsenosides and fermented Panax notoginseng leaf total saponins in a green and efficient manner. It is easy to industrialize and has excellent blood lipid lowering, non-alcoholic fatty liver relief and antioxidant activity.

[0008] The technical solution of the present invention is as follows: In one aspect, the present invention provides a method for preparing a fermented product of Panax notoginseng leaves as described above, comprising the following steps: Step (1) Using Lactobacillus germanica subsp. bulgaricus Lactobacillus bulgaricus and Streptococcus thermophilus Streptococcus thermophilus Mixed and fermented Panax notoginseng leaves.

[0009] Lactobacillus delbrueckii subsp. bulgaricus Lactobacillus bulgaricus , deposited in Guangdong Provincial Microbiological Culture Collection Center, with the deposit number GDMCC NO: 1.2783, and the deposit date is August 25, 2021.

[0010] The thermophilic streptococcus is thermophilic streptococcus Streptococcus thermophilus , deposited in Guangdong Provincial Microbiological Culture Collection Center, the deposit number is GDMCC NO: 1.2800, and the deposit date is September 16, 2021.

[0011] Preferably, the mixing ratio of the Lactobacillus delbrueckii subspecies bulgaricus and the Streptococcus thermophilus is 5:4. The ratio refers to the ratio of the number of bacterial species.

[0012] According to a preferred embodiment, step (1) includes the following sub-steps: Step (1.1) Prepare Panax notoginseng leaf powder culture medium by sterilizing; the Panax notoginseng leaf is Panax notoginseng ( Panax notoginseng (Burk) FH Chen) leaves.

[0013] The Panax notoginseng leaf powder culture medium comprises 1 part of Panax notoginseng leaf powder and 3 to 10 parts of deionized water; Step (1.2) inoculates the Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus into the Panax notoginseng leaf powder culture medium (1.1); the inoculation amount of the Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus is such that the activated bacteria (including the culture medium) accounts for 5% to 40% of the total mass of the Panax notoginseng leaf powder culture medium.

[0014] Preferably, the inoculated Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus are activated strains in the logarithmic growth phase. The seed solution of the composite bacteria of Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus is cultured and activated in MRS medium; the activation is subcultured 2 to 5 times, each time with an inoculation volume fraction of 2.5%, and the absorbance OD value of the bacterial solution at 600nm is measured to be 1 to 1.5.

[0015] Preferably, the fermentation condition is to perform anaerobic fermentation at a suitable growth temperature for Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus for 2 to 16 days. The suitable growth temperature for Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus is 37°C to 45°C, preferably 37°C to 42°C, and more preferably 37°C.

[0016] Another aspect of the present invention provides a fermented product of Panax notoginseng leaves, prepared by the above-mentioned preparation method, containing rare saponin F 2 , Rg 3 , Rk 1 , CK and Rh 2 ; and rare saponin F 2 , Rg 3 , Rk 1 , CK and Rh 2 The sum of the contents is greater than 4 mg / g.

[0017] Another aspect of the present invention provides an application of a fermented product of Panax notoginseng leaves in preparing a product for improving, preventing and treating hyperlipidemia or non-alcoholic fatty liver disease.

[0018] The product is a medicine, food or health product, and the medicine can be in the form of capsule, tablet, oral liquid, compressed candy or brewing powder.

[0019] Another aspect of the present invention provides the use of the aforementioned Panax notoginseng leaf fermentation product in the preparation of antioxidant products.

