Composition as well as preparation method and application thereof

Abstract

The invention discloses a composition as well as a preparation method and application thereof. The invention discloses a composition which is prepared from the following components in parts by weight: 30-60 parts of fructus phyllanthi, 30-60 parts of acid docynia, 20-40 parts of Chinese olive, 20-40 parts of dendrobium officinale and 10-30 parts of black tartary buckwheat. The preparation method comprises the following steps: mixing and crushing the raw materials, adding water for soaking, performing ultrahigh pressure treatment, performing enzymolysis through alpha-amylase, pectinase and cellulase, and finally performing ultrasonic ethanol water extraction, concentration and spray drying to obtain the composition. Results of an insulin resistance test, a DPP-IV enzyme inhibition test and a xanthine oxidase inhibition test show that compared with a composition or a single raw material extract prepared by a conventional method, the composition can significantly improve liver cell insulin resistance and can effectively inhibit DPP-IV enzyme activity and xanthine oxidase activity; in addition, the composition disclosed by the invention is derived from medicinal and edible plant raw materials, and is easy to obtain and high in safety.

Description

Technical Field

[0001] The invention relates to the technical field of regulating body metabolism and health, and in particular to an application in a product for improving insulin resistance and inhibiting DPP-IV enzyme and xanthine oxidase. Background Art

[0002] In the field of human health, insulin resistance, DPP-IV enzyme (dipeptidyl peptidase-IV) and xanthine oxidase are three factors that play a key role in the body's nutritional metabolic balance. They play an important role in the regulation of blood sugar levels and purine metabolism, respectively, and together maintain the stability of the body's internal environment. Insulin resistance refers to the reduced responsiveness of body cells to insulin, resulting in a decrease in the efficiency of insulin in promoting glucose uptake and utilization. This will cause blood sugar levels to rise, and long-term existence may develop into metabolic syndromes such as type 2 diabetes and cardiovascular disease. Improving insulin sensitivity is crucial for the prevention and treatment of these diseases. The DPP-IV enzyme is an enzyme that is widely present in a variety of tissues and body fluids, especially playing a core role in regulating blood sugar homeostasis. It mainly affects insulin secretion by degrading hormones such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) released by intestinal endocrine cells. GLP-1 can enhance the biological effects of insulin, promote insulin secretion by pancreatic β cells, and inhibit the secretion of glucagon, thereby effectively lowering blood sugar. Therefore, as a new type of diabetes treatment drug, DPP-IV inhibitors can achieve the purpose of controlling blood sugar by preventing the degradation of GLP-1, increasing its in vivo activity, and improving insulin resistance. Xanthine oxidase is a key enzyme in the liver that can metabolize purine into uric acid and maintain the balance of liver purine metabolism. When the liver has insulin resistance and glucose metabolism disorders occur, it usually also leads to abnormal liver purine metabolism. Therefore, improving insulin resistance and inhibiting the activity of DPP-IV enzyme and xanthine oxidase play an indispensable role in regulating blood sugar homeostasis, balancing purine metabolism and preventing metabolic diseases; they have shown great potential in maintaining human health, especially in combating metabolic diseases such as type 2 diabetes, hyperuricemia and gout. This strategy not only helps to restore normal blood sugar and uric acid regulation mechanisms, but also reduces the risk of corresponding complications and improves the quality of life. Although significant progress has been made in drugs and therapies targeting these targets, there is currently no drug that can act on these three targets at the same time. Patients often need to take multiple drugs, which not only increases the economic burden on patients, but also because these different drugs have certain side effects, the combination of multiple drugs often further amplifies these side effects, thereby increasing the burden on the human body. Therefore, it is urgent to find some coping methods that are low in burden on the patient's body and economy, and finding some combinations from traditional botanical Chinese medicinal materials, especially Chinese medicinal materials that are both medicinal and edible, is a feasible solution. These Chinese medicinal materials that are both medicinal and edible are not only cheap, but also safe and have no side effects. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a composition. The raw materials selected in the composition of the present invention are all from plants listed in the catalogue of Chinese medicine and food homology, and are safe to eat. The composition is prepared by scientific proportioning, adopting a special preparation method and process. In vitro biochemical experiments and cell experiments have confirmed that the composition of the present invention can significantly improve the insulin resistance of liver cells, inhibit the activity of DPP-IV enzyme and xanthine oxidase, thereby regulating blood sugar and uric acid levels to return to normal, and compared with compositions prepared by conventional methods or single raw material extracts, the composition prepared by the present invention has better effects.

