Composition with effects of losing weight and reducing blood fat and preparation method thereof

Abstract

The invention discloses a composition with effects of losing weight and reducing blood fat. Every 100g of the composition comprises the following components and contents: (1) 10-30mg of adiponectin functional fragments; (2) 5 to 15 mg of PCSK9 inhibitory peptide; (3) 1-5 g of a radix puerariae extract; (4) 1-3 g of garcinia cambogia extract; (5) 0.5-2 g of a green coffee bean extract; (6) 1 to 3g of microencapsulated fish oil; (7) 0.5 to 1g of chitosan; and 5 to 10 g of erythritol. Through the synergistic effect of various bioactive components, the composition can effectively reduce fat accumulation and reduce the blood fat level, and meanwhile has good taste and use convenience. The composition disclosed by the invention provides an innovative solution for losing weight and reducing blood fat, and has potential clinical and commercial application values.

Description

Technical Field

[0001] The invention relates to the field of health food and medicine, and in particular to a composition with weight loss and blood lipid lowering effects and a preparation method thereof. Background Art

[0002] Obesity and hyperlipidemia are major health problems in today's society. They not only affect personal image, but also significantly increase the risk of cardiovascular disease, diabetes, hypertension and certain cancers. Obesity is usually caused by a combination of factors, including genetic factors, unhealthy eating habits, and a sedentary lifestyle. Hyperlipidemia is manifested as an abnormal increase in serum cholesterol, low-density lipoprotein (LDL) and triglycerides (TG), which is an important pathogenic factor of atherosclerosis and coronary heart disease.

[0003] Currently, interventions for weight loss and lipid-lowering include lifestyle intervention, drug therapy, and surgical treatment. However, these methods all have limitations. For example, the effects of lifestyle interventions are slow to manifest and compliance is low; although chemical drugs (such as orlistat and statins) are effective, long-term use may lead to side effects such as gastrointestinal discomfort and abnormal liver function; and surgical treatments such as bariatric surgery are only suitable for severely obese patients and are accompanied by certain risks. Therefore, finding a safe, efficient, and convenient natural method has become a research hotspot.

[0004] In recent years, functional compositions based on natural plant extracts have attracted much attention due to their pleiotropic effects and low side effects. Studies have shown that bitter melon saponins can regulate lipid metabolism and reduce body fat accumulation; fucoidan has the potential to inhibit adipocyte differentiation and improve intestinal absorption; and white kidney bean extract reduces carbohydrate absorption by inhibiting amylase activity. In addition, active substances such as L-carnitine, hesperidin and β-glucan also have significant effects in metabolic regulation and immune enhancement. However, these ingredients have limited effects when used alone, and there is a lack of synergistic research.

[0005] The present invention compounds a variety of active ingredients in specific proportions through scientific screening and optimization of the formula, which not only enhances the efficacy of weight loss and lipid-lowering, but also significantly reduces the risk of side effects, providing a new idea and solution for related fields. Summary of the invention

[0006] The purpose of the present invention is to provide a safe and effective weight loss and lipid-lowering composition, which can effectively reduce fat accumulation and lower blood lipid levels through the synergistic effect of multiple bioactive ingredients, while having a good taste and ease of use.

[0007] Therefore, the present invention discloses a composition with weight loss and lipid-lowering effects, wherein each 100 g of the composition comprises the following components and contents: (1) adiponectin functional fragment: 10-30 mg; (2) PCSK9 inhibitory peptide: 5-15 mg; (3) kudzu root extract: 1-5 g; (4) garcinia cambogia extract: 1-3 g; (5) green coffee bean extract: 0.5-2 g; (6) microencapsulated fish oil: 1-3 g (7) chitosan: 0.5-1 g;

[0008] (8) Erythritol: 5-10g.

[0009] In one aspect, the present invention also discloses a composition with weight loss and lipid-lowering effects, wherein each 100 g of the composition comprises the following components and contents: (1) adiponectin functional fragment: 20 mg; (2) PCSK9 inhibitory peptide: 10 mg; (3) kudzu root extract: 3 g; (4) garcinia cambogia extract: 2 g; (5) green coffee bean extract: 1 g; (6) microencapsulated fish oil: 2 g; (7) chitosan: 0.5 g; (8) erythritol: 10 g.

[0010] Preferably, the amino acid sequence of the adiponectin functional fragment of the present invention is AILQFSARPFGY.