[0020] The product is a reagent, a cosmetic or a skin care product.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. A method for preparing a fermented product of Panax notoginseng leaves, wherein the common ginsenosides in Panax notoginseng leaves are converted into rare ginsenoside F which is easy for organisms to absorb and can exert multiple medicinal values ​​by fermenting with a mixed strain of Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus. 2 , Rg 3 , Rk 1 , CK and Rh 2 ; After fermentation, rare ginsenoside F 2 , Rg 3 , Rk 1 increased by 2.45 times, 11.8 times and 4.18 times respectively; at the same time, the rare ginsenosides CK and Rh 2 , 5 kinds of rare ginsenoside F 2 , Rg 3 , Rk 1 , CK and Rh 2 The sum increased by 11.76 times; the medicinal value of Panax notoginseng leaves was significantly improved; 2. An application of a Panax notoginseng leaf fermentation product. The Panax notoginseng leaf fermentation product prepared in the present application can alleviate the symptoms of hyperlipidemia mice: slow down the weight gain of hyperlipidemia model mice, reduce the liver index, perirenal fat index and epididymal fat index of the model mice; downregulate serum total cholesterol, serum triglyceride, serum low-density lipoprotein cholesterol levels, increase serum high-density lipoprotein cholesterol levels, thereby reducing the blood lipid levels of hyperlipidemia mice; 3. An application of a Panax notoginseng leaf fermentation product. The Panax notoginseng leaf fermentation product prepared in the present application can downregulate the levels of liver alanine aminotransferase and liver aspartate aminotransferase, improve the pathological morphology of liver tissue, downregulate the levels of liver total cholesterol, liver triglycerides, and liver low-density lipoprotein cholesterol, and increase the level of liver high-density lipoprotein cholesterol, thereby reducing liver fat levels and relieving the symptoms of non-alcoholic fatty liver disease; 4. An application of a Panax notoginseng leaf fermentation product. The Panax notoginseng leaf fermentation product prepared in the present application increases the levels of catalase, total superoxide dismutase, and reduced glutathione in the liver and serum; lowers the levels of malondialdehyde in the liver and serum; and has an excellent anti-oxidative stress effect; 5. A Panax notoginseng leaf fermentation product and its preparation method and application, which effectively realizes the medicinal use of Panax notoginseng leaf resources through fermentation, fully utilizes limited Panax notoginseng resources and reduces resource waste; enhances the medicinal value of Panax notoginseng leaves, provides a source of rare ginsenosides, and reduces the production and utilization costs of rare ginsenosides; promotes the development of drugs and health foods for hyperlipidemia, non-alcoholic fatty liver and anti-oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a test diagram of common ginsenosides and rare ginsenosides in the fermented Panax notoginseng leaves prepared in Example 1 of the present invention; wherein Figure A is ginsenoside Rg 1 , Rh 1 , Rb 1 , Rc, Rb 2 , Rb 3 and notoginsenoside Fe and rare ginsenoside F 2 , Rg 3 , Rk 1 , CK, Rh 2 Detection diagram of the reference substance; Figure B is the detection diagram of the rare ginsenoside F in the sample prepared in Comparative Example 1 2 , Rg, Rk 1 , CK and Rh 2 Figure C is a rare ginsenoside F in the notoginseng leaf fermentation sample prepared in Example 1 of the present invention 2 , Rg, Rk 1 , CK and Rh 2 Detection diagram; the peak numbers and corresponding saponins in the figure are 1. Ginsenoside Rg 1 ; 2. Ginsenoside Rh 1 ; 3. Ginsenoside Rb 1 ; 4. Ginsenoside Rc; 5. Ginsenoside Rb 2 ; 6. Ginsenoside Rb 3 ; 7. Ginsenoside Rd; 8. Rare ginsenoside F 2 ; 9. Rare ginsenoside Rg 3 ; 10. Rare ginsenoside Rk 1 ; 11. Rare ginsenoside CK; 12. Rare ginsenoside Rh 2 ; Figure 2The effects of the fermented Panax notoginseng leaf total saponins obtained in Example 1 of the present invention and the Panax notoginseng leaf total saponins obtained in Comparative Example 1 on the body weight and body weight gain of mice (n=10). In the figure, compared with the normal control group, ####: P<0.0001; compared with the model group, ***: P<0.001, ****: P<0.0001; Figure 3 Effects of the fermented Panax notoginseng leaf total saponins obtained in Example 1 of the present invention and the Panax notoginseng leaf total saponins obtained in Comparative Example 1 on the food intake of mice (n=10); Figure 4 The effects of the fermented Panax notoginseng leaf total saponins obtained in Example 1 of the present invention and the Panax notoginseng leaf total saponins obtained in Comparative Example 1 on the liver index (%), perirenal fat index (%) and peritesticular fat index (%) of mice (n=10). In the figure, compared with the normal control group, ####: P<0.0001; compared with the model group, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001; Figure 5 The effects of the fermented Panax notoginseng leaf total saponins obtained in the example of the present invention and the Panax notoginseng leaf total saponins obtained in comparative example 1 on the blood lipid level of mice (n=10); in the figure, compared with the normal control group, ###: P<0.001, ####: P<0.0001; compared with the model group, **: P<0.01, ****: P<0.0001; Figure 6 The effects of the fermented Panax notoginseng leaf total saponins obtained in Example 1 of the present invention and the Panax notoginseng leaf total saponins obtained in Comparative Example 1 on serum alanine aminotransferase and serum aspartate aminotransferase in mice (n=10); in the figure, ####: P<0.0001; compared with the model group, **: P<0.01, ***: P<0.001, ****: P<0.0001; Figure 7 The effects of the fermented Panax notoginseng leaf total saponins obtained in Example 1 of the present invention and the Panax notoginseng leaf total saponins obtained in Comparative Example 1 on serum oxidative stress in mice (n=10); in the figure, #: P<0.05, ###: P<0.001, ####: P<0.0001; compared with the model group, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001; Figure 8The effects of the fermented Panax notoginseng leaf total saponins obtained in Example 1 of the present invention and the Panax notoginseng leaf total saponins obtained in Comparative Example 1 on the lipid level in mouse liver (n=10); in the figure, compared with the normal control group, ###: P<0.001, ####: P<0.0001; compared with the model group, *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001; Fig. 9 The effects of the fermented Panax notoginseng leaf total saponins obtained in Example 1 of the present invention and the Panax notoginseng leaf total saponins obtained in Comparative Example 1 on alanine aminotransferase and aspartate aminotransferase in mouse liver (n=10). In the figure, compared with the normal control group, ####: P<0.0001; compared with the model group **: P<0.01, ****: P<0.0001; Fig.10 The effects of the fermented Panax notoginseng leaf total saponins obtained in Example 1 of the present invention and the Panax notoginseng leaf total saponins obtained in Comparative Example 1 on oxidative stress in mouse liver (n=10). In the figure, compared with the normal control group, ##: P<0.01, ###: P<0.001, ####: P<0.0001; compared with the model group, **: P<0.01, ***: P<0.001, ****: P<0.0001; Fig.11 The liver tissue pathological morphology of mice in each group (×200). DETAILED DESCRIPTION

