Application of kochia saponin Ic in preparation of weight-losing and lipid-lowering medicine or functional food

Abstract

The invention discloses a new application of momordin Ic (momordin Ic) as shown in a formula I or pharmaceutically acceptable salt, ester, solvate or prodrug thereof, including application in preparation of weight-losing and / or lipid-lowering drugs or functional food and application in preparation of adipocyte differentiation inhibitors. The invention further provides a pharmaceutical composition which comprises the kochia saponin Ic shown in the formula I or pharmaceutically acceptable salts, esters, solvates or prodrugs of the kochia saponin Ic and pharmaceutically acceptable auxiliary materials, the pharmaceutical composition is used as a pharmaceutical preparation, and the medicine is a weight-losing and / or lipid-lowering medicine.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of Kochia scoparia saponin Ic in the preparation of drugs, particularly in the preparation of weight loss and / or lipid-lowering drugs. This invention also provides the application of Kochia scoparia saponin Ic in inhibiting adipocyte differentiation. Background Technology

[0002] Obesity is a global public health problem, often considered a risk factor for a variety of diseases, including diabetes, cancer, non-alcoholic fatty liver disease, cardiovascular disease, and mental health issues. Currently, in many regions and countries worldwide, the mortality rate from obesity is significantly higher than that of people maintaining a normal weight. Research by The Lancet Commission indicates that obesity affects more than 2 billion people globally, and predicts that by 2025, approximately 124 million children and adolescents will be classified as obese. The prevalence of obesity is increasing annually, with its economic impact estimated at 2.19% of global GDP in 2019, and projected to rise to 3.29% by 2060.

[0003] As the core unit of fat storage and metabolism, adipocytes are closely related to the regulation of their differentiation process in terms of fat accumulation and blood lipid balance. The core goal of weight loss is to reduce the total amount of body fat, which depends on the number of adipocytes and the volume of individual adipocytes. Studies have shown that in obese animal models, the use of specific adipocyte differentiation inhibitors significantly reduces the number of newly formed adipocytes in adipose tissue, resulting in a decrease in both adipose tissue weight and total lipid content. Compared with simply reducing adipocyte volume, reducing the number of adipocytes is more conducive to maintaining long-term weight loss. Furthermore, after preadipocyte differentiation is inhibited, they may differentiate into cell types with higher energy consumption, such as muscle cells, thereby increasing energy expenditure in the body. Inhibiting adipocyte differentiation may also enhance the lipolysis capacity of existing adipocytes, promoting the breakdown of stored triglycerides into fatty acids and glycerol, which are released into the bloodstream for use by other tissues, further reducing fat reserves. Therefore, the development of novel adipocyte differentiation inhibitors has broad application prospects and social significance. Summary of the Invention

[0004] This invention has unexpectedly discovered a new use for Kochia scoparia saponin Ic. Specifically, this invention has discovered the application of Kochia scoparia saponin Ic in the preparation of weight-loss and / or lipid-lowering drugs or functional foods. This invention has unexpectedly discovered that Kochia scoparia saponin Ic can inhibit adipocyte differentiation and can be used to prepare adipocyte differentiation inhibitors, lipid-lowering drugs or functional foods, and weight-loss drugs or functional foods.

[0005] To achieve this objective, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides the use of Kochia scoparia saponin Ic of Formula I or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof in the preparation of weight loss and / or lipid-lowering drugs or functional foods:

[0007]

[0008] Preferably, the weight loss refers to reducing the weight of obese or overweight patients, and the lipid reduction refers to lowering the levels of triglycerides, cholesterol, and low-density lipoprotein in the blood or peripheral tissues of patients with hyperlipidemia.

[0009] Preferably, the drug or functional food further comprises pharmaceutically or food-acceptable excipients, including any one or a combination of at least two of the following: carrier, diluent, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0010] Preferably, the dosage form of the drug or functional food is a tablet, capsule, injection, granule, or oral liquid.

[0011] Preferably, the daily dosage of Kochia scoparia saponin Ic is 0.01–10 mg / kg based on body weight.

[0012] This invention unexpectedly discovered that Kochia scoparia saponin Ic, represented by Formula I, can inhibit adipocyte differentiation and can be applied to the prevention and treatment of obesity, hyperlipidemia, and their corresponding complications. Through experimental research, the inventors found that Kochia scoparia saponin Ic exhibits significant inhibitory activity against adipocyte differentiation at a concentration of 20 μM, and can be used to prepare weight-loss and / or lipid-lowering drugs.

