Active peptide of black ginseng and preparation method and application thereof

Abstract

Black ginseng active peptide and its preparation method and application, belong to the field of biological medicine, in order to solve the ability to promote the transformation of white adipocytes to beige adipocytes, and increase the number of mitochondria to achieve the purpose of weight loss, the amino acid sequence is Asp-Tyr-Ala-Gly-Leu-Tyr-Thr-Lys, which can be used for preparing weight loss drugs, food or health products.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a black ginseng active peptide and its preparation method, as well as a composition based on the active peptide. Background Technology

[0002] Obesity has become a global public health problem. It is closely related to the development of many chronic diseases, such as cardiovascular disease, type 2 diabetes, osteoarthritis, and certain cancers. Numerous clinical studies have shown that obese people have a risk of cardiovascular disease several times higher than people of normal weight, and about 80% of patients with type 2 diabetes were obese before the onset of the disease.

[0003] In recent years, studies have focused on the mechanism of white fat conversion to brown fat, aiming to increase fat consumption and improve energy metabolism. Fucoxanthin can activate PPARγ in white adipocytes, thereby activating the expression of the transmembrane uncoupling protein UCP1 in the mitochondria of brown adipocytes, inducing browning of white adipocytes and forming beige adipocytes. Simultaneously, it promotes the mRNA expression of the β3-adrenergic receptor Adrb3 in white adipose tissue, increasing lipolysis and non-shivering thermogenesis. However, fucoxanthin has a weak direct effect on UCP1 in mature brown adipocytes, limiting its ability to convert white adipocytes to beige adipocytes. Summary of the Invention

[0004] In order to address the ability to promote the transformation of white adipocytes into beige adipocytes and increase the number of mitochondria to achieve fat reduction, according to some embodiments of this application, the active peptide has the amino acid sequence shown in SEQ ID NO. 1.

[0005] According to the preparation method of the active peptide in some embodiments of this application, including

[0006] S1. The mixture of black ginseng powder and water is subjected to a first enzymatic hydrolysis, wherein a complex protease is added during the first enzymatic hydrolysis. The complex protease is composed of alkaline protease and flavor protease.

[0007] S2. The liquid obtained based on the first enzymatic hydrolysate is subjected to a second enzymatic hydrolysis, wherein a complex enzyme is added to the second enzymatic hydrolysis. The complex enzyme is composed of β-glucosidase A, β-glucosidase B and α-L-arabinosidase.

[0008] S3. The liquid obtained from the second enzymatic hydrolysis was passed through a 20 mm × 100 mm Sephadex G-20 gel chromatography column, and the eluent was collected over a period of 8 to 9 minutes; wherein, 0.05 mol / L phosphate buffer was used as the eluent and the flow rate was 0.8 mL / min.

[0009] S4. The eluent is passed through a 250 mm × 4.6 mm, 5 μm C18 column, and the elution peaks with a time of 11-12 min are collected to obtain the active peptide; wherein the mobile phase is acetonitrile and water, the volume ratio of acetonitrile to water is 25:75, the detection wavelength is 220 nm, and the flow rate is 1 mL / min.

[0010] According to the preparation method of the mixture of black ginseng active peptides in some embodiments of this application, including

[0011] S1. The aqueous mixture of black ginseng powder is subjected to a first enzymatic hydrolysis, wherein a complex protease is added during the first enzymatic hydrolysis. The complex protease is composed of an alkaline protease and a flavor protease.

[0012] S2. The liquid obtained based on the first enzymatic hydrolysate is subjected to a second enzymatic hydrolysis, wherein a complex enzyme is added to the second enzymatic hydrolysis. The complex enzyme consists of β-glucosidase A, β-glucosidase B, and α-L-arabinosidase.

[0013] S3. The liquid obtained from the second enzymatic hydrolysis is filtered and dried to obtain a mixture of black ginseng active peptides, including black ginseng active peptides and ginsenoside CK.

[0014] According to the active peptide preparation method or the preparation method of a mixture of black ginseng active peptides in some embodiments of this application, the amount of compound protease added is 3% of the mass of black ginseng powder, and the mass ratio of alkaline protease to flavor protease is 4:1.

[0015] According to the method for preparing active peptides or the method for preparing a mixture of active peptides of black ginseng in some embodiments of this application, the enzymatic hydrolysis temperature of the first enzymatic hydrolysis is 45~55℃, the enzymatic hydrolysis pH is 7.5~8.5, and the enzymatic hydrolysis time is 4~6h.