[0011] Preferably, the PCSK9 inhibitory peptide of the present invention is an anti-PCSK9 single domain antibody, wherein the VHH sequence of the anti-PCSK9 single domain antibody is as shown in SEQ ID NO.1.

[0012] In one aspect, the present invention also discloses a method for preparing the composition, characterized in that the method comprises the following steps: (1) first adding powdered adiponectin functional fragments and PCSK9 inhibitory peptides, and stirring at a low speed for 10 minutes; (2) adding kudzu root extract, garcinia cambogia extract, and green coffee bean extract, and mixing evenly; (3) adding chitosan, microencapsulated fish oil and erythritol, and gradually stirring evenly; (4) transferring the mixture to a vacuum drying device, dehumidifying at 40°C, and forming a homogeneous powder; (5) according to needs, the powder can be tableted, loaded into capsules, or made into an oral liquid dosage form.

[0013] In one aspect, the present invention also discloses a use of the functional fragment of adiponectin in preparing a composition having the effects of reducing weight and lowering blood lipids.

[0014] In one aspect, the present invention also discloses a use of the anti-PCSK9 single domain antibody in the preparation of a composition having weight loss and blood lipid lowering effects.

[0015] The composition of the present invention can effectively reduce fat accumulation and lower blood lipid levels through the synergistic effect of multiple bioactive ingredients, while having a good taste and convenience in use. Therefore, the composition of the present invention provides an innovative solution for weight loss and blood lipid reduction, and has potential clinical and commercial application value.

[0016] The composition of the present invention achieves multi-target effects on fat metabolism, cholesterol regulation and appetite center by optimizing the formula, integrating protein fragments, plant extracts and small molecule compounds, thereby significantly improving the therapeutic effect. The composition of the present invention fills the deficiency of the existing composition that only acts on a single pathway through the synergistic mechanism of adiponectin functional fragments and PCSK9 inhibitory peptides. At the same time, the preparation process of each component in the formula of the composition of the present invention is optimized to ensure the stability and activity of each component, laying the foundation for actual production and transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 SDS-PAGE test results of anti-PCSK9 single domain antibodies, where 1 and 2 are both anti-PCSK9 single domain antibodies.

[0018] Figure 2 Western blot test results of anti-PCSK9 single domain antibody, where 1 and 2 are both PCSK9 proteins. DETAILED DESCRIPTION

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0020] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0021] Example 1: Formulation of the composition

[0022] 1. The composition of the present invention comprises the following components (Table 1):

[0023] Table 1 Summary of components, contents and mechanisms of action per 100 g of the composition

[0024]

[0025] According to the above results, the specific formula of the composition of the present invention is shown in Table 2:

[0026] Table 2 Specific formula of each component per 100g composition

[0027] Ingredient name content Adiponectin functional fragment 20mg PCSK9 inhibitory peptides 10mg Pueraria root extract 3g Garcinia Cambogia Extract 2g Green Coffee Bean Extract 1g Microencapsulated fish oil 2g Chitosan 0.5g Erythritol 10g

[0028] 2. Design and preparation of functional fragments of adiponectin

[0029] (1) Design of functional fragments of adiponectin

[0030] Adiponectin is a multifunctional endocrine protein, and its functional fragment is considered to be its active region. Based on existing research and analysis of functional active regions, the active fragment of human adiponectin (based on its C-terminal globular domain) was selected and the peptide sequence designed and synthesized is as follows: AILQFSARPFGY.

[0031] The sequence selected amino acid residues with high affinity and biological activity, and retained the key region for binding to the adiponectin receptor. Compared with the sequence of the natural adiponectin globular domain, the stability and receptor binding activity of the peptide were increased by introducing mutations (Ala replacing Leu, etc.). Through the design of this sequence, we expect it to effectively activate the adiponectin receptor and promote the activation of downstream signaling pathways such as AMPK, thereby exerting lipid-lowering and weight-loss effects.

[0032] (2) Preparation of functional fragments of adiponectin

[0033] The peptide chain was synthesized using solid phase peptide synthesis. The synthesized peptide was purified by reverse phase high performance liquid chromatography (RP-HPLC). The structure and purity (greater than 95%) of the peptide were verified by mass spectrometry (MS) and amino acid analysis.