[0004] In order to achieve the above object, the present invention provides the following technical solutions: The composition of the present invention is composed of Yunnan olive ( Phyllanthus emblica )、Acid Duoyi( Docynia delavayi ), green fruit ( White Canary )、Dendrobium candidum( Dendrobium officinalis ) and black buckwheat ( Buckwheat Tatar ) mixed extraction; wherein 30-60 weight parts of Yunnan olives, 30-60 weight parts of sour polysaccharide, 20-40 weight parts of green fruit, 20-40 weight parts of Dendrobium officinale and 10-30 weight parts of black bitter buckwheat; The preparation method of the above composition is as follows: The Yunnan olive, sour polysaccharide, green fruit, Dendrobium officinale and black bitter buckwheat are mixed and crushed to a particle size of 50-80 meshes, and the mixed raw material powder is mixed with water, the mass volume ratio of the mixed raw material powder to water is 1:2-4 g:mL, and after soaking for 2-4 hours, it is subjected to ultra-high pressure treatment at 100-600MPa for 10-30 minutes, and then α-amylase, pectinase and cellulase are added in sequence under stirring (100-200rpm), and each enzyme is incubated at the optimal temperature of the enzyme for 2-4 hours after addition. After the incubation, edible alcohol with a volume concentration of 50-95% is added at a mass volume ratio of the mixed raw material powder to edible alcohol g:mL of 1:2-1:4, and extracted for 30-60 minutes under the assistance of 200-500W ultrasonic wave, filtered, and the residue is repeatedly extracted 2-3 times, and the combined extract is collected, concentrated, and finally spray-dried to obtain a composition.

[0005] The added amount of α-amylase is 0.5-3% of the mass of the mixed raw material powder, the added amount of pectinase is 0.5-2.5% of the mass of the mixed raw material powder, and the added amount of cellulase is 1-5% of the mass of the mixed raw material powder.

[0006] The present invention applies the above-mentioned composition to prepare a preparation that can simultaneously improve hepatocyte insulin resistance, inhibit DPP-IV enzyme activity, and inhibit xanthine oxidase activity. The ingredients (or active ingredients) of the product are the above-mentioned composition, and one or more excipients acceptable in product production can also be added to improve the absorption effect of the active ingredients or facilitate use, such as capsules or pills, powders, tablets, granules, oral liquids and injections, etc., that is, suitable dosage forms are prepared.

[0007] The composition provided by the present invention can significantly improve hepatocyte insulin resistance, and can also effectively inhibit DPP-IV enzyme activity and xanthine oxidase activity, thereby playing the role of regulating blood sugar level and purine uric acid metabolism.

[0008] Compared with the prior art, the present invention has the following advantages: 1. Compared with the existing conventional preparation method, the effect of the composition prepared by the method of the present invention is more significant and stronger; 2. The composition of the present invention can not only significantly improve insulin resistance and increase insulin sensitivity of hepatocytes, but also effectively inhibit the activity of DPP-IV enzyme and xanthine oxidase, showing multi-active target characteristics; 3. The compositions of the present invention are all derived from plant raw materials that are both medicinal and edible, and are safe and convenient to eat, and have no toxic side effects. DETAILED DESCRIPTION