[0016] According to the method for preparing active peptides or a mixture of active peptides from black ginseng in some embodiments of this application, the first enzymatic hydrolysis is performed at a temperature of 45°C, a pH of 7.5, and a time of 5 hours.

[0017] According to the active peptide preparation method or the preparation method of a mixture of black ginseng active peptides in some embodiments of this application, the amount of enzyme added to the complex enzyme is 1% of the mass of black ginseng powder, and the mass ratio of β-glucosidase A, β-glucosidase B and α-L-arabinosidase is 4:3:2.

[0018] According to the active peptide preparation method or the preparation method of a mixture of black ginseng active peptides in some embodiments of this application, the enzymatic hydrolysis temperature of the second enzymatic hydrolysis is 45~50℃, the enzymatic hydrolysis pH is 4.5~5.5, and the enzymatic hydrolysis time is 4~6h.

[0019] According to the method for preparing active peptides or the method for preparing a mixture of active peptides from black ginseng in some embodiments of this application, the enzymatic hydrolysis temperature for the second enzymatic hydrolysis is 50°C, the enzymatic hydrolysis pH is 5.5, and the enzymatic hydrolysis time is 6 hours.

[0020] According to the methods for preparing active peptides or mixtures of black ginseng active peptides in some embodiments of this application, a liquid is obtained based on a first enzymatic hydrolysate, including...

[0021] The first enzymatic hydrolysate obtained from the first enzymatic hydrolysis, or

[0022] The first enzymatic hydrolysate is inactivated, cooled to room temperature, and centrifuged to obtain the supernatant.

[0023] According to the active peptide preparation method or the preparation method of the mixture of black ginseng active peptides in some embodiments of this application, the enzyme inactivation temperature is 90~105℃, the enzyme inactivation time is 10~15min, the centrifugation speed is 80000~10000rpm, and the centrifugation time is 15~25min.

[0024] According to the methods for preparing active peptides or mixtures of black ginseng active peptides in some embodiments of this application, the liquid obtained from the second enzymatic hydrolysis includes...

[0025] The second enzymatic hydrolysate obtained from the second enzymatic hydrolysis, or

[0026] The second enzyme hydrolysate is inactivated, cooled to room temperature, and filtered to obtain the third enzyme hydrolysate; or

[0027] A mixture of peptide powder and deionized water, wherein the mass-to-volume ratio of peptide powder to deionized water is 1:10 (w / v), and the peptide powder is prepared by drying the second or third enzymatic hydrolysate.

[0028] According to the active peptide preparation method or the preparation method of the mixture of black ginseng active peptides in some embodiments of this application, the enzyme inactivation temperature is 85~100℃ and the enzyme inactivation time is 10~15min.

[0029] The application of the active peptides in the preparation of weight-loss drugs according to some embodiments of this application, or the application of the method in the preparation of black ginseng active peptides and / or ginsenoside CK.

[0030] Beneficial effects:

[0031] In the preparation of active peptides in this invention, the saponin components in black ginseng are transformed by β-glucosidase A, β-glucosidase B, and α-L-arabinosidase. Specifically, β-glucosidase A can specifically hydrolyze the glycosyl groups of ginsenoside precursor saponins such as Rb1, Rb2, and RC to generate Rd, α-L-arabinosidase hydrolyzes arabinose groups to assist in the generation of Rd, and β-glucosidase B hydrolyzes saponin Rd to F2 and converts it into ginsenoside CK, thereby greatly increasing the yield of ginsenoside CK in the product.

[0032] In the preparation of the active peptide of this invention, the active peptide SEQ ID NO.1 was obtained from the black ginseng protein component through protease hydrolysis and further separation and purification. Its amino acid sequence is Asp-Tyr-Ala-Gly-Leu-Tyr-Thr-Lys. The active peptide can activate PRDM16 downstream of the NF-κB signaling pathway. Experiments show that its combination with fucoxanthin can synergistically promote the transformation of white adipocytes into beige adipocytes. It can also upregulate the expression of mitochondrial fusion protein (MFN2). Experiments show that it increases the number of mitochondria and can improve the fat-burning efficiency of beige adipocytes. Detailed Implementation

