CN116712445A

CN116712445A - Timosaponin enzymolysis product and application of timosaponin AIII in preparation of medicines for treating hyperlipidemia

Info

Publication number: CN116712445A
Application number: CN202310497206.9A
Authority: CN
Inventors: 丁越; 赵文军; 张彤; 王小颖; 路璐; 江旻
Original assignee: Shanghai University of Traditional Chinese Medicine
Current assignee: Shanghai University of Traditional Chinese Medicine
Priority date: 2023-05-05
Filing date: 2023-05-05
Publication date: 2023-09-08

Abstract

The application relates to the field of biological medicine, in particular to an application of timosaponin enzymolysis products and timosaponin AIII in preparing medicines for treating hyperlipidemia. According to the research of the application, after timosaponin enzymolysis products and timosaponin AIII which is a main component thereof are adopted to interfere a C57BL/6 mouse, the timosaponin enzymolysis products can obviously improve the dyslipidemia of the mouse, improve the brown fat at the shoulder blade, the beige fat at the inguinal region and the fat morphological abnormality of adipose tissue at the epididymis, reduce the generation of lipid by the timosaponin enzymolysis products, enhance the fatty acid oxidation and mitochondrial functional gene expression of adipocytes, and reduce the lipid generation gene expression by the timosaponin AIII which is a main component thereof. The timosaponin enzymolysis product or the timosaponin AIII which is the main component thereof can effectively treat the hyperlipidemia, has good effect and has wider application prospect in the field of treating the hyperlipidemia.

Description

Timosaponin enzymolysis product and application of timosaponin AIII in preparation of medicines for treating hyperlipidemia

The application relates to the field of biological medicine, in particular to an application of timosaponin enzymolysis products and timosaponin AIII in preparing medicines for treating hyperlipidemia.

Background

Obesity (Obesity) is a chronic metabolic disease that is manifested by excessive body weight and body type hypertrophy due to excessive fat accumulation, and is related to various factors such as genetics, environment, eating habits, endocrine, etc. Obese patients are often associated with metabolic disorders, dyslipidemia, and thus obese people are at very high risk of hyperlipidemia (hyperlipomia), manifested as excessive overall blood lipid levels, and serious people can cause some diseases that are serious health hazards, such as coronary heart disease, atherosclerosis, and the like. Various researches have shown that obesity is one of important factors for hyperlipidemia, and obesity patients usually have symptoms of elevated blood lipid, so that the control of the blood lipid level of the obesity patients has important significance for preventing and treating serious diseases caused by hyperlipidemia.

Rhizoma anemarrhenae is a dry rhizome of rhizoma anemarrhenae (Anemarrhena asphodeloides Bge.) belonging to the family Liliaceae, has bitter taste, cold property, enters lung, stomach and kidney channels, has the effects of clearing heat and purging fire, nourishing yin and moistening dryness, has a long clinical application history, and is also widely applied to modern traditional Chinese medicine diagnosis and treatment. Timosaponin is the main active ingredient in rhizoma anemarrhenae, and accounts for about 6% of rhizome of rhizoma anemarrhenae, wherein timosaponin BII content is higher, timosaponin AII content is lower and is only 1/20 of timosaponin BII content, so timosaponin AII is difficult to directly separate from rhizoma anemarrhenae medicinal materials for preparation, and researches show that timosaponin BII is converted into timosaponin AII and timosaponin under the action of intestinal flora in vivo, wherein timosaponin AII is the main metabolite. Timosaponin AIII has various biological activities, has the effects of resisting platelet aggregation, improving learning ability and memory, resisting tumor, reducing blood sugar and the like, and for example, the patent of the issued application (issued publication number: CN 107412245B) discloses timosaponin AIII nano-liposome capable of inhibiting proliferation of tumor cells; the patent of the application (publication number CN 1513463A) discloses that timosaponin AIII extracted from rhizoma anemarrhenae can treat type II diabetes and reduce blood sugar, and can be used for preparing medicines for treating type 2 diabetes and improving hyperglycemia; the patent of the application (publication number CN 1265797C) discloses application of timosaponin AIII in preparation of medicines for reducing blood sugar and treating type II diabetes, and the timosaponin AIII can reduce blood fat of KKAy diabetic mice at a dosage of 400mg/kg, but the blood fat does not refer to specific indexes and is regarded as complications of type II diabetes.