[0009] The following are specific implementation examples of the present invention. The examples are intended to further describe the present invention, rather than to limit the present invention. The specific experimental procedures and results described in the examples are intended only to illustrate the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified: Example 1: Composition prepared by the method of the present invention 40 parts of Yunnan olives, 40 parts of sour polyyi, 20 parts of green fruit, 30 parts of Dendrobium officinale and 10 parts of black buckwheat were mixed and crushed to 50-60 meshes. The mixed raw material powder was mixed with water at a mass volume ratio of g:mL of 1:3. After soaking for 2.5 hours, it was treated with an ultra-high pressure of 500MPa for 20 minutes, and then 1.5% of α-amylase, 2.0% of pectinase and 4.0% of cellulase by mass of the mixed raw material powder were added in sequence under stirring (150rpm). After each enzyme was added, it was incubated at the optimal temperature of the enzyme for 3 hours. After the incubation, edible alcohol with a volume concentration of 80% was added to the incubation product at a mass volume ratio of g:mL of 1:3 between the mixed raw material powder and edible alcohol. The mixture was extracted for 40 minutes under the assistance of 300W ultrasound, filtered, and the residue was extracted twice. The combined extract was collected, concentrated at 45°C, and finally spray-dried to obtain a composition.

[0010] Example 2: Preparation of the composition using conventional methods 40 parts of Yunnan olives, 40 parts of sour polysaccharide, 20 parts of green fruit, 30 parts of Dendrobium officinale and 10 parts of black buckwheat were mixed and crushed to 50-60 mesh, and the mixed raw material powder was mixed with water at a mass volume ratio of g:mL of 1:3. After soaking for 2.5 hours, edible alcohol with a volume concentration of 80% was added to the soaked mixture at a mass volume ratio of g:mL of 1:3 of the mixed raw material powder and edible alcohol, and extracted for 40 minutes under the assistance of 300W ultrasonic wave, filtered, and repeatedly extracted the filter residue twice, collected and combined the extracts, concentrated at 45°C, and finally spray-dried to obtain a composition prepared by a conventional method.

[0011] Example 3: Single raw material extract prepared by the method of the present invention The method of Example 1 was used to extract Yunnan olive, sour poly, green fruit, Dendrobium officinale and black buckwheat respectively to obtain Yunnan olive extract, sour poly, green fruit extract, Dendrobium officinale extract and black buckwheat extract.

[0012] Example 4: Experimental evaluation of the effects of the extracts from Examples 1-3 on insulin resistance in HepG2 liver cancer cells HepG2 liver cancer cells in the logarithmic growth phase were inoculated in a 96-well plate with an inoculation volume of 200 μL (4×10 4 Cells / mL), after culturing for 24 hours, 200 μL of ordinary DMEM medium was added to the normal group, and 200 μL of DMEM medium containing 30.0 μmol / L insulin was added to the insulin resistance group, the positive drug group, and each sample group for 24 hours, the medium was aspirated and rinsed twice with PBS, and then the normal group and the insulin resistance group were added with incomplete medium, and the other groups were added with the corresponding samples dissolved with incomplete medium, and continued to incubate for 24 hours, wherein the positive drug was rosiglitazone, with a concentration of 20.0 μmol / L, and the sample concentration of other sample groups was 80 μg / mL. After the culture was completed, the glucose content in the culture medium was detected with the glucose determination kit of Nanjing Jiancheng Bioengineering Research Institute, and the glucose consumption was calculated. The glucose consumption was used to evaluate the effect of the composition on improving the insulin resistance of HepG2 cells, and the results are shown in Table 1.

[0013] Table 1 Cell glucose consumption in each treatment group in the HepG2 cell insulin resistance model

[0014] Note: The results in the table are expressed as mean ± SD ( n =3); Values ​​with different superscript letters indicate significant differences ( p<0.05); the positive drug was rosiglitazone (20.0µM), and the concentration of each composition or extract was 80μg / mL.