[0013] Secondly, the present invention provides the use of Kochia scoparia saponin Ic of Formula I or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof in the preparation of adipocyte differentiation inhibitors:

[0014]

[0015] Thirdly, the present invention provides a pharmaceutical composition comprising one or more of the following: Kochia scoparia saponin Ic of Formula I or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof, and a pharmaceutically acceptable excipient, for use as a pharmaceutical preparation, wherein the pharmaceutical preparation is a weight-loss or lipid-lowering drug:

[0016]

[0017] Preferably, the composition contains 0.001 to 99 wt% of the said Kochia scoparia saponin Ic or its pharmaceutically acceptable salt, ester, solvate or prodrug, based on the total weight of the composition;

[0018] Preferably, the daily dosage of Kochia scoparia saponin Ic is 0.01–10 mg / kg based on body weight.

[0019] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carrier, diluent, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer, such as a combination of binder and diluent, a combination of disintegrant and filler, a combination of disintegrant, filler, and lubricant, etc. Other arbitrary combinations will not be elaborated here.

[0020] Preferably, the pharmaceutical preparation is a tablet, capsule, injection, granule, or oral liquid. The Kochia scoparia saponin Ic or its pharmaceutically acceptable salt, ester, solvate, or prodrug of this invention can also be used in combination with other drugs to achieve better weight loss and lipid-lowering effects.

[0021] In this invention, the route of administration of the drug can be selected from any one of oral administration, sublingual administration, intravenous injection, intramuscular injection or subcutaneous injection, depending on actual needs.

[0022] This invention unexpectedly discovered that Kochia scoparia saponin Ic, as shown in Formula I, or its pharmaceutically acceptable salts, esters, solvates, or prodrugs, can inhibit adipocyte differentiation and can be used to prepare weight-loss and / or lipid-lowering drugs or functional foods, especially suitable for preparing drugs to prevent or treat obesity and hyperlipidemia, and beneficial to lipid metabolism. The compound exhibits significant inhibitory activity against adipocyte differentiation at a concentration of 20 μM. This compound is widely found in traditional Chinese medicines such as Kochia scoparia and has high safety. Therefore, Kochia scoparia saponin Ic holds promise for development into a new generation of clinical weight-loss and / or lipid-lowering drugs or functional foods. Attached Figure Description

[0023] Figure 1 (A) A representative image of 3T3-L1 adipocytes at the end of differentiation. (B) Cell differentiation process.

[0024] (C) Relative TG content in cell supernatant collected 72 hours after MIX addition (Day 5). (D) Relative TG content in cell supernatant collected 24 hours after insulin addition (Day 6). (E) Absorbance of stained cells measured at 492 nm after Oil Red O extraction. Each value is shown as the mean ± SD of three independent tests; significant differences between groups and the model group are represented by: ***P < 0.001.

[0025] Figure 2(A) Time-based mouse body weight comparison analysis results. (B) Mouse body weight change rate results. Note:

[0026] Compared with the model control group, *P<0.05, **P<0.01; ***P<0.001.

[0027] Figure 3 (A) Serum cholesterol (TC) in mice. (B) Triglyceride (TG) levels. Note: Compared with the model control group, *P<0.05, **P<0.01; ***P<0.001.

[0028] Figure 4 : Pathological section of liver tissue.

[0029] Figure 5 (A) Oil Red O staining of mouse liver tissue. (B) Data analysis results of Oil Red O staining of mouse liver tissue. Note: *P<0.05 compared with the model control group. Detailed Implementation

[0030] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0031] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as mentioned below, are all common knowledge and general knowledge in the field, and this invention does not have any particular limitations. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer.

[0032] Unless otherwise stated, all technical terms and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. However, in the event of any conflict, the specification containing the definitions shall prevail.

[0033] Kochia scoparia was purchased from Yunnan Lvsheng Pharmaceutical Co., Ltd.; all chemical reagents were chromatographic, analytical, or industrial grade, purchased from Kunming Fuhaida Chemical Glass Instrument Co., Ltd. (China) and Beijing Mairuida Technology Co., Ltd. (China); 3T3-L1 preadipocytes were purchased from American Type Culture Collection; high-glucose DMEM, low-glucose DMEM, penicillin / streptomycin (P / S), insulin, fetal bovine serum (FBS), newborn calf serum (NBCS), and phosphate-buffered saline (PBS) were purchased from Biological Industries; 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone (DEX) were purchased from Sigma-Aldrich; rosiglitazone (Rosi) was purchased from Meilun Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Solarbio; and Oil Red O dye was purchased from Nanjing Jiancheng Technology Co., Ltd.