Modern pharmacological researches show that timosaponin has good blood lipid reducing effect, however timosaponin reduces blood lipid of high-fat dietary rats, the dosage is 100-400 mg/kg, the dosage of timosaponin BII which is the main component in timosaponin for improving hyperlipidemia is about 100mg/kg, the dosage is large, and the lipid reducing effect is difficult to reach satisfactory level.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, in order to solve the technical problems of large dosage and poor lipid-lowering effect of timosaponin AIII in the existing medicines in the prior art, the application aims to provide timosaponin enzymolysis products and application of timosaponin AIII in preparation of medicines for treating hyperlipidemia. The traditional Chinese medicine has the main ways of inhibiting the absorption of exogenous lipid, inhibiting the synthesis of endogenous lipid, promoting the secretion and excretion of bile acid, improving the activity and content of high-density lipoprotein cholesterol, and the like. The application discovers that the action mechanism of the timosaponin enzymolysis product for reducing blood fat is to reduce lipid generation, increase fatty acid oxidation and promote lipid consumption, thereby achieving the effect of reducing blood fat; the action mechanism of timosaponin AIII for reducing blood lipid is mainly through reducing the generation of lipid, and the lipid-lowering mechanism is one of innovations of the application.

To achieve the above and other related objects, in one aspect, the present application provides a use of an enzymatic hydrolysate of timosaponin selected from any one or more of the following:

1) Preparing a medicament for treating hyperlipidemia;

2) Preparing a medicament for reducing serum total cholesterol;

3) Preparing a medicament for reducing serum triglyceride;

4) Preparing a medicament for reducing serum low density lipoprotein;

5) Preparing the medicine for increasing serum high density lipoprotein.

The second aspect of the present application provides a medicament for treating hyperlipidemia, comprising timosaponin AIII; preferably, the medicament comprises timosaponin enzymatic hydrolysate.

In a third aspect, the application provides a method of treating hyperlipidemia by administering to a subject an effective amount of the aforementioned medicament.

Compared with the prior art, the application has the beneficial effects that:

1. the application improves the new application of timosaponin enzymolysis products and timosaponin AIII which is a main component thereof, in particular to the application of timosaponin enzymolysis products in preparing medicines for treating hyperlipidemia.

2. According to the research of the application, after timosaponin enzymolysis products and timosaponin AIII which is a main component thereof are adopted to interfere a C57BL/6 mouse, the timosaponin enzymolysis products can obviously improve the dyslipidemia of the mouse, improve the brown fat at the shoulder blade, the beige fat at the inguinal region and the fat morphological abnormality of adipose tissue at the epididymis, reduce the generation of lipid by the timosaponin enzymolysis products, enhance the fatty acid oxidation and mitochondrial functional gene expression of adipocytes, and reduce the lipid generation gene expression by the timosaponin AIII which is a main component thereof. The timosaponin enzymolysis product or the timosaponin AIII which is the main component thereof can effectively treat the hyperlipidemia, has good effect and has wider application prospect in the field of treating the hyperlipidemia.

Drawings

FIG. 1 is a schematic diagram showing the effect of timosaponin enzymolysis product and timosaponin AIII as main component on weight of mice before and after administration.

Wherein, FIG. 1A is the weight before modeling, FIG. 1B is the weight after modeling and administration of a high fat diet, and FIG. 1C is the weight gain percentage.

FIG. 2 is a schematic diagram showing the effect of timosaponin enzymolysis product and timosaponin AIII as main component on serum blood lipid level of mice.

Wherein, fig. 2A shows the effect of timosaponin enzymolysis product on mouse serum triglyceride, fig. 2B shows the effect of timosaponin enzymolysis product on mouse low density lipoprotein content, fig. 2C shows the effect of timosaponin enzymolysis product on mouse serum total cholesterol, and fig. 2D shows the effect of timosaponin enzymolysis product on mouse high density lipoprotein content.

FIG. 3 is a schematic diagram showing the results of H & E staining of mouse adipose tissue with timosaponin enzymolysis product and timosaponin AIII as main component.

FIG. 4 is a schematic diagram showing the effect of timosaponin enzymolysis product and timosaponin AIII as main component on lipid production, fatty acid beta oxidation and mitochondrial function gene level of mouse beige adipose tissue. Wherein, fig. 4A is the effect of timosaponin enzymolysis product on lipid production gene level of mouse beige adipose tissue, fig. 4B is the effect of timosaponin enzymolysis product on fatty acid beta oxidation gene level of mouse beige adipose tissue, and fig. 4C is the effect of timosaponin enzymolysis product on mitochondrial function gene level of mouse beige adipose tissue.

FIG. 5 is a schematic diagram showing the effect of timosaponin enzymolysis product and timosaponin AIII as main component on triglyceride and total cholesterol content of fat cells. Wherein, fig. 5A shows the effect of timosaponin enzymolysis product on fat cell triglyceride, and fig. 5B shows the effect of timosaponin enzymolysis product on total cholesterol of fat cell.

Detailed Description

In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application will be further described with reference to examples. It is to be understood that the examples are provided for the purpose of illustrating the application and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.

