CN113615756A - Preparation method and application of compound Pu' er tea functional composition - Google Patents
Preparation method and application of compound Pu' er tea functional composition Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a compound Pu' er tea functional composition, and belongs to the field of biotechnology and medicine. The effective parts of Pu' er tea, dendrobium and morus plants are subjected to water extraction, alcohol precipitation, organic solvent extraction, concentration, drying and other steps to obtain extracts, and the composition for improving high fat and high sugar diet induced diseases is obtained through compounding. The composition can improve hyperglycemia, hyperlipidemia and non-alcoholic fatty liver induced by high-fat high-sugar diet; compared with the single use of the Pu' er tea extract, the effect is obvious, and the application value is obvious. The raw materials in the compound functional food belong to homologous plants of medicine and food, are green and healthy, have low side effect and good safety, are easy to realize industrialization, and have obvious social benefit and economic value.
Description
Technical Field
The invention belongs to the field of biotechnology and medicine, and discloses a preparation method of a compound Pu 'er tea functional composition and application of the compound Pu' er tea functional composition in improvement of high-fat high-sugar diet induced diseases.
Background
With the social development and the improvement of living standard of people, the intake of high-fat high-sugar diet is increased year by year, and the long-term consumption of high-fat high-sugar food is easy to generate comprehensive influence on human bodies, so that the human bodies are in a sub-health state. High fat and high sugar diet can cause fat accumulation, form obesity, and increase the content of triglyceride, total cholesterol and low density lipoprotein in blood, and can cause hyperlipidemia such as atherosclerosis or cardiovascular and cerebrovascular diseases. High-fat and high-sugar diet can also cause the increase of blood sugar level, so that the load of organs such as liver, pancreas and the like is increased, insulin resistance is caused, and type 2 diabetes is caused; this process is also accompanied by metabolic disorders of carbohydrates, proteins, fats, etc., damages blood vessels of the whole body, affects tissues and organs important to the body such as nerves, heart and kidney, and induces various diabetic complications. High-fat and high-sugar diet is also the main cause of non-alcoholic fatty liver, and the main pathological features of the diet are in hepatic lobules, and the accumulation of triglyceride in hepatic cells, the fatty degeneration of diffuse hepatic cells and the fatty vacuole (Journal of Gastroenterology and Hepatology,2010,22: 293-.
At present, the medicines for clinically treating the high-fat high-sugar induced diseases mainly take western medicines, such as biguanides, thiazolidinediones, sulfonylureas, meglitinides, alpha-glycosidase inhibitors and the like for treating hyperglycemia; fibrates and statins for the treatment of hyperlipidemia; insulin sensitizer for treating non-alcoholic fatty liver, antioxidant, microecological preparation, and medicines for reducing blood fat and body weight. However, the medicines for treating the diseases induced by high-fat and high-sugar diets have toxic and side effects, for example, Western medicines for reducing blood sugar often have intestinal reactions such as abdominal distension, abdominal pain, nausea, vomiting and the like, and have side effects such as metabolic poisoning, weight gain, hypoglycemia, hypotension, secondary failure and the like; the lipid-lowering western medicine has the defects of gastrointestinal side effects, liver function influence and the like; meanwhile, liver injury and gastrointestinal side effects are also the main side effects of the non-alcoholic liver disease treatment drugs.
The natural product is commonly used for treating diseases such as hyperglycemia, hyperlipidemia, non-alcoholic fatty liver disease and the like, has the characteristics of mild drug property, small toxic and side effects and the like, can play the roles of reducing blood sugar and blood fat and improving the non-alcoholic fatty liver disease in a multi-path and multi-target mode of regulating and controlling the expression and secretion of in-vivo protein and hormone, regulating and controlling intestinal flora and the like, and has important application in the field of improving high-fat and high-sugar diet induced diseases.