[0015] As can be seen from Table 1, compared with the normal group, the glucose consumption of HepG2 cells in the insulin resistance group was significantly reduced ( p < 0.05), which was reduced by about 53%, indicating that HepG2 cells had a more serious insulin resistance and could not absorb glucose in the culture medium normally. Treatment with 20.0 µM positive drug rosiglitazone can significantly increase the glucose consumption of HepG2 cells ( p < 0.05), which increased by about 78%, indicating that rosiglitazone effectively improved the insulin resistance of HepG2 cells. For each sample treatment group, the composition prepared by the conventional method significantly increased the glucose consumption of HepG2 cells at 80 μg / mL ( p < 0.05), which was increased by about 29%, indicating that at this concentration, the composition prepared by the conventional method in Example 2 has a certain effect on improving the insulin resistance of HepG2 cells; compared with the composition prepared by the conventional method, the composition prepared by the method of the present invention in Example 1 at a concentration of 80 μg / mL significantly increased the glucose consumption of HepG2 cells ( p < 0.05), an increase of about 91%, and comparable to the positive drug group, with no significant difference ( p > 0.05). This result clearly shows that the composition prepared by the method of Example 1 is much better than the composition prepared by the conventional method in improving the insulin resistance of HepG2 cells and improving their insulin sensitivity, and the effect is also better than that of a single raw material extract.

[0016] Example 5: In vitro DPP-IV enzyme inhibition assay This experiment used a DPP-IV enzyme inhibitor screening kit, which was purchased from Cayman Chemical Company in the United States. The specific operation is as follows: According to the instructions for use of the kit, the entire reaction system consists of sample solution (10μL), DPP-IV (10μL), buffer solution (30μL) and substrate (Gly-Pro-AMC, 50μL), and the sample solution of the control group is replaced with buffer solution. After incubation at 37°C for 30 minutes, the fluorescence value (F) of free AMC was measured using a microplate reader (Spectra Max M5, Molecular Device, USA), and the excitation and emission wavelengths of fluorescence were 350nm and 450nm, respectively. Three parallels were set for each concentration; the calculation formula for the DPP-IV enzyme inhibition rate is: DPP-IV enzyme inhibition rate (%) = (F control -Fsample ) / F control ×100, where F control and F sample are the fluorescence values ​​of the control group and the sample group, IC 50 It represents the concentration when the inhibition rate of the inhibitor on the enzyme reaches 50%. The results are shown in Table 2.

[0017] Table 2 IC of each experimental group for inhibition of DPP-IV enzyme activity 50 value

[0018] Note: The results in the table are expressed as mean ± SD ( n =3); Values ​​with different superscript letters indicate significant differences ( p <0.05); the positive drug was sitagliptin.

[0019] As can be seen from Table 2, the positive drug sitagliptin has a very strong inhibitory effect on the DPP-IV enzyme, and its IC 50 The value was only 0.34 μg / mL. The composition prepared by the conventional method in Example 2 also had a certain inhibitory effect on the DPP-IV enzyme. 50 The value was 81.63 μg / mL. Compared with the composition prepared by the conventional method, the composition prepared in Example 1 had a better and stronger inhibitory effect on the DPP-IV enzyme. 50 The value is only about one-fourth of that, about 21.45μg / mL. And the effect is better than that of a single raw material extract; IC 50 The lower the value, the stronger the inhibitory effect on the DPP-IV enzyme. These results show that the composition prepared in Example 1 has a strong inhibitory effect on the DPP-IV enzyme.