[0033] This invention relates to a mixture of black ginseng active peptides and its preparation. The method involves enzymatic hydrolysis using a complex protease and selective protease. In the preparation method, β-glucosidase A, β-glucosidase B, and α-L-arabinosidase convert the saponin components in black ginseng, increasing the content of ginsenoside CK in the final product. Black ginseng active peptides are then obtained from the black ginseng protein components through proteolytic hydrolysis, resulting in a product containing both black ginseng active peptides and ginsenoside CK. In this mixture, ginsenoside CK can activate brown adipocytes, while the black ginseng active peptides promote the transformation of white adipocytes into beige adipocytes. Furthermore, it increases the number of mitochondria, enhances the fat-burning efficiency of beige adipocytes, and increases energy consumption. Further separation and purification using chromatographic columns yield the specific black ginseng active peptide SEQ ID NO.1, which promotes the transformation of white adipocytes into beige adipocytes, with the amino acid sequence Asp-Tyr-Ala-Gly-Leu-Tyr-Thr-Lys. This peptide can activate PRDM16 downstream of the NF-κB signaling pathway and synergize with fucoxanthin to promote the transformation of white adipocytes into beige adipocytes. This peptide can also increase the number of mitochondria and enhance the fat-burning efficiency of beige adipocytes by upregulating the expression of mitochondrial fusion protein (MFN2). Based on this, the present invention combines one or more of these peptides with fucoxanthin to achieve fat reduction by activating brown adipocytes, promoting browning of white adipocytes, and enhancing mitochondrial function in beige adipocytes. The present invention further combines at least one of α-amylase inhibitors, prickly pear cactus extract, and allicin to comprehensively and synergistically target multiple points such as inhibiting sugar absorption and feedback regulation of appetite, thereby achieving the purpose of weight regulation.

[0034] The method for preparing the bioactive peptide disclosed herein includes:

[0035] S1. The mixture of black ginseng powder and water is subjected to a first enzymatic hydrolysis, wherein a complex protease is added during the first enzymatic hydrolysis. The complex protease is composed of alkaline protease and flavor protease.

[0036] S2. The liquid obtained based on the first enzymatic hydrolysate is subjected to a second enzymatic hydrolysis, wherein a complex enzyme is added to the second enzymatic hydrolysis. The complex enzyme is composed of β-glucosidase A, β-glucosidase B and α-L-arabinosidase.

[0037] S3. The liquid obtained from the second enzymatic hydrolysis was passed through a 20 mm × 100 mm Sephadex G-20 gel chromatography column, and the eluent was collected over a period of 8 to 9 minutes; wherein, 0.05 mol / L phosphate buffer was used as the eluent and the flow rate was 0.8 mL / min.

[0038] S4. The eluent was passed through a 250 mm × 4.6 mm, 5 μm C18 column, and the elution peak was collected at a time of 11-12 min to obtain the active peptide. The mobile phase was acetonitrile and water, with a volume ratio of acetonitrile to water of 25:75. The detection wavelength was 220 nm, and the flow rate was 1 mL / min.

[0039] In some embodiments, the amount of compound protease added is 3% of the mass of black ginseng powder, and the mass ratio of alkaline protease to flavor protease is 4:1. The enzymatic hydrolysis temperature for the first enzymatic hydrolysis is 45~55℃, the enzymatic hydrolysis pH is 7.5~8.5, and the enzymatic hydrolysis time is 4~6h. Preferably, the enzymatic hydrolysis temperature for the first enzymatic hydrolysis is 45℃, the enzymatic hydrolysis pH is 7.5, and the enzymatic hydrolysis time is 5h.

[0040] In some embodiments, the amount of the compound enzyme added is 1% of the mass of the black ginseng powder, and the mass ratio of β-glucosidase A, β-glucosidase B, and α-L-arabinosidase is 4:3:2. The enzymatic hydrolysis temperature for the second enzymatic hydrolysis is 45-50°C, the pH is 4.5-5.5, and the hydrolysis time is 4-6 hours. Preferably, the enzymatic hydrolysis temperature for the second enzymatic hydrolysis is 50°C, the pH is 5.5, and the hydrolysis time is 6 hours.

[0041] In some embodiments, the liquid obtained based on the first enzymatic hydrolysate includes the first enzymatic hydrolysate obtained from the first enzymatic hydrolysis, or the supernatant obtained by inactivating the enzyme in the first enzymatic hydrolysate, cooling it to room temperature, and centrifuging it; preferably, the enzyme inactivation temperature is 90~105℃, the enzyme inactivation time is 10~15min, the centrifugation speed is 80000~10000rpm, and the centrifugation time is 15~25min.