The Pu' er tea is a tea product deeply popular with the masses, and has a long history of planting and application. The traditional Chinese medicine and pharmacy research shows that the Pu' er tea has the effects of warming stomach and nourishing spleen, clearing and nourishing balance, and avoiding excessive body fluid damage and fluid consumption. Modern scientific researches find that main active substances of the Pu' er tea comprise flavonoid components such as tea polyphenol, catechin, flavandiol and the like, tea pigment, phenolic acid and the like. Recent researches also show that a series of enzymatic and non-enzymatic reactions can be generated in the fermentation processing and storage processes of the Pu 'er tea, so that the content of tea pigment is changed, for example, the content of theaflavin and thearubigins is reduced after fermentation, the content of theabrownin is increased, and the change of the content of the tea pigment can endow the Pu' er tea with the effects of improving diseases induced by high fat and high sugar, for example, the theabrownin has the health-care effects of reducing fat and losing weight. However, the weight-losing effect of the Pu ' er tea used alone is weaker, for example, researches show that 3000mg of instant Pu ' er tea can show the effect of reducing the weight of a mouse after the mouse is perfused with the Pu ' er tea every day. Human experiments show that 50 mg/(kg. d) of instant Pu 'er tea is orally taken for 4 months (equivalent to 3g of the instant Pu' er tea is orally taken by adults with 60kg of body weight every day), and the body weight is not obviously reduced (Nature Communications,2019,10, 4971).
Dendrobium nobile is a classic medicinal plant, and Dendrobium officinale is approved as a medicinal and edible homologous substance trial variety by the Weijian Commission of China in 1 month in 2020. Traditional Chinese medicine research shows that the dendrobium has sweet, light and slightly salty and cold property, and enters stomach, kidney and lung channels; can be used for treating yin deficiency and fluid deficiency, dry mouth, polydipsia, anorexia, retching, asthenic fever after disease, dim and unclear vision, and has the effects of benefiting stomach, promoting fluid production, nourishing yin and clearing heat. Modern researches find that the active ingredients of dendrobium have the highest polysaccharide content and unique structure, and are the material basis of various biological activities such as antioxidant activity, immunoregulation effect, anti-tumor effect and the like (food science, 2020,41: 8). The mulberry plant is also a medicine-food homologous plant, and the effective parts of the mulberry plant comprise mulberry leaves, mulberry twigs, mulberry bark and the like, are bitter and sweet in taste and cold in nature, enter lung and liver channels, and have the effects of dispelling wind, clearing heat, calming liver and improving eyesight. The flavone component of the effective part of the mulberry plant has wide biological and pharmacological activities, such as antioxidation, oxygen free radical scavenging, anti-inflammatory and bactericidal effects and the like, and has extremely high medicinal value (silkworm science, 2005,31: 351-. At present, no report exists for improving high-fat and high-sugar diet induced diseases by compounding a Pu' er tea extract, a dendrobium polysaccharide extract and a mulberry plant effective part flavone extract.
Disclosure of Invention
The invention aims to solve the problem that the effect of Pu 'er tea on reducing blood sugar and blood fat and non-alcoholic fatty liver is weak, provides a compound functional composition containing extracts of effective parts of Pu' er tea, dendrobium and mulberry, is used for improving diseases induced by high-fat and high-sugar diet, is a composition with good effect and low side effect, and has wide application prospect.
The technical purpose of the invention is realized by the following technical scheme:
the invention provides a preparation method of a compound composition, which mainly comprises the following steps:
(1) adding water into Pu 'er tea, refluxing for 1-5h at 50-90 deg.C, filtering, repeating the above operation for more than two times, mixing filtrates, concentrating, and drying to obtain Pu' er tea extract;
(2) adding water into dried dendrobe according to the mass ratio of material liquid of 1 (10-60), stirring at the speed of 1000rpm for 1-4h, carrying out 100-500W ultrasonic crushing for 5-30min, adding trichloroacetic acid into the obtained extracting solution until the final concentration is 1.5% (v/v), precipitating protein, regulating the pH value to 5-8 by NaOH, centrifuging for 10-30min at the speed of 5000r/min at 3000-;
(3) adding an ethanol water solution into the dried effective parts of the Morus plants according to the mass ratio of the material liquid to 1 (3-15), leaching for 12-48h at room temperature, filtering to remove residues, evaporating to remove the solvent, adding petroleum ether for extraction, removing oil, adding an ethyl acetate-water solution with the volume ratio of 1:1 into the obtained suspension for extraction for more than two times, combining the obtained ethyl acetate phases, dehydrating by using anhydrous sodium sulfate, and evaporating to remove the solvent to obtain a flavone extract of the effective parts of the Morus plants;
(4) and (3) mixing the Pu' er tea extract obtained in the step (1), the dendrobium polysaccharide extract obtained in the step (2) and the effective part flavone extract of the mulberry plant obtained in the step (3) to obtain a compound composition.