The "range" disclosed herein is defined in terms of lower and upper limits, with the given range being defined by the selection of a lower and an upper limit, the selected lower and upper limits defining the boundaries of the particular range. Ranges that are defined in this way can be inclusive or exclusive of the endpoints, and any combination can be made, i.e., any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 to 120 and 80 to 110 are listed for a particular parameter, it is understood that ranges of 60 to 110 and 80 to 120 are also contemplated. Furthermore, if the minimum range values 1 and 2 are listed, and if the maximum range values 3,4 and 5 are listed, the following ranges are all contemplated: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present application, unless otherwise indicated, the numerical ranges "a-b" represent a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed throughout, and "0-5" is only a shorthand representation of a combination of these values. When a certain parameter is expressed as an integer of 2 or more, it is disclosed that the parameter is, for example, an integer of 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or the like.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present application may be used to practice the present application according to the knowledge of one skilled in the art and the description of the present application.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present application employ conventional techniques in biomedical engineering, biophysics, pharmacy, pharmaceutical analytics, pharmaceutical chemistry, analytical chemistry, molecular biology, biochemistry, and related fields, which are conventional in the art. These techniques are well described in the prior art.

The inventor of the present application has found that timosaponin enzymolysis product and timosaponin AIII are applied in preparing medicine for treating hyperlipemia through great amount of research and research, and the present application is completed on this basis.

The hyperlipidemia is dyslipidemia, and the timosaponin enzymolysis product and the timosaponin AIII which is the main component thereof have at least one of the following effects: 1) Reducing serum triglyceride levels; 2) Reducing serum total cholesterol content; 3) Reducing the content of the blood fat, the low density lipoprotein cholesterol; 4) Raise serum high density lipoprotein cholesterol content; 5) Reducing lipid production of beige fat; 6) Increasing fatty acid oxidation in beige or brown adipocytes; 7) Increase mitochondrial function in beige or brown adipocytes. The medicine for preventing and treating hyperlipidemia can be used as not only medicine for improving dyslipidemia, but also promoter for fatty acid oxidation in beige or brown fat cells.

Timosaponin AIII has molecular formula of C ₃₉ H ₆₄ O ₁₃ The structure is as follows:

in one aspect, the application provides the use of an enzymatic hydrolysate of timosaponin selected from any one or more of the following:

1) Preparing a medicament for treating hyperlipidemia;

2) Preparing a medicament for reducing serum total cholesterol;

3) Preparing a medicament for reducing serum triglyceride;

4) Preparing a medicament for reducing serum low density lipoprotein;

5) Preparing the medicine for increasing serum high density lipoprotein.

In the application provided by the application, the timosaponin enzymolysis product comprises timosaponin AIII.

The timosaponin enzymolysis product is obtained by extracting timosaponin decoction pieces with alcohol and hydrolyzing with beta-glucosidase.

The application provided by the application further comprises any one or more of the following:

1) Preparing an inhibitor of a lipid production gene;

2) Preparing an accelerator of a fatty acid beta oxidation gene;

3) Preparing promoter of mitochondrial gene;

4) Lipid consumption promoter is prepared.

In the application provided by the application, the lipid-producing gene is selected from one or more of Fasn, acc1 and Scd 1. The fatty acid beta oxidation gene is selected from Cpt1α and/or PPARα. The mitochondrial gene is selected from Cytc, ATPsyntβ, cox4 β. The application further discovers that timosaponin enzymolysis products can ensure the normal lipid metabolism function by reducing lipid generation and promoting fatty acid beta oxidation, and the timosaponin AIII, the main component of which, is mainly used for reducing lipid generation, thereby achieving the effect of preventing and treating hyperlipidemia.

In another aspect, the present application provides a medicament for treating hyperlipidemia, comprising an effective dose of timosaponin AIII. Preferably, the medicament comprises timosaponin enzymatic hydrolysate.

An effective dose refers to a dose at which the drug may exhibit efficacy. Because the medicine can reach a certain medicine concentration only after a certain dosage is absorbed by the organism, the medicine effect can be realized only when the medicine reaches a certain medicine concentration. If the dosage is too small, an effective concentration cannot be obtained in the body, and the drug cannot exert its effective effect. However, if the dosage is too large, beyond a certain limit, the effect of the drug may change substantially, possibly causing different degrees of toxicity to the body. Therefore, in order to exert the effective action of the medicine and avoid adverse reactions, the dosage range of the medicine must be strictly controlled.

The medicine provided by the application also comprises a pharmaceutically acceptable carrier or auxiliary materials.

By "pharmaceutically acceptable" is meant that the molecular entity and composition do not produce adverse, allergic or other untoward reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier or adjuvant" should be compatible with the lysosomal modulating factor, i.e. capable of being blended therewith without substantially reducing the efficacy of the drug in the usual manner. Specific examples of some substances which may be pharmaceutically acceptable carriers or excipients are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as malondiol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as Tween; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting, stabilizing agent and antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc. These substances are used as needed to aid stability of the formulation or to aid in enhancing the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration.

The medicament provided by the application can be adapted to any form of administration, be it oral or parenteral, for example, be it pulmonary, nasal, rectal and/or intravenous, more particularly intradermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, pulmonary, buccal, sublingual, nasal, transdermal, vaginal, oral or parenteral.