[0042] In some embodiments, the liquid obtained from the second enzymatic hydrolysis includes a second enzymatic hydrolysate obtained from the second enzymatic hydrolysis, or a third enzymatic hydrolysate obtained by inactivating the enzyme in the second enzymatic hydrolysate, cooling it to room temperature, and filtering it; or a mixture of peptide powder and deionized water, wherein the mass-to-volume ratio of peptide powder to deionized water is 1:10 (w / v), and the peptide powder is prepared by drying the second or third enzymatic hydrolysate; preferably, the enzyme inactivation temperature is 85~100℃, and the enzyme inactivation time is 10~15min.

[0043] The method for preparing a mixture of black ginseng active peptides disclosed herein includes:

[0044] S1. The aqueous mixture of black ginseng powder is subjected to a first enzymatic hydrolysis, wherein a complex protease is added during the first enzymatic hydrolysis. The complex protease is composed of an alkaline protease and a flavor protease.

[0045] S2. The liquid obtained based on the first enzymatic hydrolysate is subjected to a second enzymatic hydrolysis, wherein a complex enzyme is added to the second enzymatic hydrolysis. The complex enzyme consists of β-glucosidase A, β-glucosidase B, and α-L-arabinosidase.

[0046] S3. The liquid obtained from the second enzymatic hydrolysis is filtered and dried to obtain a mixture of black ginseng active peptides, including black ginseng active peptides and ginsenoside CK.

[0047] In some embodiments, the amount of compound protease added is 3% of the mass of black ginseng powder, and the mass ratio of alkaline protease to flavor protease is 4:1. The enzymatic hydrolysis temperature for the first enzymatic hydrolysis is 45~55℃, the enzymatic hydrolysis pH is 7.5~8.5, and the enzymatic hydrolysis time is 4~6h. Preferably, the enzymatic hydrolysis temperature for the first enzymatic hydrolysis is 45℃, the enzymatic hydrolysis pH is 7.5, and the enzymatic hydrolysis time is 5h.

[0048] In some embodiments, the amount of the compound enzyme added is 1% of the mass of the black ginseng powder, and the mass ratio of β-glucosidase A, β-glucosidase B, and α-L-arabinosidase is 4:3:2. The enzymatic hydrolysis temperature for the second enzymatic hydrolysis is 45-50°C, the pH is 4.5-5.5, and the hydrolysis time is 4-6 hours. Preferably, the enzymatic hydrolysis temperature for the second enzymatic hydrolysis is 50°C, the pH is 5.5, and the hydrolysis time is 6 hours.

[0049] In some embodiments, the liquid obtained based on the first enzymatic hydrolysate includes the first enzymatic hydrolysate obtained from the first enzymatic hydrolysis, or the supernatant obtained by inactivating the enzyme in the first enzymatic hydrolysate, cooling it to room temperature, and centrifuging it; preferably, the enzyme inactivation temperature is 90~105℃, the enzyme inactivation time is 10~15min, the centrifugation speed is 80000~10000rpm, and the centrifugation time is 15~25min.

[0050] In some embodiments, the liquid obtained from the second enzymatic hydrolysis includes a second enzymatic hydrolysate obtained from the second enzymatic hydrolysis, or a third enzymatic hydrolysate obtained by inactivating the enzyme in the second enzymatic hydrolysate, cooling it to room temperature, and filtering it. Preferably, the enzyme inactivation temperature is 85~100℃, and the enzyme inactivation time is 10~15min.

[0051] Example 1: Preparation of black ginseng peptide SEQ ID NO.1

[0052] Step 1: Black ginseng pretreatment. Select 5-year-old black ginseng, wash and dry it, then crush it through a 100-mesh sieve to obtain black ginseng powder. The preferred moisture content is ≤5%.

[0053] Step 2: Mix black ginseng powder and deionized water at a mass-to-volume ratio of 1:20 (w / v), add a compound protease at a mass ratio of 3% of the black ginseng powder, wherein the ratio of alkaline protease to flavor protease in the compound protease is 4:1 by mass. Enzymatically hydrolyze for 5 hours at 45℃ and pH 7.5. After hydrolysis, inactivate the enzyme at 90℃ for 15 minutes, cool to room temperature, centrifuge at 10,000 rpm for 20 minutes, and collect the supernatant to obtain crude black ginseng peptide solution. This crude black ginseng peptide solution contains protein peptides and various ginsenosides, such as Rb1, Rg3, and CK.

