Extract composition for improving lipid metabolism as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicament preparation, in particular to an extract composition for improving lipid metabolism as well as a preparation method and application thereof, and the composition is obtained by mixing a CO2 supercritical extract of myristica fragrans and a 70% ethanol extract of wild jujube; wherein the extraction conditions of the CO2 supercritical extract of the myristica fragrans are as follows: the extraction pressure is 10-15 Mpa, the temperature is 40-50 DEG C, the pressure of the separation 1 is 6-8 Mpa, and the pressure of the separation 2 is 4-6 Mpa; the extraction condition of the 70% ethanol extract of the wild jujube is as follows: extraction is performed twice with 70% ethanol, and extraction is performed for 2 hours each time. The composition provided by the invention can significantly reduce the weight of obese mice and promote the browning change of adipose tissues of the obese mice; meanwhile, the composition and the effective monomer thereof can significantly reduce 3T3-L1 fat cell lipid metabolism disorder, and can be used for adjuvant therapy of obesity, fatty liver and metabolic disorder related diseases.

Description

Technical Field

[0001] The invention relates to the technical field of medicament preparation, and in particular to an extract composition for improving lipid metabolism, a preparation method and use thereof. Background Art

[0002] Lipid metabolism disorders are closely related to metabolic disorders such as obesity, type 2 diabetes, hypertension, dyslipidemia and cardiovascular disease (CVD). Traditional Chinese medicine is widely used in the study of lipid metabolism due to its advantages such as multiple targets, multiple pathways and relatively few adverse reactions. Traditional Chinese medicine has a long history and definite clinical efficacy in treating obesity and other diseases related to metabolic disorders.

[0003] Nutmeg is the seed of the plant Myristica fragrans of the Myristicaceae family. It has a pungent, bitter and warm taste. It enters the spleen, stomach and large intestine meridians, and has the effects of warming the middle and promoting qi, promoting digestion, and stopping diarrhea. The chemical components of nutmeg mainly include volatile oil, fatty oil, phenylpropanoids, lignans and flavonoids, among which the volatile oil content of nutmeg is 8%-15%, mainly monoterpene hydrocarbons, sesquiterpenes, aromatic ethers, monoterpene alcohols, esters, etc.; the fatty oil content is 25%-46%, mainly diaryl nonanones, the characteristic components of the Myristicaceae family.

[0004] The sour jujube is a deciduous tree of the genus Sophora japonica in the Anacardiaceae family. It is sweet, sour, slightly astringent, and flat in nature, and enters the spleen and liver meridians. It has the effects of promoting qi and blood circulation, nourishing the heart, calming the nerves, digesting food, detoxifying, sobering up, and killing insects. The chemical components of sour jujube mainly include organic acids, inorganic acids, ellagic acid, flavonoids, amino acids, sterols, etc. Among them, the content of citric acid, succinic acid, quinic acid and other components is very high, and ellagic acid components are its characteristic components. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an extract composition for improving lipid metabolism, a preparation method and use thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] An extract composition for improving lipid metabolism, the composition is obtained by mixing a CO2 supercritical extract of nutmeg with a 70% ethanol extract of jujube; wherein the extraction conditions of the CO2 supercritical extract of nutmeg are:

[0008] The extraction pressure is 10-15 MPa, the temperature is 40-50°C, the separation 1 pressure is 6-8 MPa, and the separation 2 pressure is 4-6 MPa.

[0009] The extraction conditions of the 70% ethanol extract of sour jujube are as follows: 70% ethanol extraction for 2 times, each time for 2 hours.

[0010] Furthermore, the mass ratio of nutmeg to wild jujube is 1:1 to 1:2, preferably, the mass ratio of nutmeg to wild jujube is 1:1.