The skilled artisan can select a suitable formulation depending on the mode of administration, for example, a formulation suitable for oral administration may be a formulation including, but not limited to, a pill, tablet, chew, capsule, granule, drop, or syrup, etc., and for further example, a formulation suitable for parenteral administration may be a formulation including, but not limited to, a solution, suspension, reconstitutable dry preparation, spray, etc., and for further example, a suppository may be generally suitable for rectal administration.

In another aspect, the present application provides a method of treating hyperlipidemia by administering to a subject an effective amount of the aforementioned medicament.

In the treatment method provided by the application, the effective dose of timosaponin AIII exceeds 10mg/kg. The minimum dose used in the present application was 1/10 of the prior art (CN 1265797C). The medicine of the application can greatly reduce the cost of raw materials and the dosage.

In the treatment method provided by the application, the subject comprises a patient with hyperlipidemia and/or a patient with lipid metabolism disorder caused by obesity, fatty liver, diabetes and other metabolic syndrome related diseases.

The application is further illustrated by the following examples, which are not intended to limit the scope of the application. In the examples which follow, unless otherwise indicated, the techniques employed are conventional and well known to those skilled in the art, and the reagents and materials of the application are available commercially or from other common sources.

The percentages are by weight unless otherwise indicated.

Example 1

1. Experimental reagent and material

Rhizoma anemarrhenae decoction pieces, no.210113, shanghai Kangqiao Chinese medicinal decoction pieces, inc.; timosaponin AIII control, lot number: R12S8F43634, purity >98%, shanghai Yuan Ye Biotechnology Co., ltd; timosaponin BII, lot number R14D9F77658, purity >98%, shanghai Yuan Ye Biotechnology Co., ltd; beta-glucosidase, shanghai Feng Biochemical Co., ltd; methanol (chromatographic purity), acetonitrile (chromatographic purity), absolute ethanol (analytical purity), national pharmaceutical chemicals, inc.

2. Experimental method

(1) Preparation of anemarrhena rhizome ethanol extract and its enzyme hydrolysate

Weighing rhizoma anemarrhenae decoction pieces 60g, adding 6 times of 50% ethanol, and extracting under reflux for 2 hr for 2 times. Filtering, mixing the extractive solutions, rotary evaporating under reduced pressure, recovering ethanol, evaporating in water bath, transferring to vacuum drying oven, drying at 60deg.C under reduced pressure overnight to obtain mother ethanol extract, and weighing.

Weighing 4g of each of the anemarrhena ethanol extract and the beta-glucosidase, placing into a conical flask with a plug, adding 200ml of acetic acid-sodium acetate buffer solution with pH of 5.0, placing into a constant temperature oscillator, and shaking for 3h at 55 ℃. Extracting the extract with water saturated n-butanol with equal volume for 3 times, washing with ammonia solution for 3 times, mixing n-butanol extractive solutions, rotary evaporating under reduced pressure, recovering n-butanol, vacuum drying at 60deg.C, and drying under reduced pressure to obtain enzymolysis product of ginsenoside. And measuring the contents of timosaponin BII and timosaponin AIII after enzymolysis of the extract by HPLC.

Example 2

1. Experimental reagent and material

(1) Experimental animals: male C57BL/6 mice, 60, weighing 20+ -2 g, were purchased from Shanghai Ji Hui laboratory animal feeding Co. Animal feeding is carried out at an animal experiment center of Shanghai traditional Chinese medicine university, and the experimental animals use a production license number; SYXK (Shanghai) 2020-0009. The raising temperature is 25+/-1 ℃, the humidity is 60+/-2%, the free diet is adopted, the cage raising is carried out, the artificial light and dark circulation is carried out for 12 hours, and all animal experiment operations accord with the animal center regulation system of Shanghai Chinese medicine university.

(2) Reagent: timosaponin AIII (timosaponin AIII, TA III, shanghai Seiyaka leaf Biotechnology Co., ltd., lot: R12S8F 43634); timosaponin BII (Timosapon BII, TB II, shanghai Yuan leaf Biotechnology Co., ltd., lot: R14D9F 77658) timosaponin enzymatic hydrolysis product: example 1 preparation obtained, lot number: 20190922, 62.5% pure (HPLC); metformin hydrochloride (sangon biotech, lot F823BA 007). qPCR premix kit (next san Biotech (Shanghai) Co., ltd., lot H7212920); the following reagents were purchased from institute of bioengineering with south Beijing: TC kit (lot number: 20220108), TG kit (lot number: 20220108), HDL-C (lot number: 20220111) kit, LDL-C (lot number: 20220111) kit; ethanol (national drug group, 20210901); isopropyl alcohol (national drug group, lot number 20210816); TRIzol reagent (TaKaRa). The mouse feed is a normal maintenance feed and is purchased from Jiangsu province collaborative medical bioengineering Limited liability company.