[0023] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0025] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0026] Example 1 This embodiment provides a method for preparing a fermented Panax notoginseng leaf product and a fermented Panax notoginseng leaf total saponin, and the specific steps are as follows: (1) The preparation method of Panax notoginseng leaf powder culture medium is as follows: take dried Panax notoginseng leaves, crush them, pass them through a 60-mesh sieve, stir and mix them evenly according to the ratio of 1 part Panax notoginseng leaf powder to 10 parts deionized water, sterilize them at 121°C for 20 minutes, and place them at room temperature to prepare Panax notoginseng leaf powder culture medium.

[0027] (2) The preparation method of the composite bacterial seed liquid of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus is as follows: Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus are fermented and inoculated in MRS medium, the inoculation amount of Lactobacillus delbrueckii subsp. bulgaricus is 1.0% by volume, and the inoculation amount of Streptococcus thermophilus is 0.8% by volume, and the solution is incubated at 37°C for 24 hours to obtain a preliminary seed liquid, the preliminary seed liquid is inoculated in MRS medium for activation, the inoculation amount is 2.5% by volume, and the solution is incubated at 37°C for 24 hours to obtain a preliminary seed liquid, the preliminary seed liquid is activated, the inoculation amount is 2.5% by volume, and the solution is incubated at 37°C for 24 hours to obtain a preliminary seed liquid, the activation is performed 2 to 5 times, and the inoculation amount is 2.5% by volume each time, and the absorbance OD value of the bacterial solution at 600 nm is measured to be 1 to 1.5, thereby obtaining the composite bacterial seed liquid of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0028] (3) Add the composite bacterial seed liquid of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to the Panax notoginseng leaf powder culture medium of step (1) at an inoculation amount of 40% (g / g), that is, the composite bacterial seed liquid accounts for 40% (g / g) of the total mass of the Panax notoginseng leaf powder culture medium, and place it in an oven at 37°C for anaerobically cultured for 2 days.

[0029] (4) After the fermentation in step (3) is completed, the obtained Panax notoginseng leaf fermentation liquid is placed in an oven and dried at 80° C. until the moisture content is 12%. The liquid is dispersed and mixed evenly using a high-speed disperser at 5000 rpm / min to obtain a Panax notoginseng leaf fermentation product.

[0030] (5) The Panax notoginseng leaf fermentation product obtained in step (4) was reflux-extracted three times with 10-fold amount of 47% (v / v) ethanol aqueous solution, with extraction times of 1 hour, 0.5 hour and 0.5 hour respectively. The extracts were combined, concentrated under reduced pressure, and freeze-dried to obtain freeze-dried powder of fermented Panax notoginseng leaf total saponin extract.

[0031] Weigh 0.6g of freeze-dried powder of total saponin extract of fermented Panax notoginseng leaves accurately, dilute to 50ml with methanol, weigh, ultrasonicate for 30min at 100W50kHz, weigh again after cooling, make up with methanol, shake well, filter with 0.22μm microporous filter membrane, and the filtrate is the test solution. Use high performance liquid chromatography, Agilent C18 column (250mm×4.6mm, 5μm), mobile phase acetonitrile-water solution for gradient elution, flow rate of 1mL / min, column temperature of 40℃, detection wavelength of 203nm, injection volume of 10μL, to determine rare ginsenoside F in total saponin extract of fermented Panax notoginseng leaves. 2 , Rg 3 , Rk 1 , CK and Rh 2 The content.

[0032] Test results: After the Panax notoginseng leaves were processed through the above steps (1)-(5), the saponin composition changed, and the rare ginsenoside F 2 , Rg 3 , Rk 1 , CK and Rh 2 0.76mg / g, 0.59mg / g, 0.46mg / g, 1.93mg / g and 1.19mg / g, respectively, 5 kinds of rare ginsenoside F 2 , Rg 3 , Rk 1 , CK and Rh 2 The total content is 4.93 mg / g.