[0011] The present invention also provides a method for preparing the above-mentioned extract composition for improving lipid metabolism, comprising the following steps:

[0012] S1. Preparation of CO2 supercritical extract of nutmeg:

[0013] After crushing the nutmeg, extract it for 2 hours under the conditions of pressure 10 MPa, extraction temperature 40°C, separation 1 pressure 7 MPa, separation 2 pressure 5 MPa; then, extract it for 1 hour under the conditions of pressure 15 MPa, extraction temperature 40°C, separation 1 pressure 7 MPa, separation 2 pressure 5 MPa, and collect the extract to obtain;

[0014] S2. Preparation of 70% ethanol extract of wild jujube:

[0015] Crush the sour jujube, soak it in 70% ethanol overnight, extract it twice with 8 times the amount of 70% ethanol, each time for 2 hours, filter it, combine the filtrates, concentrate it under reduced pressure to a thick paste with a relative density of 1.15-1.2, dry it under reduced pressure, crush it, and pass it through an 80-mesh sieve to obtain the product;

[0016] S3. Preparation of the composition:

[0017] The obtained CO2 supercritical extract of nutmeg and 70% ethanol extract of sour jujube were mixed in a ratio of 1:1 based on the mass ratio of nutmeg to sour jujube.

[0018] The present invention provides use of the extract composition for improving lipid metabolism in preparing medicines for preventing and treating obesity and / or non-alcoholic fatty liver.

[0019] The present invention provides use of the extract composition for improving lipid metabolism in medicines for lowering blood lipids.

[0020] The present invention provides use of the extract composition for improving lipid metabolism in a medicine for reducing lipid metabolism disorder in 3T3-L1 adipocytes.

[0021] The composition of the present invention uses nutmeg and sour jujube as raw materials, and has great advantages in regulating lipid metabolism disorders. Animal experimental results show that the composition of the present invention can significantly reduce the body weight of obese mice and promote browning of adipose tissue of obese mice; at the same time, the composition and its effective monomers can significantly reduce lipid metabolism disorders in 3T3-L1 adipocytes, and can be used for the auxiliary treatment of diseases related to obesity, fatty liver and metabolic disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0023] Figure 1 This is the LC-MS total ion chromatogram of extract A from nutmeg extracted by supercritical CO2 extraction.

[0024] Figure 2 This is the LC-MS total ion chromatogram of ethanol extract B of Jujube.

[0025] Figure 3 The effects of nutmeg extract A, jujube ethanol extract B, and A+B combination on body weight and eWAT of C57BL / 6 obese mice.

[0026] Figure 4 The effects of nutmeg extract A, jujube ethanol extract B, and A+B combination on body weight and BAT in C57BL / 6 obese mice.

[0027] Figure 5 The effects of nutmeg extract A, jujube alcohol extract B, and A+B combination on the body weight of C57BL / 6 obese mice.

[0028] Figure 6 The effects of nutmeg extract A, jujube alcohol extract B, and A+B combination drug-containing intestinal fluid on 3T3-L1 adipocytes;

[0029] In the figure: A. Typical intuitive picture of cells in each group; B. Typical picture of Oil Red O staining of cells in each group; C. Statistical picture of Oil Red O staining of cells in each group. DETAILED DESCRIPTION

[0030] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0031] Example

[0032] 1.1 Instrument

[0033] Supercritical extraction equipment (Shanghai Depu Machinery Equipment Co., Ltd., model: GKSF220-50-6L), HPLC-Q-Exactive-MS / MS (Thermo Fisher Scientific, USA), TB-214 electronic balance (Beijing Sartorius Instrument System Co., Ltd.), ultrasonic cleaning machine (Ningbo Xinzhi Biotechnology Co., Ltd.), centrifugal concentration device (cold trap UT200 and centrifugal concentration CVE-3110 combination, Tokyo Rika EYELA), GC-MS (EXPEC model 5250 triple quadrupole mass spectrometer-GC2000) (Room 316, 3rd floor, Building H, Jinshan Campus, Inner Mongolia Medical University), gas chromatography column (Agilent-HP5MS, 0.25mm), supercritical extraction equipment (Shanghai Depu Machinery Equipment Co., Ltd. GKSF220-50-6L), CO2 incubator (Thermo HERACELL 150i), centrifuge (Hunan Xiangyi, TDZ5-WS), microplate reader (Thermo), inverted microscope (OLYMPUS, CKS53), etc.