Further, the mass ratio of the material liquid when the Pu' er tea is extracted and dried in the step (1) is 1 (1-6).
Further, the dendrobium in the step (2) is selected from dendrobium officinale, dendrobium nobile, dendrobium huoshanense, dendrobium chrysotoxum, dendrobium fimbriatum, dendrobium purpureum, dendrobium moniliforme, dendrobium loddium, dendrobium loddigesii, dendrobium nobile and dendrobium loddigesii.
Further, the final concentration of ethanol is 60-80% (v/v) when the dendrobium polysaccharides are precipitated in the step (2).
Further, the effective parts of the plants of the genus Morus in the step (3) include ramulus Mori, folium Mori and cortex Mori.
Further, the concentration of the ethanol aqueous solution when the effective parts of the Morus plants are extracted in the step (3) is 60-90% (v/v).
Further, the content of theabrownin in the Pu' er tea extract obtained in the step (1) is not less than 5%.
Further, the polysaccharide content in the dendrobium polysaccharide extract obtained in the step (2) is not less than 40%.
Further, the content of flavone in the effective part flavone extract of the mulberry plants obtained in the step (2) is not less than 13%.
The invention also provides a compound composition obtained by the preparation method, wherein the mass part ratio of the Pu' er tea extract, the dendrobium polysaccharide extract and the effective part flavone extract of the mulberry is (0.5-5): 1.
The invention also provides a functional food which comprises the compound composition.
The invention also provides application of the compound composition in food and/or medicines for improving high-fat high-sugar diet-induced diseases.
Further, the dosage forms of the food and/or the medicine comprise tablets, capsules, oral liquid, granules, powder for taking by water, powder, solution and the like; oral dosage forms are preferred, such as: powder, oral liquid, etc. are taken with water.
Further, the high-fat high-sugar diet-induced diseases are hyperglycemia, hyperlipidemia and non-alcoholic fatty liver.
Further, the food and/or the medicine can enhance the hypoglycemic effect by inhibiting the activity of alpha-glucosidase and improving insulin sensitivity.
Further, the food and/or pharmaceutical product enhances the lipid-lowering effect by increasing adsorption of fats and oils, losing weight, preventing adipocyte hypertrophy, and lowering total cholesterol level.
Further, the food and/or pharmaceutical product can enhance the effect of improving non-alcoholic fatty liver disease by preventing hepatomegaly and hepatonecrosis, reducing fat vacuoles, etc.
Advantages and positive effects of the invention
The invention provides a functional composition compounded by Pu 'er tea, dendrobium and mulberry plant effective part extracts, which is obtained by efficiently and selectively extracting effective components in the Pu' er tea, the dendrobium and the mulberry plant effective parts, and the extracts show stronger effect of improving high-fat high-sugar diet induced diseases through compounding. The main points are as follows: can obviously reduce the blood sugar level of the organism, enhance the insulin sensitivity and further improve the hyperglycemia symptom; can obviously enhance the adsorption effect of grease, reduce the content of total cholesterol and reduce the weight, thereby improving the symptom of hyperlipidemia; can remarkably prevent hepatomegaly and hepatonecrosis and reduce fat vacuole, thereby improving non-alcoholic fatty liver induced by high-fat high-sugar diet; compared with the effect of single use of Pu' er tea, the tea has obvious synergistic effect and obvious application value. The raw materials in the compound functional food belong to homologous plants of medicine and food, and the compound functional food has the advantages of greenness, health, low side effect, good safety and the like, is suitable for industrial application, and has obvious social benefit and economic value.
Drawings
FIG. 1: the functional compositions with different proportions have the inhibition effect on the activity of alpha-glucosidase; FIG. A: the inhibition effect of the composition compounded by the Pu' er tea extract, the dendrobium polysaccharide extract and the mulberry leaf flavone extract on the activity of alpha-glucosidase is achieved; and B: the inhibition effect of the compound composition of the Pu' er tea extract, the dendrobium polysaccharide extract and the mulberry twig flavone extract on the activity of alpha-glucosidase is achieved; wherein C1-C3 is the single-use group of Pu' er tea extract; MLF: a Pu' er tea extract, a dendrobium polysaccharide extract and a mulberry leaf flavone extract; MF: a Pu' er tea extract, a dendrobium polysaccharide extract and a mulberry twig flavone extract; MLF1-7 and MF1-7 are compound groups with different ratios, and the ratios of the components are shown in example 9.