[0034] (3) Comparative study of functional fragments of adiponectin

[0035] 3T3-L1 adipocytes were cultured, and after the cells were cultured to fusion, differentiation induction solution (DMEM+10% FBS+0.5mM IBMX, 1μM dexamethasone and 10μg / mL insulin) was added for differentiation for 5 days. Adiponectin functional fragment (50μg / mL), adiponectin (50μg / mL) and PBS buffer were then added and cultured for 48 hours. Oil red O staining was used to evaluate the amount of intracellular lipid accumulation, and the Oil red O in the cells after staining was extracted, and the absorbance was measured with a spectrophotometer (OD510). The results are shown in Table 1. The lipid accumulation in the adiponectin functional fragment treatment group was significantly reduced, compared with the PBS buffer group, it decreased by 40.5% (p<0.01), and it was also better than the adiponectin group (p<0.05).

[0036] The above results show that the adiponectin functional fragment prepared by the present invention has a better effect on reducing fat accumulation than adiponectin. Therefore, the adiponectin functional fragment prepared by the present invention has a significant effect of reducing fat and improving lipid metabolism, and its mechanism may involve lipolysis promotion and gene regulation.

[0037] Table 1 Comparison results of absorbance value (OD510)

[0038] Group PBS buffer group Adiponectin group Adiponectin functional fragment group Absorbance (OD510) 1.00±0.05 0.68±0.03 0.59±0.02

[0039] 3. Preparation of PCSK9 inhibitory peptide (anti-PCSK9 single domain antibody)

[0040] (1) Recombinant PCSK9 protein (ab198471) and FCA were emulsified to prepare a vaccine, wherein the PCSK9 protein content in the vaccine was 0.5 mg / ml, and the prepared vaccine was subcutaneously injected into the shark, 0.5 ml / time / animal. Two weeks later, the recombinant PCSK9 protein (ab198471) and FIA emulsified vaccine were used for the second immunization at the same dose and method. After an interval of two weeks, the third immunization was carried out at the same dose and method. Within 7 days after the third immunization, peripheral blood was collected from the shark and lymphocytes were isolated. Total RNA of B cells was extracted using the TRIzol method. The extracted RNA was reverse transcribed into cDNA according to the instructions of the kit. At the same time, specific primers for the shark single-domain antibody VHH fragment were designed, and specific primers (upstream primer: 5'-gtcctggctcagggagagcgg t-3', downstream primer: 5'-ggtacggttggcctgttgagg-3') were used to amplify the VHH fragment in cDNA (PCR conditions: 94°C pre-denaturation for 5 minutes; 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute, 35 cycles; 72°C extension for 5 minutes).

[0041] (2) The VHH fragment amplified by PCR was cloned into the pGEM-T vector. DH5α E. coli competent cells were transformed and positive clones were picked. The positive clone plasmid was extracted and sent for sequencing to determine the VHH sequence of the antibody. After sequencing, the VHH sequence of the prepared anti-PCSK9 single domain antibody was shown in SEQ ID NO.1. The sequence analysis results of the single domain antibody are as follows:

[0042] FR1: ADQTPTTTTKEAGEESLTIN; CDR1: CVDSKCELSSTYWY;

[0043] FR2: FTKKGATKKARISIGGRYSG; CDR2: TKNTASSKSFS;

[0044] FR3:LRISDLRVEDKASLSTGGLRPH;

[0045] CDR3:TWGTCDGGSDTKNTASSYEGGGTIL.

[0046] (3) The VHH sequence of the anti-PCSK9 single domain antibody determined above was cloned into the pET28a vector and transformed into E. coli BL21 (DE3) competent cells. When the OD600 was 0.6 at 37°C, 1 mM IPTG was added for induction, and the cells were collected after 4 hours of induction. The cells were disrupted by ultrasound and the soluble protein was collected. The anti-PCSK9 single domain antibody was purified by nickel column through His-tag, and after dialysis and liquid exchange, it was sterilized by 220 nm filter membrane, and then aliquoted and frozen in a -80°C refrigerator for use.

[0047] (4) The purity of the anti-PCSK9 single domain antibody was detected by SDS-PAGE. Figure 1 The single domain antibody was used to perform western blot detection on the recombinant PCSK9 protein (ab198471). The results are shown in Figure 2 As shown, the single domain antibody can specifically bind to the PCSK9 protein (the molecular weight of the PCSK9 protein is about 72kDa). At the same time, according to the above purification records, the expression amount of the purified single domain antibody is calculated, and its expression amount reaches more than 1g / L. Therefore, the single domain antibody has a high expression yield and is suitable for large-scale application.