[0033] Example 2 This embodiment provides a method for preparing a fermented product of Panax notoginseng leaves, and the specific steps are as follows: (1) Preparation of Panax notoginseng leaf powder culture medium: Take dried Panax notoginseng leaves, crush them, pass them through a 60-mesh sieve, stir and mix them evenly according to the ratio of 1 part Panax notoginseng leaf powder to 8 parts deionized water, sterilize them at 121°C for 20 minutes, and let them cool to room temperature to prepare Panax notoginseng leaf powder culture medium.

[0034] (2) Preparation of seed solution of composite bacteria of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus: Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus were fermented and inoculated in MRS medium at a volume fraction of 1.0% for Lactobacillus delbrueckii subsp. bulgaricus and 0.8% for Streptococcus thermophilus. The solution was incubated at 37°C for 24 hours to obtain a preliminary seed solution. The preliminary seed solution was inoculated in MRS medium for activation at a volume fraction of 2.5%. The solution was incubated at 37°C for 24 hours to obtain a preliminary seed solution. The preliminary seed solution was activated 2 to 5 times with a volume fraction of 2.5% each time. The absorbance OD value of the bacterial solution at 600 nm was measured to be 1 to 1.5 to obtain a seed solution of composite bacteria of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0035] (3) Add the composite bacterial seed liquid of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to the Panax notoginseng leaf powder culture medium of step (1) at an inoculation rate of 20% (g / g), i.e., the composite bacterial seed liquid accounts for 20% (g / g) of the total mass of the Panax notoginseng leaf powder culture medium, and place it in an oven at 37°C for anaerobically cultured for 8 days.

[0036] (4) After the fermentation in step (3) is completed, the obtained Panax notoginseng leaf fermentation liquid is placed in an oven and dried at 60° C. until the moisture content is 12%. The liquid is dispersed and mixed evenly using a high-speed disperser at 8000 rpm / min to obtain a Panax notoginseng leaf fermentation product.

[0037] (5) The Panax notoginseng leaf fermentation product obtained in step (4) was extracted three times by reflux with 10-fold amount of 47% (v / v) ethanol aqueous solution, with extraction times of 1 hour, 0.5 hour and 0.5 hour respectively. The extracts were combined, concentrated under reduced pressure and freeze-dried to obtain the fermented Panax notoginseng leaf total saponin extract.

[0038] The rare ginsenoside F in the total saponin extract of Panax notoginseng leaves was determined by high performance liquid chromatography using an Agilent C18 column (250 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile-water for gradient elution at a flow rate of 1 mL / min, a column temperature of 40 °C, a detection wavelength of 203 nm, and an injection volume of 10 μL. 2 , Rg 3 , Rk 1 , CK and Rh 2 The content.

[0039] Test results: After the Panax notoginseng leaves were processed through the above steps (1)-(5), the saponin composition changed, and the rare ginsenoside F 2 , Rg 3 , Rk 1 , CK and Rh 2 0.94mg / g, 0.53mg / g, 0.44mg / g, 1.4mg / g and 1.1mg / g, respectively, 5 kinds of rare ginsenoside F2 , Rg 3 , Rk 1 , CK and Rh 2 The total content is 4.41 mg / g.

[0040] Example 3 This embodiment provides a method for preparing a fermented product of Panax notoginseng leaves, and the specific steps are as follows: (1) Preparation of Panax notoginseng leaf powder culture medium: Take dried Panax notoginseng leaves, crush them, pass them through a 60-mesh sieve, mix 1 part of Panax notoginseng leaf powder with 3 parts of deionized water, and sterilize them at 121°C for 20 minutes, then let them cool to room temperature to prepare Panax notoginseng leaf powder culture medium.

[0041] (2) Preparation of seed solution of composite bacteria of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus: Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus were fermented and inoculated in MRS medium at a volume fraction of 1.0% for Lactobacillus delbrueckii subsp. bulgaricus and 0.8% for Streptococcus thermophilus. The solution was incubated at 37°C for 24 hours to obtain a preliminary seed solution. The preliminary seed solution was inoculated in MRS medium for activation at a volume fraction of 2.5%. The solution was incubated at 37°C for 24 hours to obtain a preliminary seed solution. The preliminary seed solution was activated 2 to 5 times with a volume fraction of 2.5% each time. The absorbance OD value of the bacterial solution at 600 nm was measured to be 1 to 1.5 to obtain a seed solution of composite bacteria of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus.

[0042] (3) Add the composite bacterial seed liquid of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to the Panax notoginseng leaf powder culture medium of step (1) at an inoculation amount of 5% (g / g), that is, the composite bacterial seed liquid accounts for 5% (g / g) of the total mass of the Panax notoginseng leaf powder culture medium, and place it in an oven at 37°C for anaerobic culture for 16 days.