[0034] Example 1

[0035] Preparation of Kochia saponin Ic

[0036] 20 kg of dried Kochia scoparia fruit was extracted four times at room temperature using 80% industrial ethanol for 7 days, 3 days, 3 days, and 1 day respectively. The extracts were combined and concentrated under reduced pressure to obtain 2 kg of Kochia scoparia fruit ethanol extract. The 2 kg of Kochia scoparia fruit ethanol extract was dissolved in warm water by stirring until completely dissolved. The mixture was then stirred with 1:1 volume of 80-100 mesh silica gel, allowed to air dry, and ground until it reached a slurry consistency. The solution was then loaded onto a column. Elution was performed sequentially using petroleum ether, ethyl acetate, and ethanol until all the sample was flushed off. The solution was concentrated under reduced pressure to obtain 150 g of the petroleum ether fraction, 260 g of the ethyl acetate fraction, and 1400 g of the ethanol fraction. These three fractions were stored for later use. 1.4 kg of the ethanol fraction was dissolved in a 20% ethanol aqueous solution and centrifuged at 5000 rpm for 15 min. The supernatant was removed, and the lower sediment (100 g) was used as the crude extract for the subsequent preparation of Kochia scoparia fruit saponin Ic. The lower sediment sample (100g) was fully dissolved in industrial ethanol by heating and then filtered. The filtrate was evaporated to dryness, ground into powder, and finely sieved to obtain the sample to be purified (86g) and insoluble impurities that could not be filtered out (13g).

[0037] The sample to be purified (86g) was dried and ground into powder, then sieved through a 60-mesh sieve. Five times its volume of anhydrous ethanol (86×5mL) was added to dissolve the powder, and recrystallization was performed. The mixture was then filtered to obtain a mother liquor and a filter residue (50.7g). This recrystallization process was repeated four times, followed by final filtration to obtain a mother liquor and a filter residue (30g). The purity of Kochia scoparia saponin Ic in the 30g filter residue was analyzed by liquid chromatography, and the purity was found to be 79.10%.

[0038] 30g of the sample to be purified was dissolved in 1L of anhydrous ethanol in a water bath at 65℃ for 1 hour, followed by ultrasonic cooling. The solution was then centrifuged at 5000rpm for 10 minutes. The supernatant was mixed with 80-100 mesh silica gel. The lower sediment was subjected to the same dissolution, centrifugation, and mixing process until all 30g of sample was dissolved and a final 60g mixed sample was obtained. The 60g mixed sample was air-dried, ground to a slurry consistency, and then loaded onto a column. The polarity ratio of the eluents was determined using thin-layer chromatography, and V was selected as the initial eluent. 正己烷 V 乙酸乙酯 V 乙酸 A gradient elution of 1.2:0.8:0.1 (elution volume 23.5 L) was performed to remove impurities of low polarity; then V... 正己烷 V 乙酸乙酯 V 乙酸 A gradient elution of 1:1:0.1 (elution volume 28 L) was performed to remove impurities on Kochia scoparia saponin Ic. Finally, a gradient elution was performed using the same elution solvent from the second elution (2 L) + ethyl acetate (200 mL) + acetic acid (40 mL) (total elution volume 62 L). Kochia scoparia saponin Ic began to appear at elution volume 8 L; a large amount of Kochia scoparia saponin Ic appeared at elution volume 22 L, until Kochia scoparia saponin Ic was completely eluted. The eluted fraction was concentrated under reduced pressure using a rotary evaporator to obtain a Kochia scoparia saponin Ic sample (17 g, purity 92.26%).

[0039] A 92.26% purity sample of Kochia scoparia saponin Ic was recrystallized, filtered, and the resulting mother liquor and filter residue were obtained. The filter residue was dissolved in redistilled anhydrous ethanol and concentrated under reduced pressure to obtain 5.69 g of purified Kochia scoparia saponin Ic sample, with a purity of 97.14% according to liquid chromatography analysis.