FIG. 2: the in vivo hypoglycemic effect of the compound functional composition is realized; FIG. A: the effect of a functional composition on postprandial blood glucose (OGTT) in prediabetic mice; and B: effect of functional composition on area under the postprandial blood glucose (OGTT) curve of pre-diabetic mice. Statistical analysis: there were no significant differences between the same letters in panel B, and significant differences between different letters (p < 0.05); wherein, NC: a normal control group; pre: a pre-diabetic group; MET: a metformin positive control group; t: the Pu' er tea extract is used singly; MLF: the folium Mori flavone extract is used singly; DP: the dendrobium polysaccharide extract is singly used; MLF + T: and compounding a functional composition 1 group, wherein the mass ratio of the Pu' er tea extract, the dendrobium polysaccharide extract and the mulberry leaf flavone extract is 5:2: 2.
FIG. 3: the compound functional composition improves insulin resistance in vivo; FIG. A: the influence of the compound functional composition on the insulin content of the prediabetic mouse; and B: the effect of the compound functional composition on insulin resistance index HOMA-IR of the prediabetic mouse. Statistical analysis: graph a and graph B data are shown as mean ± standard deviation, with no significant difference between the same letters and significant difference between different letters (p < 0.05); the abbreviations for the respective sample names in the figures have the same meanings as those in FIG. 2.
FIG. 4: the effects of the built functional composition on body weight and total cholesterol content in vivo. FIG. A: the influence of the Pu' er tea compound functional composition on the weight of the prediabetic mouse; and B: the influence of the Pu' er tea compound functional composition on the total cholesterol content of the prediabetic mouse. Statistical analysis: graph a and graph B data are shown as mean ± standard deviation, with no significant difference between the same letters and significant difference between different letters (p < 0.05); wherein, MF: using the single mulberry twig flavone extractive; and MF + T, Pu 'er tea compounding functional composition 2 groups, wherein the mass ratio of the Pu' er tea extract to the dendrobium polysaccharide extract to the mulberry leaf flavone extract is 5:2:2, and the abbreviations of other sample names have the same meanings as those in figure 2.
FIG. 5: the influence of the compound Pu' er tea functional composition on the change of the adipose tissue form in vivo; the abbreviations for the respective sample names in the figures have the same meanings as those in FIG. 2.
FIG. 6: the influence of the compound Pu' er tea functional composition on liver tissues in vivo; the abbreviations for the respective sample names in the figures have the same meanings as those in FIG. 2.
Detailed Description
For further explanation of the contents, features and effects of the present invention, the following embodiments are described in detail with reference to the accompanying drawings. It should be noted that the following examples are non-limiting and do not limit the invention in any way.
Materials and methods for use in the invention
Unless otherwise specified, the concentration of the liquid solution in the present specification means mass concentration. The solvents used for extraction in the invention are all analytical pure reagents.
(I) test materials
1. Experimental animals: SPF grade C57BL/6 mice, purchased from Liaoning Biotechnology Ltd.
2. The main reagents are as follows: streptozotocin (STZ) was purchased from solibao limited; the insulin detection kit is purchased from Wuhan Huamei Co., Ltd; the Total Cholesterol (TC) detection kit is purchased from Nanjing construction Co., Ltd; high-fat high-sugar feed, type: d12451, available from Changzhou mouse-mouse two Biotech Co., Ltd
(II) Experimental analysis method
1. Analyzing the content of tea pigment in the Pu' er tea extract:
weighing 3g of Pu' er tea extract, adding 125mL of water, carrying out boiling water bath for 10min, filtering with 102 filter paper, and cooling the filtrate to room temperature; weighing 50mL of the filtrate, adding 50mL of ethyl acetate, shaking for 5 minutes, separating the filtrate, and diluting 4mL of ethyl acetate layer (upper layer) to 25mL (solution A) by using 95% ethanol; measuring 2mL of water layer, and adding 6mL of water, 2mL of saturated oxalic acid and 15mL of 95% ethanol to form a total system of 25mL (solution D); then, 25mL of ethyl acetate layer (upper layer) was measured and 25mL of NaHCO was added3(2.5g/100mL), shake for 30 s; diluting the 4mL ethyl acetate layer with 95% ethanol to a constant volume of 25mL (solution C); in addition, 25mL of tea filtrate is measured, added with 25mL of butanol, shaken for 3 minutes, and after the mixture is layered, 2mL of water layer is taken, and 6mL of water, 2mL of saturated oxalic acid solution and 15mL of 95% ethanol are added to form 25mL of total system (solution B). The absorbance of each group was measured at 380nm, and the contents of Theaflavins (TFs), Thearubigins (TRs) and Theabrownins (TBs) were calculated according to the formulas (1) to (3).