[0034] 1.2 Drugs

[0035] Nutmeg (Inner Mongolia Beiyu Pharmaceutical Co., Ltd., batch number: 19092603), Ziziphus jujuba (Inner Mongolia Beiyu Pharmaceutical Co., Ltd., batch number: 1901010519).

[0036] 1.3 Reagents and consumables

[0037] 60% high-fat and high-sugar feed (Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., XTHF60-1), DMEM high-glucose culture medium (Dalian Meilun Biotechnology Co., Ltd., MA0212-2), insulin

[0038] [CAS#11070-73-8](Sigma 1-5500, Yuanye S12033), dexamethasone [CAS#50-02-2](SigmaD4902), 3-isobutyl-1-methylxanthine [CAS#28822-58-4](Sigma 15879, Meilun MB5226), palmitic acid [CAS#57-10-3](Sigma P5585), BSA [CAS#9048-46-8](Coolable CA1382), Oil Red Dye (Beijing Solebold Technology Co., Ltd., G1260), PBS (Dalian Meilun Biotechnology Co., Ltd., MA0015), etc.

[0039] Reference substances: dehydrodiisoeugenol (Beijing Saibaicao Technology Co., Ltd., 111838-201403), gallic acid (China Food and Drug Inspection Institute, 110831-201906), quercetin (China Food and Drug Inspection Institute, 100081-201610), myristic ether (China Food and Drug Inspection Institute, 190180-201701), citric acid (China Food and Drug Inspection Institute, 110831-201906), eugenol (China Food and Drug Inspection Institute, 110831-201906). =The results were as follows: 1. The results were as follows: 1. The results were as follows: 2. The results were as follows: 3. The results were as follows: 4. The results were as follows: 1. The results were as follows: 2. The results were as follows: 3. The results were as follows: 4. The results were as follows: 5. The results were as follows: 6. The results were as follows: 7. The results were as follows: 8. The results were as follows: 9. The results were as follows: 10. The results were as follows: 11. The results were as follows: 12. The results were as follows: 13. The results were as follows: 14. The results were as follows: 15. The results were as follows: 16. The results were as follows: 17.

[0040] 1.4 Animals

[0041] C57BL / 6JN mice (8 weeks old, 22.5 g ± 1.5 g), male, were purchased from Beijing Sibeifu Biotechnology Co., Ltd., production license number: SCXK (Beijing) 2021-0006.

[0042] 1.5 cells

[0043] 3T3-L1 cells were purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences (Catalog No. SCSP-5038).

[0044] 2 Methods and Results

[0045] 2.1 Preparation of test substances

[0046] 2.1.1 Preparation of Nutmeg CO2 Supercritical Extract A

[0047] Crush nutmeg, extract at 40°C, under a pressure of 10 MPa for 2 h, then at 15 MPa for 1 h. Combine the two extracts to obtain nutmeg extract A.

[0048] 2.1.2 Preparation of ethanol extract B of Ziziphus jujuba

[0049] Crush the wild jujube, soak it in 70% ethanol overnight, extract it twice with 8 times the amount of 70% ethanol, each time for 2 hours, filter, combine the filtrate, and concentrate it under reduced pressure to a thick paste with a relative density of 1.15-1.2, dry it under reduced pressure, grind it, and pass it through an 80-mesh sieve to obtain the wild jujube alcohol extract paste B.

[0050] 2.2 Identification and attribution of components of nutmeg extract A and jujube alcohol extract B

[0051] Total ion currents of nutmeg extract A and jujube ethanol extract B are shown in Figure 1 , Figure 2 The identification results of the components of nutmeg extract A and jujube alcohol extract B are shown in Tables 1 and 2. The results of LC-MS experiments showed that 11 components were identified in nutmeg extract A, including 6 phenylpropanoids and 5 lignans. A total of 7 components were identified in jujube alcohol extract B, including 4 phenolic acids, 2 flavonoids, and 1 organic acid. Among them, 11 components were confirmed by comparison with standards.