2. Experimental method

C57BL/6 mice were randomly divided into 6 groups (10 per group), the blank group was fed with normal feed, and the rest were fed with high-fat feed: high fat group, timosaponin alcohol extraction group, timosaponin BII group, timosaponin AIII group, timosaponin enzymolysis product group. Feeding with high fat for 7 weeks, starting administration of rhizoma anemarrhenae ethanol extract (4 g crude drug/kg), rhizoma anemarrhenae enzymolysis product (15.8 mg/kg, suspended in CMC-Na), timosaponin group AIII (10 mg/kg, suspended in CMC-Na) and timosaponin group BII (100 mg/kg, suspended in CMC-Na) the mice were free to eat and drink water, and the drug was intervened for 10 weeks.

All mice were fed last, fasted for 12h without water withdrawal, and after anesthesia, the mice were bled and serum was isolated for detection of Total Cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) levels in serum. Mice were sacrificed by cervical vertebra removal after blood collection, three adipose tissues of scapula brown fat (BAT), inguinal beige fat (iWAT) and epididymis white fat (eWAT) were taken out, one part of tissues was soaked in 4% paraformaldehyde and then subjected to staining section examination, the other part of tissues was used for extracting RNA, and the other tissues were stored in a refrigerator at-80 ℃ for later use.

3. Index detection

(1) Weight measurement

Determining the body weight of the mice after the experimental administration is finished; the food intake was measured once every three days for the last two weeks after the end of the experiment, four times in total. The results are shown in FIG. 1.

(2) Serum blood lipid level detection

Determination of Triglycerides (TG): taking 2 mu L of serum and distilled water as blank, taking a standard substance as a standard hole, adding 200 mu L of working solution, incubating for 10min at 37 ℃, and detecting TG absorbance at a wavelength of 510 nm. Serum TG content (mmol/L) = (sample OD value-blank OD value)/(standard cone OD value-blank OD value) ×standard concentration (2.26 mmol) was calculated according to the formula.

Total Cholesterol (TC) was measured as above with a standard concentration of (5.17 mmol/L).

Measurement of Low Density lipoprotein cholesterol (LDL-C): taking 2.5 mu L of serum and distilled water as blank, taking a standard substance as a standard hole, adding 180 mu L of first working solution, incubating for 5min at 37 ℃, detecting LDL-C absorbance A1 at 546nm wavelength, adding 60 mu L of second working solution, incubating for 5min at 37 ℃, and detecting LDL-C absorbance A2 at 546nm wavelength. Serum LDL-C content (mmol/L) = ((sample A2-sample A1) - (blank A2-blank A1))/(standard A2-standard A1) - (blank A2-blank A1)) ×standard concentration (2.20 mmol/L) as calculated by the formula.

The procedure for high density lipoprotein cholesterol (HDL-C) determination was as above with a standard concentration of 1.05mmol/L.

The results are shown in FIG. 2.

(3) Adipose tissue sections and observations

Fresh adipose tissues were sent for paraffin section treatment and hematoxylin-eosin (HE) staining was performed. The dyeing method comprises the following steps: firstly, paraffin sections are put into dimethylbenzene for dewaxing; taking out the slices, rehydrating the slices by 100%, 95%, 85%, 75% and 50% ethanol in sequence, and cleaning the slices with distilled water for 3 times; firstly, placing the slices into hematoxylin dye solution, passing for 10min, taking out the slices, immersing the slices in distilled water for reverse blue, and washing the slices with distilled water after about 5min; then putting into 0.5% acetic acid ethanol for differentiation, and washing with distilled water once after success; then placing the slice into eosin dye solution for 30sec, taking out, and soaking in 95% ethanol for 10sec for color separation treatment; finally, the slices are dehydrated, transparent, then are sealed by a sealing tablet, and observed and left under an optical microscope.

The results are shown in FIG. 3.

(4) Inguinal beige adipose tissue gene detection

Tissue RNA extraction: after weighing the mouse adipose tissue iWAT, rapidly adding into magnetic beads (pre-soaked with 75% ethanol for more than 30 min) and 1.5RNAiso Plus, using a tissue lysis instrument, setting the frequency at 28.0/s, and homogenizing for 9min. After homogenization, the mixture was allowed to stand at room temperature (15-30 ℃) for 5min, centrifuged at 12,000 g at 4℃for 5min, the supernatant carefully aspirated, and transferred to a new centrifuge tube. Adding chloroform (RNAiso Plus 1/5 volume) into RNAiso Plus containing tissue or cell, mixing upside down, emulsifying to milky white, standing at room temperature for 5min, centrifuging at 4deg.C for 15min at 000 g; carefully taking out the centrifuged homogenate, wherein the homogenate is divided into three layers, RNA is present in the colorless supernatant layer, carefully sucking the colorless supernatant, and transferring the supernatant into a new centrifuge tube; isopropanol, the volume of which is 0.5 times that of RNAiso Plus, is added into the sucked supernatant, the mixture is inverted upside down or vortexed into a centrifuge tube, the mixture is fully mixed, the mixture is kept stand at room temperature for 10min, if the RNA content in the sample is small, the mixture can be kept stand at-20 ℃ for 2h or overnight and kept stand at 4 ℃, and 12,000 g of the mixture is centrifuged for 10min, so that RNA precipitation appears at the bottom of the centrifuge tube.