[0054] Step 3: Add a compound enzyme to the crude black ginseng peptide solution. The mass of the compound enzyme added is 1% of the mass of the black ginseng powder. The compound enzyme, by mass, has a ratio of β-glucosidase A:β-glucosidase B:α-L-arabinosidase = 4:3:2. React at 50℃ and pH 5.5 for 6 hours. After the reaction, raise the system temperature to 85℃ to inactivate the enzyme for 15 minutes, cool to room temperature (25℃), filter to remove the precipitate, and obtain a secondary enzymatic hydrolysate. After drying the secondary enzymatic hydrolysate, black ginseng peptide powder bgp1 containing a high content of saponin CK is obtained. HPLC analysis shows that the saponin CK content is 2.3%. β-glucosidase A specifically hydrolyzes the glycosyl groups of ginsenosides Rb1, Rb2, RC, and other precursor saponins to generate Rd. α-L-arabinosidase hydrolyzes arabinosyl groups to assist in the generation of Rd. β-glucosidase B hydrolyzes saponin Rd to F2 and converts it into ginsenoside CK, thereby increasing the yield of ginsenoside CK in the product.

[0055] Step 4: Purification of black ginseng peptide. 100g of black ginseng peptide powder from step 3 was mixed with deionized water at a ratio of 1:10 (w / v) and loaded onto a 20mm × 100mm Sephadex G-20 gel chromatography column. 0.05mol / L phosphate buffer was used as the eluent, preferably pH 7.0, at a flow rate of 0.8mL / min. The eluent with a retention time of 8-9min was collected. The eluent was then passed through a 250mm × 4.6mm, 5μm C18 column. The mobile phase was acetonitrile:water at a volume ratio of 25:75. The detection wavelength was 220nm, the flow rate was 1mL / min, and the elution peak with a retention time of 11-12min was collected.

[0056] Step 5: Freeze-dry the elution peak to obtain 1.28g of SEQ ID NO.1 peptide component with a purity of 96.3%. According to the calculation, the mass content of SEQ ID NO.1 peptide in black ginseng peptide powder is 1.23%.

[0057] Example 2: A composition for weight loss, by weight, comprising 0.8%–2.5% fucoxanthin, 0.6%–1.2% ginsenoside CK, 0.3%–0.6% black ginseng active peptides, 12%–30% α-amylase inhibitor, 6%–15% prickly pear cactus extract, 0.2%–0.6% allicin, with the balance being excipients such as maltodextrin and / or hydroxypropyl methylcellulose. Preferably, the composition contains 45.09%–72% maltodextrin and 5.01%–8% hydroxypropyl methylcellulose.

[0058] Fucoxanthin, with a preferred purity of ≥95%, has metabolites that are abundantly stored in white adipocytes. Fucoxanthin can upregulate the expression of brown adipose tissue-related genes (UCP1, PGC-1α), initiating browning. Ginsenoside CK can activate existing brown adipocytes; black ginseng active peptides synergistically promote the conversion of white adipose tissue to beige adipose tissue, increasing the number and size of mitochondria; α-amylase inhibitors, with a preferred enzyme activity of ≥2200 U / g, inhibit starch breakdown and reduce the synthesis of glycogenic fats. Prickly pear cactus extract, with a preferred polysaccharide content of ≥65%, regulates intestinal flora and enhances browning through short-chain fatty acids (SCFAs). Allicin, with a preferred purity of ≥95%, inhibits harmful intestinal bacteria and synergistically improves the metabolic microenvironment. Maltodextrin and hydroxypropyl methylcellulose are used as excipients to improve formulation stability and oral bioavailability.

[0059] The preferred embodiment of this product includes 2.0% fucoxanthin, 1.2% ginsenoside CK, 0.6% black ginseng active peptide, 25% α-amylase inhibitor, 12% prickly pear cactus extract, 0.5% allicin, 52.83% maltodextrin, and 5.87% hydroxypropyl methylcellulose.