EA, EB, EC and ED are respectively the corresponding absorbance values of the solutions A, B, C and D
m-percentage of dry matter content of the sample%
2. Analyzing the content of the dendrobium extract polysaccharide:
preparation of a standard curve, accurately weighing 0.5g of analytically pure glucose, dissolving the analytically pure glucose in 10mL of distilled water to prepare a 50mg/mL standard glucose solution, diluting the standard solution into 0, 0.05, 0.1, 0.15, 0.2, 0.25 and 0.5mg/mL solutions respectively, adding 100 mu L of each solution into a test tube, adding 200 mu L of 50mg/mL phenol solution, mixing uniformly, immediately adding 1mL of concentrated sulfuric acid into each tube, mixing uniformly by vortex, standing for 30min, and measuring the absorbance at 490 nm. Taking the mass concentration of the glucose as an abscissa (x) and the absorbance as an ordinate (y), linear fitting is performed to obtain a standard curve (in terms of glucose) of the polysaccharide (formula 4).
y＝0.7291x+0.1395R2＝0.9928 (4)
The determination method of the total polysaccharide content of the dendrobium in the sample is basically the same as the determination method of the standard curve, and only the analytically pure glucose is replaced by the dendrobium extract sample.
3. The method for analyzing the total flavonoids of the effective parts of the mulberry plants comprises the following steps:
drawing a standard curve: precisely sucking 0mL, 0.2mI, 0.4mL, 0.6mL, 0.8mL and 1mL of rutin standard substance working solution (0.2mg/mL) respectively and placing in a test tube. Adding 3mL of 60% ethanol solution and 5% NaNO in sequence2Solution 1mL, 10% Al (NO)3)31mL of the solution and 4mL of a 4% NaOH solution, adding water to the system to be 10mL, and shaking up. Absorbance was measured at 510 nm. Taking rutin mass concentration (mg/mL) as a horizontal coordinate (x),and (5) performing linear fitting to obtain a standard curve (in terms of rutin) of the flavone by taking the light absorption value as a vertical coordinate (y) (formula 5).
y＝2.9268x+0.0031 R2＝0.9957 (5)
The method for measuring the content of total flavonoids in effective parts of the mulberry plants is basically the same as the standard curve measuring method, and rutin is replaced by an extract sample of the effective parts of the mulberry plants.
4. The method for measuring the alpha-glucosidase inhibitory activity comprises the following steps:
the mice were killed by neck-breaking after fasting for 24h without water prohibition, the small intestine sections were weighed, minced, placed in PBS buffer at 4 ℃ at a ratio of 0.02g/L, and ground to homogeneity on ice with a glass homogenizer. The obtained small intestine homogenate is centrifuged at 12000g/min at 4 ℃ for 5min, and the supernatant is the alpha-glucosidase solution and stored at-20 ℃. The reaction was carried out in PBS buffer in a total volume of 250. mu.L (125. mu.L of LPBS buffer + 50. mu.L of enzyme solution + 25. mu.L of sample solution + 50. mu.L of 1g/20mL of maltose substrate), and the enzyme solution of the blank was replaced with an equal volume of PBS buffer. The sample solution of the proenzyme group was replaced with an equal volume of PBS buffer. The system was incubated at 37 ℃ for 20min and the reaction was stopped in a boiling water bath for 5 min. The reaction product was cooled to room temperature, the concentration of glucose produced was measured by the glucose oxidase method in the glucose assay kit, the OD value at 505nm was measured to show the concentration of produced glucose, the alpha-glucosidase inhibition rate was calculated according to the formula (6), and this was repeated three times.