Washing and dissolving of RNA precipitate: the supernatant was carefully aspirated using a micropipette, and a small amount of isopropanol could remain without contact or impact to the pellet. Preparing 75% ethanol by using absolute ethyl alcohol and non-ribozyme water, adding 75% ethanol equivalent to RNAiso Plus into each centrifuge tube, reversing the centrifuge tube upside down, washing the tube wall of the centrifuge tube, centrifuging at 4 ℃ for 5min at 500g, carefully sucking the supernatant by a pipettor, and likewise, not contacting or impacting the supernatant to precipitate; and opening a centrifugal tube cover, drying and precipitating at room temperature for a few minutes, and adding a proper amount of non-ribozyme water into the mixture to swirl the mixture after the precipitate is dried, so that the non-ribozyme water is fully dissolved. 1. Mu.L of the RNA was assayed and quantified (about 500 ng) and the remainder was kept at-80℃for further use.

Reverse Transcription (RT) reaction: according to Table 1, the corresponding components were added to the eight-pipe, and RT reaction solution was prepared (the preparation of the reaction solution was carried out at a low temperature as much as possible), and after the reaction solution was mixed uniformly, the mixture was centrifuged to carry out the reverse transcription reaction: 15min at 37 ℃, 5sec at 85 ℃ and 4 ℃.

TABLE 1RT reaction liquid formulation table

Amplification and calculation: preparing PCR reaction liquid (shown in Table 2), mixing, centrifuging, performing PCR amplification reaction (shown in Table 3), and calculating the change of the relative amount of gene expression according to the formula relative quantification=2-DeltaCt. (Ct is the number of cycles when a fluorescence value is detected).

TABLE 2 preparation of PCR reaction solutions

TABLE 3PCR amplification procedure

(5) Statistical analysis

All results data were treated with mean±sem using GraphPad mapping and statistics, one-way ANOVA with One-way ANOVA to check for variance alignment, and Student's-test for analysis, p <0.05 indicated significant differences.

The results are shown in FIG. 4.

Example 3

1. Experimental materials

Rhizoma anemarrhenae decoction pieces, no.210113, shanghai Kangqiao Chinese medicinal decoction pieces, inc.; timosaponin enzymolysis product, prepared in example 1, lot number: 20190922, 62.5% pure (HPLC); timosaponin AIII (from source leaf organisms), timosaponin BII (from source leaf organisms), lovastatin (from source leaf organisms); 3T3-L1 cells (from the cell bank of the national academy of sciences); the following materials were purchased from the sameidie technology: DMEM, fetal bovine serum, 0.25% trypsin, isobutylmethylxanthine, dexamethasone, insulin; oleic acid (from merck life sciences), palmitic acid (from merck life sciences).

2. Experimental method

(1) Preparation of experimental medicine

100g of rhizoma anemarrhenae is taken, 50% ethanol with the amount of 6 times is added, reflux extraction is carried out for 2 times, each time for 2 hours, and the dried rhizoma anemarrhenae crude extract is obtained after the extraction is finished. Timosaponin AIII, timosaponin BII, timosaponin enzymolysis product and timosaponin ethanol crude extract are dissolved in DMSO and filtered with 0.22 μm filter membrane to obtain concentration of 4mg/L, and stored (-20deg.C) for use.

(2) Culture conditions

3T3-L1 preadipocytes were passaged resuscitated in DMEM medium containing 10% Fetal Bovine Serum (FBS), and inoculated into 100mL glass culture flasks at 37℃with 5% CO ₂ And (5) standing and culturing in an incubator. The 3T3-L1 preadipocytes and the culture medium were visually observed under an inverted microscope every 1-2 days, and if there was a characteristic change, such as a color change, the culture medium was replaced with fresh medium and the cells were again placed in the incubator for culturing.

(3) Cell passage

3-4 days after cell inoculation, after 3T3-L1 preadipocyte monolayer grows densely and the fusion degree reaches 80-90%, 0.25% trypsin is used for digestion, after the cells are observed under an inverted microscope until cytoplasmatic retraction and cell gaps are increased, pancreatin is immediately poured out, 2mL of DMEM medium containing 10% FBS is added, digestion is stopped, suspended cells are gently blown and collected, and the cells are centrifuged, resuspended, counted and uniformly inoculated in a 12-well plate.

(4) Cell differentiation

After selection of the DMEM medium containing 10% FBS, which is well grown after passage and has a fusion density of 60-70%, the differentiation medium, i.e., the DMEM medium containing 0.5mM isobutyl methylxanthine (IBMX), 1mM dexamethasone (Dex) and 10. Mu.g/mL insulin, is replaced, after 48 hours of culture, the differentiation medium, i.e., the DMEM medium containing only 10. Mu.g/mL insulin, is replaced, and finally replaced with the DMEM medium containing 10% FBS for 8-12 days, and is used for the experiment after the cells differentiate to maturity.