[0043] (4) After the fermentation in step (3) is completed, the obtained Panax notoginseng leaf fermentation liquid is placed in an oven and dried at 70° C. until the moisture content is 12%. The liquid is dispersed and mixed evenly using a high-speed disperser at 10,000 rpm / min to obtain a Panax notoginseng leaf fermentation product.

[0044] (5) The Panax notoginseng fermentation product obtained in step (4) was subjected to reflux extraction three times with 10-fold amount of ethanol aqueous solution, during which the concentration of ethanol aqueous solution was controlled to be 47% (v / v), and the extraction times were 1 hour, 0.5 hour and 0.5 hour, respectively. The extracts were combined, concentrated under reduced pressure, and freeze-dried to obtain the fermented Panax notoginseng leaf total saponin extract.

[0045] The rare ginsenoside F in the total saponin extract of Panax notoginseng leaves was determined by high performance liquid chromatography using an Agilent C18 column (250 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile-water for gradient elution at a flow rate of 1 mL / min, a column temperature of 40 °C, a detection wavelength of 203 nm, and an injection volume of 10 μL. 2 , Rg 3 , Rk 1 , CK and Rh 2 The content.

[0046] Test results: After the above steps (1)-(5) of Panax notoginseng leaves, the saponin composition changed, and rare ginsenoside F 2 , Rg 3 , Rk 1 , CK and Rh 2 The contents of 5 rare ginsenosides F were 1.45mg / g, 0.49mg / g, 0.48mg / g, 0.86mg / g and 1.26mg / g respectively. 2 , Rg 3 , Rk 1 , CK and Rh 2 The total content is 4.54 mg / g.

[0047] Comparative Example 1: A method for preparing a total saponin extract from Panax notoginseng leaves The difference between this comparative example and Example 1 is that the Panax notoginseng leaves are not subjected to the preparation of Panax notoginseng leaf powder culture medium, the composite bacteria seed liquid of Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus is not inoculated for fermentation, and the fermentation liquid is not dried. The specific steps are as follows: (1) Take dried Panax notoginseng leaves, crush them and pass through a 60-mesh sieve.

[0048] (2) The notoginseng leaf powder obtained in step (1) was subjected to reflux extraction three times with a 10-fold amount of ethanol aqueous solution. During the reflux extraction, the concentration of the ethanol aqueous solution was controlled to be 47% (v / v). The extraction times were 1 hour, 0.5 hour and 0.5 hour, respectively. The extracts were combined, concentrated under reduced pressure and freeze-dried to obtain a notoginseng leaf total saponin extract.

[0049] The rare ginsenoside F in the total saponin extract of Panax notoginseng leaves was determined by high performance liquid chromatography using an Agilent C18 column (250 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile-water for gradient elution at a flow rate of 1 mL / min, a column temperature of 40 °C, a detection wavelength of 203 nm, and an injection volume of 10 μL. 2 , Rg 3 , Rk 1 , CK and Rh 2 The content.

[0050] Test results: Rare ginsenoside F in total saponin extract of Panax notoginseng leaves 2 , Rg 3 , Rk 1 , CK and Rh 2 0.31mg / g, 0.05mg / g, 0.11mg / g, 0.00mg / g and 0.00mg / g, 5 kinds of rare ginsenoside F 2 , Rg 3 , Rk 1 , CK and Rh 2 The sum of the contents is 0.47 mg / g; the comparison of the contents of the five rare ginsenosides in the total saponin extract of Panax notoginseng leaves after fermentation in Examples 1-3 is shown in Table 1.

[0051] Table 1 Detection results of 5 kinds of rare human saponin contents in Examples and Comparative Examples

[0052] Figure 1 The peak number and the corresponding saponin are 1. Ginsenoside Rg 1 ; 2. Ginsenoside Rh 1 ; 3. Ginsenoside Rb 1 ; 4. Ginsenoside Rc; 5. Ginsenoside Rb 2 ; 6. Ginsenoside Rb 3 ; 7. Notoginsenoside Fe; 8. Rare ginsenoside F 2 ; 9. Rare ginsenoside Rg 3 ; 10. Rare ginsenoside Rk 1 ; 11. Rare ginsenoside CK; 12. Rare ginsenoside Rh 2 .from Figure 1 As can be seen from Table 1, the composition of total saponins in the Panax notoginseng leaf fermentation product prepared by the method of the present invention has changed, the original saponin content of total saponins in Panax notoginseng leaf has been greatly reduced, and the rare ginsenoside F 2 , Rg 3 , Rk 1 Significantly increased rare ginsenoside F 2 , Rg 3 , Rk 1 The content of ginsenosides increased by 2 times, 9 times, and 4 times respectively, and two rare ginsenosides CK and Rh were added. 2 , 5 kinds of rare ginsenoside F 2 , Rg 3 , Rk 1 , CK and Rh 2 The sum increased by more than 9 times compared with the unfermented ones.