Inhibition (%) - (OD proenzyme-OD sample)/(OD proenzyme-OD blank) × 100% (6)
5. Oil adsorption test:
accurately weighing Pu 'er tea extract or compound Pu' er tea composition (W1) in a 50mL centrifuge tube, adding 6.0g of edible peanut oil, standing for 1h in a constant temperature environment at 37 ℃, then centrifuging at 4500r/min for 20min, discarding an upper oil layer, sucking the free peanut oil on the precipitate with paper, weighing to obtain W2, and calculating the oil absorption according to formula (7):
oil absorption (g/g) ═ W2-W1)/W1 (7)
6. Oral glucose tolerance test (OGTT test):
mice were fasted overnight without water deprivation, and each mouse was injected intraperitoneally with 2g/Kg glucose solution. The concentration of the glucose solution is 200mg/ml, and the injection dosage of the mouse with the weight of 20g is 0.2 ml. And measuring the blood sugar value of the mouse by adopting a tail vein blood sampling mode at 0min, 30min, 60min, 90min and 120min after the glucose is injected.
Example 1: preparation of Pu' er tea extract 1
Weighing 100g of dried Pu 'er tea, mixing with 400mL of distilled water, refluxing at 70 ℃ for 2h, filtering, repeating for three times, combining the filtrates, concentrating and drying to obtain the Pu' er tea extract. According to the determination method of the tea pigment in the formulas (1) to (3), the content of theabrownin is measured to be 6.47 percent, which is higher than 0.27 percent of theaflavin and 1.91 percent of thearubigin, and the theabrownin is the main tea pigment in the Pu' er tea extract.
Example 2: preparation of dendrobe polysaccharide 1
Mixing dried Dendrobium devonianum 10g with 200mL of distilled water, stirring at 500rpm for 2h, carrying out ultrasonic crushing at 400W for 20min, adjusting pH to 7.0 by citric acid, adding trichloroacetic acid until the final concentration (v/v) of precipitated protein is 1.5%, adjusting pH to 7 by NaOH, centrifuging at the rotation speed of 4000r/min for 10min, collecting supernatant, carrying out vacuum concentration at 65 ℃ to 0.2 time of the original volume, adding absolute ethyl alcohol into the concentrated solution until the final concentration is 80%, standing overnight at 4 ℃, centrifuging at 4000r/min for 10min, taking precipitate, and carrying out freeze drying at-50 ℃ to obtain the dendrobium polysaccharide extract. The total polysaccharide content was found to be 51% according to the polysaccharide assay described above.
Example 3: preparation of ramulus mori flavones 1
Weighing 50g of dried mulberry twig tender skin, crushing, adding 500mL of 80% ethanol aqueous solution, and leaching for 24h at room temperature. And (3) carrying out suction filtration on the crude extract for 2 times by using filter paper, evaporating the solvent by using a rotary evaporator, adding petroleum ether to remove grease, adding ethyl acetate-water solution with the volume ratio of 1:1 into the suspension for extraction for three times, combining ethyl acetate phases, dehydrating by using anhydrous sodium sulfate, and evaporating the solvent to dryness to obtain the mulberry twig flavone extract. The content of total flavonoids was found to be 34% according to the above method for determining total flavonoids.
Example 4: preparation of flavones from mulberry leaves 1
Weighing 50g of dry mulberry leaves, crushing, adding 500mL of 80% ethanol water solution, and leaching for 24h at 4 ℃ to obtain an extracting solution. Filtering the extract to remove residues, evaporating to remove ethanol, adding petroleum ether for extraction to remove oil, adding ethyl acetate-water solution with volume ratio of 1:1 into the suspension for extraction for three times, combining ethyl acetate phases, dehydrating with anhydrous sodium sulfate, and evaporating to remove solvent to obtain folium Mori total flavone; the total flavone content was found to be 15.7% according to the flavone assay described above.
Example 5: preparation of Pu' er tea extract 2
The procedure of example 1 was followed except that the amount of water added was adjusted to 200mL (feed-to-liquid ratio: 1: 2). According to the method for measuring the tea pigment, the contents of theaflavin, thearubigin and theabrownin of the Pu' er tea extract are measured. The result shows that the theabrownin content is 5.78 percent, is higher than 0.30 percent of theaflavin and 1.79 percent of thearubigin, and is the main tea pigment in the Pu' er tea extract.
Example 6: preparation of dendrobe polysaccharide 2
According to the method of the embodiment 2, only the dendrobium devonianum is replaced by the dendrobium officinale, the adding amount of the distilled water is changed to 400ml (the material-liquid ratio is 1:40), and the final ethanol concentration of the precipitated polysaccharide is adjusted to 60%. The total polysaccharide content was measured to be 45% according to the polysaccharide assay described above.