[0060] The prickly pear cactus extract can be prepared as follows: Take prickly pear cactus dry powder, add deionized water at a material-to-liquid ratio of 1:18 (mass ratio), stir evenly to fully wet the powder; extract at a constant temperature of 80-90℃ for 2-3 hours, rapidly cool the extract to room temperature, collect the clear filtrate through a 0.22μm aqueous filter membrane, discard the residual filter residue, concentrate the clear filtrate under reduced pressure to a solid content of 15%-20%, and spray dry or freeze dry to obtain prickly pear cactus extract dry powder.

[0061] Among them, α-amylase inhibitors can be derived from white kidney beans.

[0062] Experiment 1. Verification of the conversion effect of ginsenoside CK.

[0063] In step 3 of Example 1, the secondary enzymatic hydrolysate was dried to obtain black ginseng peptide powder bgp1 containing a high content of saponin CK. In step 2 of Example 1, the crude black ginseng peptide solution was dried to obtain black ginseng peptide powder bgp2 (protease hydrolysis only). Following steps 1 and 2 of Example 1, in step 3, the complex enzyme combination was replaced with the addition of only β-glucosidase B, finally obtaining black ginseng peptide powder bgp3 (single enzyme conversion). The contents of ginsenoside CK and total saponins in black ginseng peptide powders bgp1, bgp2, and bgp3 were detected by HPLC. Table 1 shows the total saponin content and the ginsenoside CK content.

[0064] Table 1

[0065]

[0066] The results showed that the solubility of the precursor saponins was greatly increased after adding the specific invertase of the present invention to convert them into CK, and the extraction rate of total saponins was improved to a certain extent. The specific compound enzymatic hydrolysis conversion scheme used in the present invention has a better conversion effect.

[0067] Experiment 2. In vitro cell experiment - Verification of the synergistic effect of fucoxanthin and black ginseng peptide on browning.

[0068] C57BL / 6 mouse white adipocyte precursor cells (3T3-L1) were cultured in vitro and induced for 10 days with insulin (10 μg / mL), dexamethasone (1 μmol / L), and IBMX (0.5 mmol / L) to differentiate into mature white adipocytes. Primary brown adipocytes (iBAT cells) between the scapulae of C57BL / 6 mice were isolated and cultured to the 5th generation for use. The two cell lines were divided into three replicates each, and cultured at 37°C in a 5% CO2 incubator for 7 days. The drug-containing medium was changed every 2 days.

[0069] Detection indicators: Total RNA was extracted from cells, and the relative mRNA expression level of the browning-related gene UCP was detected (with GAPDH as the internal reference gene); total protein was extracted from cells, and the expression level of UCP1 protein was detected (with β-actin as the internal reference protein); oxygen consumption rate (OCR, reflecting thermogenesis capacity) was detected using a Seahorse XF cell energy metabolism analyzer, and the number of mitochondria (number of mitochondria / cell) was observed using transmission electron microscopy.

[0070] Table 2 shows the detection results of browning indicators in white adipocytes (3T3-L1), and Table 3 shows the detection results of activation indicators in brown adipocytes (iBAT).

[0071] Table 2

[0072]

[0073] Table 3

[0074]

[0075] The results showed that in white adipocytes, the fucoxanthin group exhibited a significant browning-promoting effect, and this effect was further enhanced in the fucoxanthin + peptide group and the fully synergistic group. SEQ ID NO.1 peptide showed an effect of promoting an increase in the number of mitochondria per unit cell. Ginsenoside CK did not show a significant effect on browning in white adipocytes. However, in brown adipocytes, the fucoxanthin group did not show a significant activation effect, while cells treated with ginsenoside CK showed significant increases in UCP1 protein expression and basal oxygen consumption, indicating that it has an activating effect on brown adipocytes.

[0076] Experiment 3: Animal Experiments on Weight Loss Effects

[0077] SPF-grade male C57BL / 6 mice (6 weeks old, weighing 18-20g) were selected and induced into an obesity model (weight ≥32g, body fat percentage ≥40%) for 8 weeks using a high-fat diet (60% fat content). Mice were randomly divided into 8 groups (n=5 per group), with a normal control group fed a standard diet. The intervention period was 12 weeks. Indicator measurements:

[0078] (1) Weigh yourself weekly. At the end of the experiment, use nuclear magnetic resonance to measure body fat percentage and inguinal white fat (iWAT) weight.

[0079] (2) Western blot was used to detect the expression level of UCP1 protein in iWAT;

[0080] (3) Observe the mitochondrial density (number of mitochondria per 100 μm²) in iWAT using transmission electron microscopy.