[0052] Table 1 Identification and attribution of chemical components in nutmeg extract A under positive ion mode

[0053]

[0054] Table 2 Identification and attribution of chemical components in ethanol extract B of Jujube jujube under negative ion mode

[0055]

[0056] Note: A = lignans, B = phenylpropanoids, C = phenolic acid, D = organic acid, F = flavonoids. Sz = jujube, Rdk = nutmeg. Superscript "#" = compared with the standard.

[0057] 2.3 Effects of the composition on body weight and adipose tissue in obese mice

[0058] 2.3.1 Methods

[0059] (1) Animal models

[0060] 50 5-week-old male C57BL / 6JN mice. Adaptively fed with ordinary feed for 5 days, free access to water, 10 mice were selected as blank control group and 40 mice as high-fat group according to body weight. The blank control group was fed with ordinary feed, and the obesity model group was fed with high-fat and high-sugar feed (60% fat energy supply). The body weight was measured every week. After 10 weeks, the body weight was considered to be successful when it exceeded the average body weight of the blank control group by 20%. Subsequently, the obesity model group was randomly divided into model control group, drug intervention group: nutmeg A group (nutmeg extract A 0.2g / kg), sour jujube B group (sour jujube alcohol extract B 0.08g / kg), and combination group (nutmeg extract A 0.2g / kg + sour jujube alcohol extract B 0.08g / kg) 10 mice each. Except for the blank control group given ordinary feed, the other groups still maintained a high-fat and high-sugar diet. The drug intervention group and the combination group were gavaged with the corresponding drugs, and the blank control group and the model control group were gavaged with an equal volume of distilled water. After 8 weeks of continuous administration, the epididymal white fat and dorsal brown fat tissue were collected without food or water, fixed with 4% paraformaldehyde, and subjected to pathological analysis.

[0061] (2) Pathological analysis

[0062] Preparation of paraffin sections: ① Tissue fixation: After fixation with 4% paraformaldehyde for 24 hours, the tissue was flattened and rinsed with running water for 1 hour; ② Dehydration and permeabilization: placed in 50% ethanol (2.5 hours) → 60% ethanol (1.5 hours) → 75% ethanol (1.5 hours) → 80% ethanol (overnight) → 90% ethanol (1 hour) → 95% ethanol (1 hour) → 100% ethanol I (1 hour) → 100% ethanol II (1 hour) → xylene I (10 minutes) → xylene II (10 minutes); ③ Wax immersion : low melting point Ⅰ (1h) → low melting point Ⅱ (1h); ④ Embedding: preheat the metal embedding box for 1h, add a little paraffin, place the fat tissue at the bottom of the embedding box, embed the tissue, and separate the tissue wax block from the embedding box after sufficient cooling on the cold table; ⑤ Sectioning and spreading: precool the wax block at -20℃ for 40min, repair the block with a slicer, the slice thickness is 0.5μm, and place the wax slice on the water surface of the 55℃ spreader to fully unfold the wax slice; ⑥ Drying: scoop the wax slice onto a glass slide, control the moisture with filter paper, and store at room temperature.

[0063] HE staining of paraffin sections: ① Section preparation: bake at 60℃ for 15min; ② Dewaxing and rehydration: place in xylene I (10min) → xylene II (10min) → 100% ethanol I (10min) → 100% ethanol II (10min) → 95% ethanol (5min) → 90% ethanol (5min) → 80% ethanol (5min); ③ Staining: hematoxylin staining (2min) → rinse with running water → 1% hydrochloric acid alcohol color separation → wash with water and reverse blue (2min) → eosin staining (5min); ④ Dehydration and fixation: 80% ethanol (10-20s) → 90%-95% ethanol (10-20s) → 100% ethanol I (10-20s) → 100% ethanol II (10-20s) → xylene I (1-2min) → xylene II (1-2min); ⑤ Sealing: use neutral gum to seal the slides.

[0064] 2.3.2 Results

[0065] The results of animal experiments can be found in Figure 3-Figure 5 After being fed a high-fat diet for 12 consecutive weeks, the mice gained weight significantly compared with the blank group, with an average weight increase of 21.93% compared with the blank group. The nutmeg group A, the sour jujube group B and the combination group all significantly reduced the weight of obese mice (P < 0.01 or P < 0.05), among which the combination group had the largest reduction, which was 19.8% lower than the model group.