Example 7: preparation of ramulus mori flavones 2
The procedure of example 3 was followed except that the ethanol concentration of the extraction was adjusted to 70%. The content of total flavonoids was found to be 33.5% according to the above method for measuring total flavonoids.
Example 8: preparation of folium Mori flavone 2
The procedure of example 4 was followed except that the amount of the extracted ethanol water added was adjusted to 250mL (feed-to-liquid ratio: 1: 5). The total flavone content was found to be 14.9% according to the flavone assay described above.
Example 9: compounding of Pu' er tea extract, dendrobium polysaccharide extract and effective part flavone extract of mulberry
Compounding Pu 'er tea extract, dendrobe polysaccharide extract and mulberry plant effective part flavone extract according to different proportions, wherein MF1-MF7 are the compounds of Pu' er tea extract, dendrobe polysaccharide and mulberry branch flavone; MLF1-MLF7 is a compound of Pu' er tea extract, dendrobium polysaccharide and mulberry leaf flavone. The proportion of Pu' er tea extract, dendrobium total polysaccharide and effective part flavone of mulberry in each compound composition is shown in table 1.
Table 1: mass ratio of compounded functional composition
Example 10: inhibiting effect of compound combinations with different proportions on alpha-glucosidase activity in vitro
According to the method for determining the alpha-glucosidase inhibitory activity, the inhibitory effect of the compound composition with different proportions on the alpha-glucosidase activity in example 9 is determined, and three single doses of Pu 'er tea extracts, namely C1, C2 and C3, are set, wherein the final concentration of C1 is 200 mug/mL, the final concentration of the Pu' er tea extracts in a compound group of MF1, MF 4-MF 7, MLF1, MLF 4-MLF 7 is the same, the final concentration of C2 is 500 mug/mL, the final concentration of the Pu 'er tea extracts in a compound group of MF2 and MLF2 is the same, and the final concentration of C3 is 1000 mug/mL, the final concentration of the Pu' er tea extracts in a compound group of MF3 and MLF3 is the same. The results of the samples for inhibiting the activity of the alpha-glucosidase are shown in figure 1, and the results show that the inhibition rates of the alpha-glucosidase activity of the Pu ' er tea extract, the MF1-MF7 compounded by the dendrobe polysaccharide and the mulberry leaf flavone, or the alpha-glucosidase activity inhibition rates of the Pu ' er tea extract, the MLF1-MLF7 compounded by the dendrobe polysaccharide and the mulberry leaf flavone are obviously higher than those of a corresponding Pu ' er tea extract single-use group. For example, the alpha-glucosidase inhibition rate of the compound groups MF1, MF 4-7, MLF1 and MLF 4-7 is more than 10 times that of the single group C1; the inhibition rate of alpha-glucosidase of the compound groups MF2 and MLF2 is more than 7 times that of the single group C2; the inhibition rate of alpha-glucosidase of the compound groups MF3 and MLF3 is more than 2 times of that of the single group C3. The compound composition with different proportions shows better hypoglycemic activity than that of the Pu' er tea extract when being used alone.
Example 11: adsorption of compound combinations with different proportions on grease in vitro
Research shows that the lipid absorption capacity of the compound puer tea can reflect the lipid reduction effect of drugs or functional Foods (Chem Foods 2003,80,221-229), the invention takes MLF1, MLF2 and MLF3 prepared in example 9 to investigate the lipid absorption effect of the compound puer tea functional composition, and similarly, the puer tea extract is used for controlling a group C1 alone, and the content of the puer tea extract is the same as the content of the puer tea extract in MLF 1. The results are shown in table 2, the oil absorption per gram of each group of the Pu ' er tea composition MLF1, MLF2 and MLF3 compounded at different ratios is respectively 1.58 times, 2.34 times and 4.62 times of that of the Pu ' er tea extract used alone, and the Pu ' er tea compound functional food at different ratios obviously improves the oil adsorption effect, so that the lipid-lowering effect is improved.
Table 2: adsorption effect of compound Pu' er tea functional composition on grease
Example 12: hypoglycemic effect of compound Pu' er tea functional composition 1
According to the in vitro activity result, the compounding ratio of the compound composition represented in the example 9 is selected to carry out the pharmacodynamic function research of in vivo experimental animals.