[0040] Example 2

[0041] Evaluation of adipocyte differentiation inhibition activity

[0042] Experimental Methods: Cultured 3T3-L1 preadipocytes were incubated with appropriate amounts of trypsin and collagenase at 37°C in a 5% CO2 incubator for 3 min. After adding an appropriate amount of NBSC culture medium and shaking to mix the cells, the cells were seeded into 24-well plates and cultured overnight. Samples were added to the cell culture plates (500 μL), and the blank control group was treated with the corresponding concentration of DMSO. Each sample was replicated in 3 wells. Once the 3T3-L1 preadipocytes had completely differentiated into adipocytes, the adipocytes were stained with Oil Red O. First, the culture medium for the 3T3-L1 cells was removed, and the cells were washed twice with PBS. The cells were then fixed in 10% formaldehyde at room temperature for 10 min, the 10% formaldehyde was discarded, and fresh 10% formaldehyde was added for 1 h of fixation. After fixation, the cells were washed once with 60% isopropanol. After washing, the cells were stained with Oil Red O working solution (V... 储备液 V 稀释液 3T3-L1 adipocytes were stained at room temperature for 30 min using a 5:2 ratio. After staining, the adipocytes were washed five times with water to remove non-specifically bound dye, and then photographed under a microscope. Finally, to quantify the number of lipid droplets, the stained 3T3-L1 adipocytes were washed with 100% isopropanol, and the dissolved oil red O was measured at 492 nm using a microplate reader.

[0043] After the glucose uptake assay, all the culture medium was aspirated, and 15 μL of CellTiter was added to each well. Aqueous solution for cell proliferation assay. After incubation at 37°C and 5% CO2 for 8 hours, absorbance is measured at 490 nm using an ELISA reader to determine the cytotoxicity of the compound to adipocytes. Relative cell viability is calculated using the following formula:

[0044]

[0045] Where A1 represents the absorbance of the blank control; A2 represents the absorbance of the positive control or sample group.

[0046] See results Figure 1 After treating 3T3-L1 preadipocytes with 20 μM Kochia scoparia saponin Ic, the number of yellow lipid droplets in the cells was significantly reduced. Figure 1 A). This phenomenon can be observed more directly by staining lipid droplets in adipocytes with Oil Red O at the end of differentiation. Figure 1 B). By detecting the relative content of triglycerides (TG) in the supernatant of adipocytes, we found that during cell differentiation, the relative content of TG in the supernatant of adipocytes treated with 20 μM Kochia scoparia saponin Ic was significantly lower than that in the model control group (P < 0.001). Figure 1(C,D). Oil Red O was used to stain mature adipocytes that had completed differentiation. After extracting Oil Red O, the absorbance of the stained cells was measured at 492 nm. We found that the absorbance of cells treated with 20 μM Kochia scoparia saponin Ic was significantly lower than that of the model group, indicating that Kochia scoparia saponin Ic at 20 μM has an inhibitory effect on adipocyte differentiation (P<0.001). Figure 1 E).

[0047] Example 3

[0048] In vivo animal experiments: Using an obese db / db mouse model, Kochia scoparia saponin Ic was administered as an intervention to observe the effects of Kochia scoparia saponin Ic on body weight, serum triglyceride and cholesterol levels, and liver tissue lipid metabolism in db / db mice, thereby comprehensively evaluating the effect of Kochia scoparia saponin Ic on glucose and lipid metabolism in db / db mice.

[0049] Experimental Results: The results showed that the body weight of mice in both the Kochia scoparia saponin Ic-30 mg / kg BW and Kochia scoparia saponin Ic-60 mg / kg BW groups was lower than that of the model control group, especially the Kochia scoparia saponin Ic-60 mg / kg BW group. Notably, the body weight of mice in the Kochia scoparia saponin Ic-60 mg / kg BW group decreased significantly during the administration period. Figure 2 A). In addition, through Figure 2 As shown in Figure B, at week 4 of treatment, the body weight of mice in the model group, metformin group, Kochia scoparia saponin Ic-30 mg / kg BW dose group, and Kochia scoparia saponin Ic-60 mg / kg BW dose group increased by 14.09%, 22.02%, 8.91%, and 1.74%, respectively (compared to day 0). This indicates that Kochia scoparia saponin Ic can play a role in controlling body weight in obese diabetic mice.

[0050] To evaluate the effect of Kochia scoparia saponin Ic on improving lipid metabolism disorders, we measured serum cholesterol (TC) in db / db mice. Figure 3 A) and triglycerides (TG) Figure 3 B) levels. Compared with the model group, metformin and Kochia scoparia saponin Ic both significantly reduced the level of TG in mouse serum (P<0.05).