[0066] 2.4 Effects of the composition on abnormal lipid metabolism in 3T3-L1 adipocytes

[0067] 2.4.1 Methods

[0068] (1) 3T3-L1 cell culture

[0069] 3T3-L1 cells were cultured using 10% FBS+1% PP+89% high-glucose DMEM culture medium. When the cell confluence reached about 80%, trypsin digestion was used for subculture.

[0070] (2) Preparation of adipocyte differentiation medium

[0071] Prepare insulin solution: the concentration of the storage solution is 10 mg / ml, which is diluted 1000 times when inducing differentiation;

[0072] Preparation of dexamethasone solution (Dex): The concentration of the stock solution is 2.5 mmol / L, and it is diluted 2500 times when inducing differentiation;

[0073] Preparation of 3-isobutyl-1-methylxanthine (IBMX): The concentration of the stock solution is 0.5 mol / L, and it is diluted 1000 times when inducing differentiation;

[0074] After the above three solutions are prepared, they are filtered, sterilized, and stored in a -20°C refrigerator.

[0075] Differentiation solution A: complete medium 100 ml + Insulin (10 μg / ml) 60 μL + IBMX (0.5 mmol / L) 100 μL + Dex solution (1 μmol / L) 40 μL;

[0076] Differentiation solution B: 100 ml complete culture medium + 60 μL Insulin solution (10 μg / ml).

[0077] (3) 3T3-L1 cell differentiation induction

[0078] When the cells grew to about 90% digestion, the cell suspension was counted and inoculated into a well plate at 5*104 / ml. After 24 hours, the medium was replaced with 5% FBS+1% PP+94% high glucose DMEM to start induction.

[0079] Differentiation medium A was used for 48 hours, then replaced with new differentiation medium A for another 48 hours, followed by differentiation medium B for 48 hours, and then complete medium for 48 hours to complete the induction of differentiation. The whole process takes about 10 days.

[0080] (4) Modeling and drug administration

[0081] A. Drug preparation: BSA solution: Take 3 ml of PBS preheated to 55°C, add 0.6 g of BSA to dissolve and let stand, add 3 ml of pre-prepared NaOH solution (0.1 mol / l) to obtain a 10% BSA solution, filter and divide into packages, and store at 4°C / -20°C.

[0082] PA (palmitic acid) solution: Take 3 ml of PBS preheated to 55°C, add 1.2 g of BSA to dissolve and let stand to obtain 40% BSA (brown and clear). Take 3 ml of NaOH (0.1 mol / l), add 0.0307 g of PA, heat to 75°C, maintain the temperature for 30 min, and obtain 40 mM PA solution (colorless and clear). Maintain the temperature at 75°C, add 3 ml of 40% BSA solution, and obtain 20 mM PA solution (clear brown and yellow), cool at room temperature, filter and aliquot, and store at 4°C / -20°C.

[0083] B. Dosage and time of administration

[0084] Control group: administered with an equal volume of solvent, i.e., 10ul 10% BSA solution, for 24h.

[0085] Model group: PA administration concentration was 0.3mM, i.e. 7.5ul of 20mM PA stock solution was added, and the administration time was 24h.

[0086] Nutmeg A-containing intestinal fluid (RDK-IAS) group: 15 times the clinical equivalent dose, each intestinal segment was mixed for 2 hours and then diluted 20 times, RDK-IAS 10ul, and acted for 24 hours.

[0087] Suanzaorbitol B-containing intestinal fluid (SZ-IAS) group: 15 times the clinical equivalent dose, each intestinal segment was mixed for 2 hours and then diluted 20 times, RDK-IAS 10ul, and the administration time was 24 hours.

[0088] Combination (combination-IAS) group: 15 times clinical equivalent dose, each intestinal segment was mixed for 2 hours and then diluted 20 times, RDK-IAS 10ul, and the administration time was 24 hours.