[0020] Example 6: In vitro xanthine oxidase inhibition assay The xanthine oxidase and xanthine (purity 98%) used in this experiment were purchased from Beijing Solebow Technology Co., Ltd. The specific operation is as follows: PBS buffer was used to prepare different concentrations of each composition and the positive drug allopurinol. The entire reaction system consisted of PBS buffer (100 μL), xanthine oxidase solution (0.05 U / mL, 100 μL), sample solution (100 μL) and substrate xanthine solution (200 μmol / L, 100 μL); the sample solution of the control group was replaced with buffer solution. After incubation at 37°C for 30 minutes, the reaction was terminated with HCl (1.0 mol / L) and the absorbance was measured at 295 nm. Three parallels were set for each sample concentration, and the calculation formula for the xanthine oxidase inhibition rate was: Xanthine oxidase inhibition rate (%) = (F control -Fsample ) / F control ×100, where F control and F sample are the absorbance values ​​of the control group and the sample group, IC 50 It indicates the concentration when the inhibition rate of the combined inhibitor on the enzyme reaches 50%, and the results are shown in Table 3; Table 3 IC of each experimental group for inhibition of xanthine oxidase activity 50 value

[0021] Note: The results in the table are expressed as mean ± SD ( n =3); Values ​​with different superscript letters indicate significant differences ( p <0.05); the positive drug was allopurinol.

[0022] As can be seen from Table 3, the positive drug allopurinol has a strong inhibitory effect on xanthine oxidase, and its IC 50 The value was 8.36 μg / mL. The composition prepared by the conventional method also had a certain inhibitory effect on xanthine oxidase, but the inhibitory effect was weak. 50 The value was 103.39 μg / mL. Compared with the composition prepared by the conventional method in Example 2, the composition prepared by the method of the present invention had a better and stronger inhibitory effect on xanthine oxidase. 50 The value is about 33.48 μg / mL, and the effect is better than the inhibitory effect of a single raw material extract on xanthine oxidase. These results show that the composition prepared by the method of the present invention in Example 1 has a strong inhibitory effect on xanthine oxidase.

[0023] In summary, the composition prepared by the method of the present invention can significantly improve the insulin resistance of HepG2 cells, inhibit the activity of DPP-IV enzyme and xanthine oxidase, and compared with the composition prepared by the general conventional method or the single raw material extract, the composition prepared by the method of the present invention has better effect. Therefore, the composition prepared by the method of the present invention can be used to prepare a preparation that can simultaneously improve the insulin resistance of hepatocytes, inhibit the activity of DPP-IV enzyme, and inhibit the activity of xanthine oxidase.

Claims

1. A composition, It is characterized in that The invention is prepared from the following components: 30-60 parts by weight of Yunnan olive, 30-60 parts by weight of sour polyphenols, 20-40 parts by weight of green fruit, 20-40 parts by weight of dendrobium officinale and 10-30 parts by weight of black bitter buckwheat.

2. The composition according to claim 1, Features: The Yunnan olive, green fruit, sour duoyi, dendrobium officinale and black bitter buckwheat are mixed and crushed, the mixed raw material powder is mixed with water, and the mixture is soaked for 2-4 hours, and then subjected to ultra-high pressure treatment at 100-600MPa for 10-30 minutes, and then α-amylase, pectinase and cellulase are added in sequence under stirring. After each enzyme is added, the mixture is incubated at the optimal temperature of the enzyme for 2-4 hours. After the incubation, edible alcohol with a volume concentration of 50-95% is added, and ultrasonic-assisted extraction is performed 3-4 times. The extracts are collected and combined, and concentrated and spray-dried to obtain a composition.

3. The composition according to claim 2, Features: The mixed raw materials are crushed to a particle size of 50-80 mesh.

4. The composition according to claim 2, Features: The mass volume ratio of the mixed raw material powder to water is 1:2-4 in g:mL. The added amount of α-amylase is 0.5-3% of the mass of the mixed raw material powder. The added amount of pectinase is 0.5-2.5% of the mass of the mixed raw material powder. The added amount of cellulase is 1-5% of the mass of the mixed raw material powder.

5. The composition according to claim 2, Features: The mass volume ratio of the mixed raw material powder to edible alcohol is 1:2-1:

4.

6. The composition according to claim 2, Features: The ultrasonic power is 200-500W, and the ultrasonic extraction time is 30-60min.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of insulin sensitizers, DPP-IV enzyme inhibitors and xanthine oxidase inhibitors.

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