(5) Determination of triglyceride and Total cholesterol levels in cells

Blank (Blank) was cultured in serum-free DMEM after complete differentiation of cells. Model group (Con) was cultured in serum free DMEM with free fatty acid (10 μg/mL) (free fatty acid condition oleic acid: palmitic acid=2:1). After 4mg/L of timosaponin alcohol crude extract (ZC), timosaponin AIII (TA III), timosaponin BII (TB II), timosaponin enzymolysis product (E-TA III) and lovastatin (Lov) are respectively added into the medicine administration, cells are treated for 30min, free fatty acid (the condition of the free fatty acid is oleic acid: palmitic acid=2:1) is added, all the cells are continuously cultured for 8h, the cells are collected by splitting, and the TG and TC contents of the cells are measured according to the requirements of the specification of a commercial kit.

The results are shown in FIG. 5.

Example 4

Experimental results

1. Ethanol extract of rhizoma anemarrhenae and its enzyme hydrolysate containing TB II and TA III

Respectively precisely weighing 150.00mg of rhizoma anemarrhenae ethanol extract and 10.00mg of enzyme hydrolysate, placing in a 25ml volumetric flask, dissolving with ultrasound, fixing volume with 50% ethanol, filtering with 0.45 μm filter membrane, and determining TB II and TA III content. Calculating the conversion rate of TA III = (m1×M2)/(m2×M1) ×100%, wherein M1 represents the actual measured amount of TA III in the enzyme hydrolysate; m2 represents the actual measured amount of TB II in the ethanol extract; m1 represents TA III relative molecular weight; m2 represents the relative molecular weight of TB II.

The results show that in the prepared rhizoma anemarrhenae ethanol extract, the TB II content is (31.3+/-1.4)%, the TB II extraction rate is (95.3+/-4.9)%, and the TA III content is (3.2+/-0.3)%; in the enzyme hydrolysate, TA III content was (62.5.+ -. 0.3)% (RSD was 0.5%, n=3), TB II was not detected, and TA III conversion was (82.6.+ -. 1.7)% (RSD was 1.6%, n=3), indicating complete conversion. The main component of the enzymolysis product of the anemarrhena alcohol extract is timosaponin AIII.

2. Timosaponin enzymolysis product and influence of timosaponin AIII as main component on weight of mice

The C57BL/6 mice were weighed before molding and 10 weeks after gavage administration, respectively. Compared with the blank group, the weight of the mice in the high-fat model group is obviously increased after the administration of high-fat feed; in contrast, the dosing groups all significantly reduced the body weight of the mice given high fat compared to the high fat model group (fig. 1). Wherein the timosaponin enzymolysis product and timosaponin AIII have the ability of reducing the weight of mice 1.42 times and 1.31 times of timosaponin BII, and the administration dosage of timosaponin enzymolysis product is about 1/6 of timosaponin BII, and timosaponin AIII is 1/10 of timosaponin BII.

3. Timosaponin enzymolysis product and influence of timosaponin AIII as main component on serum blood lipid level of mice

Fig. 2 shows: the high fat control group had elevated serum TG, TC and LDL-C levels and reduced HDL-C levels, respectively, as compared to the placebo group mice, with a significant increase in TC, TG, LDL-C levels of 68%,191% and 418% respectively. The timosanol extract, timosaponin AIII, BII and timosaponin enzymolysis products remarkably reduce the TG, TC and LDL-C contents of high-fat mice. Timosaponin AIII has a 1.28-fold lower LDL-C than timosaponin BII. At the same time, timosaponin enzymolysis product group and timosaponin AIII group can obviously raise HDL-C content to blank mouse level, and timosaponin BII can not obviously improve HDL-C reduction caused by high-fat diet. This shows that the low dose of timosaponin enzymolysis product and timosaponin AIII can effectively regulate the dyslipidemia in obese mice, and can not improve the hyperlipidemia state of the mice.

4. Timosaponin enzymolysis product and influence of timosaponin AIII as main component on mouse fat morphology

Three adipose tissues of mice were extracted and separated, and then, the mice were embedded in sections and subjected to HE staining. The different adipose tissue morphologies of the mice are shown in the figure (figure 3), and compared with the blank group, fat drops in the fat of the three adipose tissues of the high-fat group mice are obviously enlarged; each dosing group significantly reduced adipocyte lipid droplet size compared to the high lipid model group. After the timosaponin enzymolysis product is used, the diameter of adipose tissue cells at epididymis is obviously reduced, the number of cells per unit area is increased, which suggests that the improvement of dyslipidemia is related to the reduction of the size of adipose tissue lipid drops, and the timosaponin enzymolysis product has an improvement effect.