[0089] (5) Oil red staining to detect lipid content

[0090] A. Pre-cool paraformaldehyde and PBS.

[0091] B. Aspirate the culture medium in the well plate into an eppendorf tube / centrifuge tube (for indirect co-culture) and rinse three times with cold PBS.

[0092] C. Fix with precooled paraformaldehyde for 30 minutes and rinse with distilled water three times.

[0093] D. Add oil red dye solution and dye for 30 minutes (the oil red dye solution is configured as saturated oil red solution: distilled water = 3:2), and rinse with distilled water 3 times.

[0094] E. After adding isopropanol to dissolve for 10 minutes, transfer the liquid to an ep tube / 15ml centrifuge tube, add 150ul / well to a 96-well plate (three replicate wells per group, make sure to mix well), and measure the OD value at a wavelength of 490nm.

[0095] 2.4.2 Results

[0096] The results of cell experiments are shown in Figure 6 The lipid content in the PA model group increased significantly, while the lipid content in adipocytes and lipid accumulation were significantly reduced in the RDK-IAS, SZ-IAS, and combination-IAS groups, with the SZ-IAS and combination-IAS groups showing the greatest reductions.

[0097] In summary, the composition of the present invention can significantly reduce the body weight of obese mice and promote browning of adipose tissue in obese mice; at the same time, the composition and its effective monomers can significantly reduce lipid metabolism disorders in 3T3-L1 adipocytes, and can be used for the auxiliary treatment of diseases related to obesity, fatty liver and metabolic disorders.

[0098] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. An extract composition for improving lipid metabolism, characterized in that: The composition is obtained by mixing CO2 supercritical extract of nutmeg with 70% ethanol extract of jujube.

2. The extract composition for improving lipid metabolism according to claim 1, characterized in that: The mass ratio of the nutmeg to the sour jujube is 1:

1.

3. The extract composition for improving lipid metabolism according to claim 1, characterized in that: The extraction conditions of the CO2 supercritical extract of nutmeg are: The extraction pressure is 10-15 MPa, the temperature is 40-50°C, the separation 1 pressure is 6-8 MPa, and the separation 2 pressure is 4-6 MPa.

4. The extract composition for improving lipid metabolism according to claim 1, characterized in that: The extraction conditions of the 70% ethanol extract of sour jujube are as follows: 70% ethanol extraction for 2 times, each time for 2 hours.

5. The method for preparing an extract composition for improving lipid metabolism according to any one of claims 1 to 4, characterized in that: The steps include: S1. Preparation of CO2 supercritical extract of nutmeg: After crushing the nutmeg, extract it for 2 hours under the conditions of pressure 10 MPa, extraction temperature 40°C, separation 1 pressure 7 MPa, separation 2 pressure 5 MPa; then, extract it for 1 hour under the conditions of pressure 15 MPa, extraction temperature 40°C, separation 1 pressure 7 MPa, separation 2 pressure 5 MPa, and collect the extract to obtain; S2. Preparation of 70% ethanol extract of wild jujube: Crush the sour jujube, soak it in 70% ethanol overnight, extract it twice with 8 times the amount of 70% ethanol, each time for 2 hours, filter it, combine the filtrates, concentrate it under reduced pressure to a thick paste with a relative density of 1.15-1.2, dry it under reduced pressure, crush it, and pass it through an 80-mesh sieve to obtain the product; S3. Preparation of the composition: The obtained CO2 supercritical extract of nutmeg and 70% ethanol extract of sour jujube were mixed in a ratio of 1:1 based on the mass ratio of nutmeg to sour jujube.

6. Use of the extract composition for improving lipid metabolism as claimed in any one of claims 1 to 4 in the preparation of medicines for preventing and treating obesity and / or non-alcoholic fatty liver disease.

7. Use of the extract composition for improving lipid metabolism as claimed in any one of claims 1 to 4 in a drug for lowering blood lipids.

8. Use of the extract composition for improving lipid metabolism as claimed in any one of claims 1 to 4 in a medicine for reducing lipid metabolism disorders in 3T3-L1 adipocytes.

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