[0048] 4. Preparation of Pueraria Extract

[0049] (1) Raw material preparation: First, select fresh and uncontaminated Pueraria lobata roots. Make sure the root surface is clean and remove impurities. If using dried products, select Pueraria lobata that has been properly dried and well preserved. Wash the Pueraria lobata roots and cut them into uniform small sections (about 5-10 cm) to expose more surface and improve the extraction effect. The dried products can be crushed as needed.

[0050] (2) Raw material processing: Add appropriate amount of deionized water to the cut Pueraria root segments, soak for about 30 minutes, and put them into the cooking equipment.

[0051] (3) Steaming extraction: Steam or directly heat to boiling to dissolve the active ingredients in Pueraria root into water. The extraction time is 60-120 minutes to ensure sufficient extraction.

[0052] (4) Separation and concentration: After filtration, a preliminary extract is obtained, and water is removed using a concentration device (such as a rotary evaporator) to finally obtain a concentrated kudzu root extract.

[0053] (5) Concentration and drying: Use a vacuum concentrator (rotary evaporation) to remove the solvent from the extract to obtain a concentrated solution. Use spray drying or freeze drying to further remove the residual solvent and convert the concentrated solution into a powder state for easy storage and use.

[0054] (6) Through detection, the puerarin content in the extract prepared by this method is 25%, which has a high yield.

[0055] 5. Preparation of Garcinia Cambogia Extract

[0056] (1) Raw material preparation: Select mature and pest-free Garcinia cambogia. Remove the peel and pulp after picking, wash the fruit, and cut it into small pieces or crush it according to experimental requirements. Add Garcinia cambogia pulp or dried fruit powder to 70% ethanol solution at a ratio of 1:10 and soak.

[0057] (2) Ultrasonic treatment: The mixture was placed in an ultrasonic cleaning machine, the power was set to 300 W, the frequency was set to 40 kHz, and the extraction time was set to 30 minutes.

[0058] (3) Filtration and separation: After the extraction is completed, solid impurities are removed by filtering through filter paper or cloth to obtain a crude extract.

[0059] (4) Concentration: The crude extract was concentrated using a rotary evaporator at 40°C to remove ethanol and obtain a concentrated extract.

[0060] (5) Through testing, the content of garcinia cambogia extract is 65%, which has a high yield.

[0061] 6. Preparation of green coffee bean extract

[0062] (1) Raw material preparation: Select fresh green coffee beans and ensure that they are free of mold and damage. Wash the green coffee beans, remove surface impurities, and dry them for later use. If using dried green coffee beans, crush them to a suitable particle size (such as coarse powder) to enhance the extraction efficiency.

[0063] (2) Soaking: Mix the crushed green coffee beans with 70% ethanol aqueous solution in a ratio of 1:10 and soak for 1 hour.

[0064] (3) Ultrasonic treatment: The mixture was placed in an ultrasonic cleaning machine, the power was set to 300 W, the frequency was set to 40 kHz, and the extraction time was set to 30 minutes.

[0065] (4) Filtration and separation: After ultrasonic extraction is completed, solid impurities are filtered out and the extract is collected.

[0066] (5) Concentration: The ethanol is removed by a rotary evaporator at 40° C. to obtain a concentrated green coffee bean extract.

[0067] (6) Through testing, the content of chlorogenic acid in green coffee bean extract is 34%, which has a high yield.

[0068] 7. Microencapsulated fish oil: The fish oil is encapsulated in gelatin / gum arabic wall material using the emulsification-spray drying method to prepare microcapsule particles.

[0069] 8. Chitosan: Dissolve in 1% acetic acid solution, filter and spray dry to make powder.

[0070] Example 2: Preparation of the composition

[0071] Weigh each active ingredient in Example 1 according to proportion and add them into the mixer in sequence:

[0072] 1. First add the powdered adiponectin functional fragment and PCSK9 inhibitory peptide and stir at low speed for 10 minutes.

[0073] 2. Add Pueraria root extract, Garcinia cambogia extract, and green coffee bean extract and mix evenly.

[0074] 3. Add chitosan, microencapsulated fish oil and erythritol, and stir gradually until evenly mixed.

[0075] 4. The mixture was transferred to a vacuum drying apparatus and dehumidified at 40°C to form a homogeneous powder.

[0076] 5. According to the needs, the powder can be compressed into tablets, filled into capsules, or made into oral liquid dosage forms.