5. Timosaponin enzymolysis product and influence of timosaponin AIII as main component on expression of mouse iWAT adipose tissue gene

Timosaponin enzymolysis product and timosaponin AIII as main component can reduce lipid drop size, and improve insulin resistance of organism, possibly through fatty acid oxidation, thereby increasing lipid drop utilization and reducing blood lipid level. Beige fat (Beige adipose tissue) is a newly discovered class of adipose tissue that not only resembles white fat with lipid droplets stored therein, but also contains more mitochondria (sites of fatty acid beta oxidation). We therefore further examined the expression of gene levels associated with lipid production, fatty acid beta oxidation and mitochondrial function in beige adipose tissue. The expression of the genes related to lipid production of the C57BL/6 hyperlipidemic mice is obviously increased, and the level of the genes related to mitochondrial function is obviously reduced. After drug administration, timosaponin AIII and timosaponin enzymatic products significantly reduced lipid production-related genes Fasn, acc1 and Scd1 compared to the high-lipid group, whereas timosaponin alcohol extract and timosaponin BII did not improve expression of lipid production genes. Administration of timosaponin enzymolysis product therapy can up-regulate expression of fatty acid beta oxidation gene. While significantly increasing the expression levels of the mitochondrial related genes Cytc, atpsyntβ, and cox4β (fig. 4). This shows that timosaponin enzymolysis product can reduce hyperlipidemia by reducing lipid generation, increasing fatty acid oxidation and enhancing mitochondrial function, and timosaponin AIII as its main component can reduce hyperlipidemia by reducing lipid generation. Although timosanol extract and timosaponin BII can increase fatty acid oxidation, they have strong effect of increasing lipid production (about 20 times of Fasn gene expression enhancement), and it is presumed that the effect of timosanol extract and timosaponin BII on fatty acid oxidation may be metabolic compensation effect after lipid overproduction.

6. Timosaponin enzymolysis product and effect of timosaponin AIII as main component on content of triglyceride and total cholesterol of fat cell

Differentiated 3T3-L1 adipocytes were cultured under free fatty acid conditions (10. Mu.g/mL) for 8h, with a 105% increase in total triglyceride levels in the cells (P < 0.001) and a similar increase in total cholesterol levels in the cells compared to the blank without fatty acids. Cell triglyceride and total cholesterol levels were reduced by 85% (P < 0.001) and 63% (P < 0.001), respectively, with the addition of 4mg/L timosaponin AIII. The addition of 4mg/L timosaponin enzymolysis product also significantly reduced triglyceride and total cholesterol levels (76% (P < 0.001) and 54% (P < 0.01)), respectively. Whereas the group supplemented with 4mg/L of timosaponin and timosaponin BII did not show the ability to lower triglyceride and total cholesterol levels. This shows that timosaponin AIII, an enzymatic product of timosaponin, has a stronger effect on reducing cell triglyceride and total cholesterol levels than timosaponin BII, an alcoholic extract of timosaponin, at the same dosage administered. (FIG. 5)

The above embodiments are merely illustrative of the principles of the present application and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the application. Accordingly, it is intended that all equivalent modifications and variations of the application be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. Use of timosaponin enzymatic hydrolysate selected from any one or more of the following:

1) Preparing a medicament for treating hyperlipidemia;

2) Preparing a medicament for reducing serum total cholesterol;

3) Preparing a medicament for reducing serum triglyceride;

4) Preparing a medicament for reducing serum low density lipoprotein;

5) Preparing the medicine for increasing serum high density lipoprotein.

2. The use according to claim 1, wherein the timosaponin enzymolysis product comprises timosaponin aiii.

3. The use according to claim 1, wherein the use of timosaponin enzymatic hydrolysate further comprises one or more of the following:

1) Preparing an inhibitor of a lipid production gene;

2) Preparing an accelerator of a fatty acid beta oxidation gene;

3) Preparing promoter of mitochondrial gene;

4) Lipid consumption promoter is prepared.

4. The use according to claim 3, wherein the lipid producing gene is selected from the group consisting of Fasn, acc1 and Scd 1.

5. The use according to claim 3, wherein the fatty acid β -oxidation gene is selected from cpt1α and/or pparα.

6. The use according to claim 3, wherein the mitochondrial gene is selected from the group consisting of Cytc, atpsyntβ, cox4β.

7. A medicament for treating hyperlipidemia, wherein the medicament comprises timosaponin aii; preferably, the medicament comprises timosaponin enzymatic hydrolysate.

8. A method of treating hyperlipidemia comprising administering to a subject an effective amount of the medicament of any one of claims 7.

9. The method of treatment of claim 8, wherein the effective dose of timosaponin aii exceeds 10mg/kg.

10. The method of treatment of claim 8, wherein the subject comprises a patient suffering from hyperlipidemia and/or a patient suffering from a disorder of blood lipid or lipid metabolism due to metabolic syndrome related diseases such as obesity, fatty liver, and diabetes.