[0053] Example 4 The fermented Panax notoginseng leaf total saponins prepared in Example 1 and the Panax notoginseng leaf total saponins prepared in Comparative Example 1 were tested for their lipid-lowering, non-alcoholic fatty liver disease relief and antioxidant activity.

[0054] The hyperlipidemia animal model was used to test the lipid-lowering, non-alcoholic fatty liver disease relief and antioxidant stress activation.

[0055] Animals: Male SPF C57BL / 6J mice, weighing 16-18 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. [Experimental Animal License No. SYXK (Xiang) 2019-0017] and were kept in an SPF animal room at 23°C ± 2°C, relative humidity (50 ± 5)%, and a light / dark cycle of 12h / 12h. This experiment was approved by the Animal Research Ethics Committee of Yunnan University of Traditional Chinese Medicine (ethics number R-062023174).

[0056] High-fat feed: sucrose 15%, egg yolk powder 12%, lard 12%, cholesterol 1.5%, pig bile salt 1%, salt 0.2%, maintenance feed 58.3%; high-fat emulsion: lard 20%, Tween-80 15%, cholesterol 6%, white sugar 5%, sodium cholate 1%, propylthiouracil 0.2%, water 52.8%.

[0057] Animal grouping and drug administration: 50 male C57BL / 6J mice were randomly selected as the normal control group and given ordinary feed, and the rest of the mice were the modeling group, which were given high-fat feed and high-fat emulsion gavage once every 3 days. After 6 weeks, the mice with successful modeling were randomly divided into the model group, the positive drug group (simvastatin, 2.570 mg / kg), the total saponin group of Panax notoginseng leaves (Comparative Example 1 11.5843 mg / kg), and the fermented total saponin group of Panax notoginseng leaves (Example 1 11.5843 mg / kg), and were gavaged once a day according to the dose for 10 consecutive weeks, with a gavage volume of 1 ml / 100 g. The normal control group and the model group were given equal doses of normal saline. During the experiment, food intake and body weight were measured every Monday morning.

[0058] After 10 weeks of administration, the levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), catalase (CAT), MDA, SOD and GSH-PX in serum and liver of rats in normal control group, model group, fermented notoginseng leaf total saponins group and notoginseng leaf total saponins group were detected.

[0059] Liver histological analysis: Freshly isolated rat liver tissue was fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin, and sliced. After HE staining, the pathological changes of liver tissue were observed under an optical microscope.

[0060] Figure 2The effects of fermented Panax notoginseng leaf total saponins in Example 1 and Comparative Example 1 on the body weight of mice are shown. Figure 2 As shown, during the test period, the weight of mice in the model group has been in a rapid growth trend. After 16 weeks of feeding, the weight of mice in the model group has been significantly higher than that of other groups, and its average weight has reached 32.16±2.86g. The normal control group, positive drug group, fermented Panax notoginseng total saponin group and Panax notoginseng total saponin group gained weight slowly. At 16 weeks, the average weight of mice in the normal control group was 22.74±0.69g, the average weight of mice in the positive drug group was 26.00±1.47g, the average weight of mice in the Panax notoginseng total saponin group in Comparative Example 1 was 25.84±0.43g, and the average weight of mice in the fermented Panax notoginseng total saponin group in Example 1 was 24.40±1.28g. During the test period, the weight gain of mice in the model group was much higher than that of other groups, and there was a significant difference with other groups; the weight gain of mice in the fermented Panax notoginseng total saponin group was less than that of mice in the unfermented Panax notoginseng total saponin group. Moreover, Figure 3 The changes in daily food intake of mice in each group are shown. Figure 3 It can be seen that there is no significant difference in the daily food intake of each group of mice, indicating that the administration of total saponins from Panax notoginseng leaves has little effect on the appetite of mice.

[0061] Figure 4 The effects of fermented Panax notoginseng leaf total saponins and Panax notoginseng leaf total saponins on the liver index, perirenal fat index and peri-epididymal fat index of mice were shown. Figure 4 As shown, compared with the normal control group, the liver index (P<0.0001), perirenal fat index (P<0.0001) and peritesticular fat index (P<0.0001) of the mice in the model group were significantly increased; compared with the model group, the liver index of the positive drug group (P<0.05), total saponin group of Panax notoginseng leaves (P<0.0001) and fermented total saponin group of Panax notoginseng leaves (P<0.0001) were significantly decreased; the perirenal fat index of the positive drug group (P<0.001), total saponin group of Panax notoginseng leaves (P<0.01) and fermented total saponin group of Panax notoginseng leaves (P<0.0001) were significantly decreased; the epididymal fat index of the positive drug group (P<0.001), total saponin group of Panax notoginseng leaves (P<0.01) and fermented total saponin group of Panax notoginseng leaves (P<0.0001) were significantly decreased. Figure 4 It can be seen that the liver index, perirenal fat index and epididymal fat index of the fermented Panax notoginseng leaf total saponin group were better than those of the unfermented Panax notoginseng leaf total saponin group.