[0051] H&E staining of paraffin sections of liver tissue samples from db / db mice revealed numerous lipid droplet vacuoles in the liver cells of the model group mice. (Lipid droplets in adipocytes are dissolved by H&E; these droplets occupy a large portion of the adipocyte volume, resulting in vacuolated cells.) After drug treatment, the number of vacuoles in the liver cells of all groups of mice significantly decreased, especially in the Kochia scoparia saponin Ic-60 mg / kg BW dose group. Comprehensive analysis indicates that Kochia scoparia saponin Ic has an effect in improving hepatic steatosis. Figure 4 ).

[0052] Oil Red O staining is mainly used to visualize steatosis and abnormal lipid deposition in tissues and organs. Oil Red O staining results in mouse livers showed large red areas in the liver cells of the model group mice. Figure 5 A). Semi-quantitative analysis of the fat content using ImageJ software revealed that, compared with the model control group, the metformin hydrochloride group and the Kochia scoparia saponin Ic-60mg / kg BW dose group showed significantly reduced hepatic steatosis, with statistically significant differences (P<0.05, P<0.001). Figure 5 B). Oil Red O staining of liver tissue further confirmed that Kochia scoparia saponin Ic improves liver lipid metabolism in diabetic mice.

[0053] Example 4

[0054] Tablets: Mix 1g of compound momordin Ic with 0.6g of lactose and 0.5g of starch, moisten with water, sieve and dry the moistened mixture, sieve again, add 0.1g of magnesium stearate, mix well, compress into tablets, and compress into 20 tablets. Tablet weight: 110mg, content: 50mg / tablet.

[0055] Example 5

[0056] Capsules: Mix 1g of compound momordin Ic with 0.5g of lactose and 0.1g of magnesium stearate until homogeneous, sieve, mix evenly, and fill the resulting mixture into 20 hard gelatin capsules. Capsule weight: 80mg, content: 50mg / capsule.

[0057] Example 6

[0058] Oral ampoules: Add 1g of compound momordin Ic to the conventional additives and purified water used in the preparation of the oral solution, make up to 0.1L, and fill into 20 ampoules under aseptic conditions, each ampoule containing 5ml, with a content of 50mg / ampoule.

[0059] Example 7

[0060] Granules: Mix 1g of compound momordin Ic with 1g of lactose and 0.6g of starch evenly, moisten with water, sieve the moistened mixture and dry it, sieve it again, and then make the mixture into granules and pack them into 20 bags, each bag weighing 130mg, with a content of 50mg / bag.

[0061] The above descriptions are merely several embodiments of this application and are not intended to limit this application in any way, i.e., they do not imply that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of raw materials for the product of the present invention, additions of auxiliary components, and selection of specific methods, all fall within the protection and disclosure scope of the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. This invention discloses the application of momordin Ic (as shown in Formula I) or its pharmaceutically acceptable salts, esters, solvates or prodrugs in the preparation of weight-loss and / or lipid-lowering drugs or functional foods:

2. The application according to claim 1, characterized in that, The weight loss refers to reducing the weight of obese or overweight patients, and the lipid reduction refers to lowering the levels of triglycerides, cholesterol, and low-density lipoprotein in the blood or peripheral tissues of patients with hyperlipidemia.

3. The application according to any one of claims 1 and 2, characterized in that, The aforementioned drugs or functional foods exert their weight loss and / or lipid-lowering effects by inhibiting adipocyte differentiation.

4. The application according to any one of claims 1 and 2, characterized in that, The drug or functional food also includes one or more pharmaceutically or food-acceptable excipients.

5. The application according to claim 4, characterized in that, The dosage form of the drug or functional food is a capsule, granule, tablet, or oral liquid preparation.

6. The use of Kochia scoparia saponin Ic as shown in Formula I or its pharmaceutically acceptable salt, ester, solvate or prodrug in the preparation of adipocyte differentiation inhibitors.

7. A pharmaceutical composition comprising one or more of the following: Kochia scoparia saponin Ic of Formula I or a pharmaceutically acceptable salt, ester, solvate or prodrug thereof, and a pharmaceutically acceptable excipient, for use as a pharmaceutical preparation, wherein the pharmaceutical preparation is a weight-loss and / or lipid-lowering drug.

8. The pharmaceutical composition according to claim 7, characterized in that, The composition contains 0.001 to 99 wt% of the said Kochia scoparia saponin Ic or its pharmaceutically acceptable salt, ester, solvate or prodrug, based on the total weight of the composition.

9. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical preparation is a tablet, capsule, injection, granule, or oral liquid.

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