[0077] Example 3: Application of the composition

[0078] 1. Experimental animals: Male SD rats, 8 weeks old, weighing 200±20 g, adapted for 7 days. Environmental conditions: 22±2℃, 12-h light / dark cycle, humidity 55±5%, free access to food and water.

[0079] 2. Experimental groups:

[0080] (1) Control group (10 rats): fed with normal feed;

[0081] (2) High-fat control group (10 mice): fed with a high-fat diet (60% standard diet + 20% fat + 20% carbohydrate);

[0082] (3) Drug control group (10 rats): high-fat diet + oral administration of orlistat (20 mg / kg, dissolved in normal saline, once a day);

[0083] (4) Test group 1 of the present invention (10 mice): high-fat diet + oral administration of the composition of Table 2 of Example 1 of the present invention (excluding PCSK9 inhibitory peptide) (50 mg / kg, once a day);

[0084] (5) Test group 2 of the present invention (10 mice): high-fat diet + oral administration of the composition of Table 2 of Example 1 of the present invention (containing PCSK9 inhibitory peptide) (50 mg / kg, once a day).

[0085] 3. Experimental period: Intervention started 4 weeks after obesity induction and lasted for 8 weeks.

[0086] 4. Experimental observation and data collection: Body weight was measured using an electronic balance every week, and the average change trend of each group was recorded. The weight of the remaining feed was measured every day, and the average food intake of each rat was calculated. At the end of the experiment (week 12), tail vein blood was collected after fasting for 12 hours. Serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were detected using an automatic biochemical analyzer.

[0087] 5. Experimental data: The weight loss effect and blood lipid regulation effect of the experimental group 2 of the present invention are better than those of the experimental group 1 of the present invention and the drug control group, among which the experimental group 1 of the present invention is equivalent to the drug control group. Compared with the high-fat control group, serum TC, TG and LDL were significantly reduced (p<0.01), and HDL was significantly increased (p<0.05). See Table 2 for details.

[0088] Table 2 Experimental results report

[0089]

[0090] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A composition having the effects of reducing weight and lowering blood lipids, characterized in that: Each 100g of the composition includes the following components and contents: (1) Adiponectin functional fragment: 10-30 mg; (2) PCSK9 inhibitory peptide: 5-15 mg; (3) Pueraria root extract: 1-5g; (4) Garcinia cambogia extract: 1-3g; (5) Green coffee bean extract: 0.5-2g; (6) Microencapsulated fish oil: 1-3g (7) Chitosan: 0.5-1 g; (8) Erythritol: 5-10g.

2. A composition having the effects of reducing weight and lowering blood lipids, characterized in that: Each 100g of the composition includes the following components and contents: (1) Adiponectin functional fragment: 20 mg; (2) PCSK9 inhibitory peptide: 10 mg; (3) Pueraria root extract: 3g; (4) Garcinia cambogia extract: 2g; (5) Green coffee bean extract: 1 g; (6) Microencapsulated fish oil: 2 g; (7) Chitosan: 0.5 g; (8) Erythritol: 10g.

3. The composition according to any one of claims 1 or 2, characterized in that The amino acid sequence of the adiponectin functional fragment is AILQFSARPFGY.

4. The composition according to any one of claims 1 or 2, characterized in that The PCSK9 inhibitory peptide is an anti-PCSK9 single domain antibody, wherein the VHH sequence of the anti-PCSK9 single domain antibody is shown in SEQ ID NO.

1.

5. A method for preparing the composition according to any one of claims 1 or 2, characterized in that: The method comprises the following steps: (1) First, add powdered adiponectin functional fragment and PCSK9 inhibitory peptide and stir at low speed for 10 minutes; (2) adding kudzu root extract, garcinia cambogia extract, and green coffee bean extract and mixing evenly; (3) adding chitosan, microencapsulated fish oil and erythritol, and gradually stirring evenly; (4) transferring the mixture to a vacuum drying device and dehumidifying at 40° C. to form a homogeneous powder; (5) Depending on the needs, the powder can be compressed into tablets, put into capsules, or made into oral liquid dosage forms.

6. Use of the functional fragment of adiponectin as claimed in claim 3 in preparing a composition having the effects of reducing weight and lowering blood lipids.

7. Use of the anti-PCSK9 single domain antibody as claimed in claim 4 in preparing a composition having weight loss and lipid-lowering effects.

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