[0062] Figure 5The effect of fermented Panax notoginseng leaf total saponins and Panax notoginseng leaf total saponins on the blood lipid level of mice is shown. As can be seen from the figure, the serum total cholesterol, serum triglyceride, and serum low-density lipoprotein cholesterol levels of the model group were significantly higher than those of the other groups, while the serum high-density lipoprotein cholesterol level was significantly lower than that of the other groups. The serum total cholesterol, serum triglyceride, and serum low-density lipoprotein cholesterol levels of the mice in the two Panax notoginseng leaf total saponins groups were significantly lower than those in the model group, and the serum high-density lipoprotein cholesterol level was significantly increased, indicating that the administration of Panax notoginseng leaf total saponins can significantly reduce the levels of serum total cholesterol, serum triglyceride, and serum low-density lipoprotein cholesterol, and increase the level of serum high-density lipoprotein cholesterol, thereby reducing the blood lipid level of hyperlipidemic mice. Among them, the fermented Panax notoginseng leaf total saponins have a better lipid-lowering effect than the Panax notoginseng leaf total saponins.

[0063] Figure 6 The results show the effects of fermented Panax notoginseng leaf total saponins and Panax notoginseng leaf total saponins on serum alanine aminotransferase and serum aspartate aminotransferase in mice. Figure 6 It can be seen that the serum alanine aminotransferase and serum aspartate aminotransferase levels in the model group were significantly higher than those in the other groups, and the serum alanine aminotransferase and serum aspartate aminotransferase levels of the two types of Panax notoginseng leaf total saponin groups were significantly lower than those in the model group, indicating that the administration of Panax notoginseng leaf total saponin can significantly reduce the serum alanine aminotransferase and serum aspartate aminotransferase levels. Among them, the fermented Panax notoginseng leaf total saponin has a better effect on reducing the serum alanine aminotransferase and serum aspartate aminotransferase levels than the Panax notoginseng leaf total saponin.

[0064] Figure 7 The effects of fermented Panax notoginseng leaf total saponins and Panax notoginseng leaf total saponins on serum oxidative stress in mice are shown. As can be seen from the figure, the levels of serum catalase (CAT), serum total superoxide dismutase (SOD), and serum reduced glutathione (GSH-PX) in the model group were significantly lower than those in other groups, while serum malondialdehyde was significantly higher than that in other groups. The levels of serum catalase, serum total superoxide dismutase, and serum reduced glutathione in the two Panax notoginseng leaf total saponins groups were significantly increased compared with the model group, and serum malondialdehyde was significantly decreased compared with the model group, indicating that the administration of Panax notoginseng leaf saponins can significantly increase the levels of serum alanine aminotransferase and serum aspartate aminotransferase, and reduce the level of serum malondialdehyde, and has a good anti-oxidative stress effect. Among them, the anti-oxidative stress effect of fermented Panax notoginseng leaf total saponins is better than that of Panax notoginseng leaf total saponins.

[0065] Figure 8The effects of fermented Panax notoginseng leaf total saponins and Panax notoginseng leaf total saponins on the liver lipid levels of mice are shown. As can be seen from the figure, the levels of liver total cholesterol, liver triglycerides, and liver low-density lipoprotein cholesterol in the model group were significantly higher than those in other groups, while the level of liver high-density lipoprotein cholesterol was significantly lower than that in other groups. The levels of liver total cholesterol, liver triglycerides, and liver low-density lipoprotein cholesterol in the two Panax notoginseng leaf total saponin groups were significantly lower than those in the model group, and the level of liver high-density lipoprotein cholesterol was significantly increased, indicating that the administration of Panax notoginseng leaf total saponin extract can significantly reduce the levels of liver total cholesterol, liver triglycerides, and liver low-density lipoprotein cholesterol, and increase the level of liver high-density lipoprotein cholesterol, thereby reducing the liver lipid level of hyperlipidemic mice. Among them, fermented Panax notoginseng leaf total saponins are better than Panax notoginseng leaf total saponins in reducing liver lipids and relieving fatty liver.

[0066] Fig. 9 The effects of fermented Panax notoginseng leaf total saponins and Panax notoginseng leaf total saponins on liver glutamic alanine aminotransferase and liver aspartate aminotransferase in mice are shown. As can be seen from the figure, the levels of liver glutamic alanine aminotransferase and liver aspartate aminotransferase in the model group were significantly higher than those in other groups, and the levels of liver glutamic alanine aminotransferase and liver aspartate aminotransferase in the two Panax notoginseng leaf total saponins groups were significantly lower than those in the model group, indicating that the administration of Panax notoginseng leaf total saponins can significantly reduce the levels of liver glutamic alanine aminotransferase and liver aspartate aminotransferase. Among them, fermented Panax notoginseng leaf total saponins have a better effect on reducing serum glutamic alanine aminotransferase and serum aspartate aminotransferase levels than Panax notoginseng leaf total saponins.