[0081] (4) Gas chromatography was used to detect the total content of short-chain fatty acids (SCFA, including acetic acid, propionic acid and butyric acid) in feces;

[0082] (5) Measure postprandial blood glucose 2 hours after gavage administration of 2g / kg glucose;

[0083] (6) Record daily food intake; detect serum 5-hydroxytryptamine (5-HT) levels by ELISA; detect the mRNA expression levels of hypothalamic appetite-related genes (NPY, appetite-promoting gene; POMC, appetite-inhibiting gene) by qPCR.

[0084] The intervention methods for each group are shown in Table 4. Table 5 shows the weight loss rate, body fat percentage, and iWAT weight for each group. Table 6 shows the UCP1 protein and mitochondrial density in iWAT for each group. Table 7 shows the total fecal SCFA, 2-hour postprandial blood glucose, average daily food intake, serum 5-HT, hypothalamic NPY mRNA, and hypothalamic POMC mRNA for each group.

[0085] Table 4

[0086]

[0087] Table 5

[0088]

[0089] Table 6

[0090]

[0091] Table 7

[0092]

[0093] The results showed that, due to the lack of energy metabolism-promoting effects of fucoxanthin and black ginseng peptide in group B, the expression level of UCP1 protein and mitochondrial density in iWAT were significantly lower than those in the full synergistic group. Meanwhile, the full synergistic group was superior to the group lacking black ginseng peptide alone, indicating that the SEQ ID NO.1 component of black ginseng peptide further promoted the browning of white adipocytes and energy consumption under the influence of fucoxanthin. Furthermore, the comparison of body weight and body fat percentage among the groups showed that the saponin CK component of black ginseng peptide compensated for the weak effect of fucoxanthin on mature brown adipose tissue, forming a complementary effect of white adipose tissue browning and brown adipose tissue activation, thereby enhancing energy consumption and improving weight loss.

[0094] The 2-hour postprandial blood glucose in group C (without α-amylase inhibitor) was significantly higher than that in the synergistic group, demonstrating that α-amylase inhibitor can reduce glucose absorption and alleviate the burden on lipid metabolism. The total SCFA in group F (without cactus and allicin) was only 34% of that in the synergistic group, and the expression level of UCP1 was lower than that in the synergistic group, confirming that the two assist browning by promoting SCFA production. The SCFA and UCP1 levels in groups D and E were both lower than those in the synergistic group, indicating that the two have a synergistic effect in regulating the microbiota-browning pathway.

[0095] Group F had significantly lower serum 5-HT levels than the full synergistic group, higher average daily food intake, and higher hypothalamic NPY expression and lower POMC expression, indicating that both reduce appetite through the 5-HT pathway. Group B, on the other hand, had higher food intake than the full synergistic group and lower POMC expression, indicating that fucoxanthin and black ginseng peptides further suppress appetite through free fatty acids produced by lipid metabolism, forming a synergistic effect with intestinal components.

[0096] The synergistic group showed significantly better results than the deficient groups in terms of weight loss rate, body fat percentage, UCP1 expression, and SCFA content, demonstrating that the formula achieves efficient weight loss through multi-target effects of "synergistic energy metabolism, synergistic glucose absorption-intestinal regulation, and synergistic appetite-fat metabolism," and that each component is indispensable.

[0097] This invention discloses a composition with weight-loss effects and its application. The formulation consists of fucoxanthin, ginsenoside CK, α-amylase inhibitor, prickly pear cactus extract, allicin, etc. Fucoxanthin, ginsenoside CK, and active peptides synergistically activate brown adipose tissue and reshape the energy metabolism network, thereby increasing energy expenditure; the α-amylase inhibitor, prickly pear cactus extract, and allicin synergistically inhibit sugar absorption and regulate intestinal flora, reducing fat synthesis; appetite-regulating components and fat metabolism components synergistically regulate appetite signals, reducing food intake. Experiments have demonstrated that this formulation has a significantly better weight-loss effect than single ingredients or simple combinations, and is highly safe, providing a new multi-target synergistic approach for obesity intervention.

[0098] Finally, it should be noted that the above examples are merely a few specific embodiments of the present invention. All derivatives that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. An active peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The use of the active peptide according to claim 1 in the preparation of a fat-reducing drug.

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