[0067] Fig.10 The effects of fermented Panax notoginseng leaf total saponins and Panax notoginseng leaf total saponins on oxidative stress in mice are shown. As can be seen from the figure, the levels of liver catalase, liver total superoxide dismutase, and liver reduced glutathione in the model group were significantly lower than those in other groups, while liver malondialdehyde was significantly higher than that in other groups. The levels of liver catalase, liver total superoxide dismutase, and liver reduced glutathione in the two Panax notoginseng leaf total saponin extract groups were significantly increased compared with the model group, and liver malondialdehyde was significantly decreased compared with the model group, indicating that the administration of Panax notoginseng leaf saponins can significantly increase the levels of liver alanine aminotransferase and liver aspartate aminotransferase, and reduce the level of liver malondialdehyde, and has a good anti-oxidative stress effect. Among them, the anti-oxidative stress effect of fermented Panax notoginseng leaf total saponins is better than that of Panax notoginseng leaf total saponins.

[0068] Fig.11The pathological morphology of liver tissues of mice in each group is shown (the scale bar in the figure is 100 μm). As can be seen from the figure, the hepatocyte cords of the normal control group are arranged regularly, the cell membrane is intact, and no fatty degeneration, ballooning degeneration and inflammatory changes are observed. In the model group, the hepatocyte cords are arranged disorderly, the hepatocyte volume is enlarged, a large number of hepatocytes have fat vacuoles, a large number of hepatocytes have obvious fatty degeneration and ballooning degeneration, and are accompanied by obvious inflammatory cell infiltration. In the positive drug group, the hepatocyte cords are arranged more regularly, a small number of hepatocytes have fat vacuoles, a small number of hepatocytes have fatty degeneration and ballooning degeneration, and the pathological morphology of liver tissue is improved compared with the model group. In the Panax notoginseng leaf total saponin group, the hepatocyte cords are arranged more regularly, a small number of hepatocytes have fat vacuoles, a small number of hepatocytes have fatty degeneration and ballooning degeneration, and the pathological morphology of liver tissue is improved compared with the model group. Fermented notoginseng leaf total saponin group: The hepatocyte cords were arranged regularly, the cell membranes were intact, and occasionally very few hepatocytes had fat vacuoles and fatty degeneration. There were no obvious abnormalities in the hepatocyte structure and hepatic cord arrangement, and the liver tissue was close to that of the normal control group.

[0069] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A method for preparing a fermented product of Panax notoginseng leaves, characterized in that: The steps include: Step (1) fermenting Panax notoginseng leaves by mixing Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus; In the step (1), the mixing ratio of Lactobacillus germanica subspecies bulgaricus and Streptococcus thermophilus is 5:

4.

2. The method for preparing a fermented product of Panax notoginseng leaves according to claim 1, characterized in that: The step (1) includes the following sub-steps: Step (1.1) preparing Panax notoginseng leaf powder culture medium from Panax notoginseng leaf and sterilizing; The Panax notoginseng leaf powder culture medium comprises 1 part of Panax notoginseng leaf powder and 3 to 10 parts of deionized water; Step (1.2) inoculates the notoginseng leaf powder culture medium prepared in step (1.1) with the Lactobacillus delbrueckii subspecies bulgaricus and the thermophilic Streptococcus; the inoculation amount of the Lactobacillus delbrueckii subspecies bulgaricus and the thermophilic Streptococcus accounts for 5% to 40% of the total mass of the notoginseng leaf powder culture medium.

3. The method for preparing a fermented product of Panax notoginseng leaves according to claim 2, characterized in that: The inoculated Lactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus are activated strains in the logarithmic growth phase.

4. The method for preparing a fermented product of Panax notoginseng leaves according to claim 1, characterized in that: The fermentation conditions of step (1) are to perform anaerobic fermentation at a suitable growth temperature for Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus for 2 to 16 days.

5. A fermented product of Panax notoginseng leaves, characterized in that: The product is prepared by the method for preparing a Panax notoginseng leaf fermentation product as described in any one of claims 1 to 4, and contains rare saponins F2, Rg3, Rk1, CK and Rh2; and the sum of the contents of F2, Rg3, Rk1, CK and Rh2 is greater than 4 mg / g.

6. Use of the Panax notoginseng leaf fermentation product as claimed in claim 5 in the preparation of products for improving, preventing and treating hyperlipidemia or non-alcoholic fatty liver disease.

7. The use according to claim 6, characterized in that: The product is a medicine, food or health product.

8. The use according to claim 7, characterized in that The medicine can be in the form of capsule, tablet, oral solution, compressed candy or brewing powder.

9. Use of the Panax notoginseng leaf fermentation product as claimed in claim 5 in the preparation of antioxidant products.

10. The use according to claim 9, characterized in that: The product is a reagent, a cosmetic or a skin care product.

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