In-vivo experiments of animals, a high-fat high-sugar auxiliary streptozotocin-induced prediabetes model is used, the prediabetes is the early stage of type 2 diabetes mellitus, has characteristics similar to those of type 2 diabetes mellitus, namely abnormal blood sugar, insulin resistance and the like, can represent dyslipidemia closely related to the abnormal blood sugar, can also represent diseases such as nonalcoholic fatty liver and the like, and is a better model for evaluating high-fat high-sugar diet-induced diseases (Journal of Functional Foods,2017,37, 339-.
1. Establishment of prediabetic mouse model
A breeding environment: the experimental animals are raised in an animal room of a barrier system at the temperature of 22 +/-2 ℃ and the relative humidity of 40-60%, the illumination time is controlled to be half of the day and the dark day, the animals are raised in cages, feed and drink water freely, padding materials are replaced at regular time, and mouse cages are cleaned. The experiment was started one week after acclimatizing feeding of the experimental animals.
The prediabetes model was made by referring to published literature and modifying appropriately (Journal of Functional Foods,2017,37,339-353), using 18-22 g of C57BL/6 mice, after continuously feeding mice with high-fat high-sugar diet as shown in Table 1 for 2 weeks, each mouse was intraperitoneally injected with 45mg/kg Streptozotocin (STZ), and after feeding with high-fat high-sugar diet for one week, the STZ-injected mice were subjected to Oral Glucose Tolerance Test (OGTT): the gastric perfusion amount of glucose is 2g/kg, the blood glucose value after 2 hours of meal is measured by a glucometer, a mouse with the blood glucose value more than or equal to 7.8mmol/L and less than 11.1mmol/L is selected, high-fat high-glucose food is continuously fed for 3 weeks to stabilize the blood glucose of the mouse, the selected mouse is subjected to an oral glucose tolerance test again, the mouse with the blood glucose value more than or equal to 7.8mmol/L and less than 11.1mmol/L after 2 hours of meal is selected as a model mouse, the model mouse and a normal mouse are randomly divided into 9 groups, 8 mice in each group are respectively labeled as male and female, and subsequent experiments are carried out.
2. Grouping of laboratory animals
Mice were divided into Normal Control (NC), model group (DC), positive control group, single use group, and compound group. The molded diabetic mice were fed with high-fat and high-sugar feed, and the normal group mice were fed with animal basal feed, fed freely, and gavaged every day at regular intervals. The compound group (compound Pu' er tea functional composition 1): the main components are Pu 'er tea extract, dendrobium polysaccharide extract and mulberry leaf flavone extract which are compounded according to the mass ratio of 5:2:2, and the specific dosage is the Pu' er tea extract (250mg/kg), the dendrobium polysaccharide (100mg/kg) and the mulberry leaf flavone (100 mg/kg); 3 single-use groups respectively comprise single-use (250mg/kg) of Pu' er tea extract and single-use (100mg/kg) of dendrobium polysaccharide; the flavonoids of mulberry leaf are singly used (100 mg/kg); the positive control was metformin (200mg/kg) and gavage was performed for 10 weeks.
After administration for 10 weeks, oral glucose tolerance test (OGTT test) is carried out, and the results are shown in figure 2, compared with a group 1 of the compound Pu 'er tea functional composition, a group with single Pu' er tea extract, a group with single dendrobium polysaccharide and a group with single mulberry flavone, the compound Pu 'er tea functional composition can obviously reduce the blood sugar level two hours after meal, and the blood sugar reduction effect of the compound Pu' er tea functional composition has no obvious difference with that of a positive control group (metformin). Simultaneously, the serum insulin (FINS) content and fasting blood glucose of each group are measured, and an insulin sensitivity index HOMA-IR value (HOMA-IR fasting blood glucose fasting insulin/22.5) is calculated, and the result shows that compared with each single-use group, the compound can obviously enhance the insulin sensitivity, has no obvious difference with a normal group (figure 3), and shows that compared with the single-use group of the Pu' er tea extract, the compound functional composition provided by the invention can obviously reduce the postprandial blood glucose, enhance the insulin sensitivity and further improve the hypoglycemic effect.
Example 13: lipid-lowering effect of compound Pu' er tea functional composition 1
The efficacy evaluation of the experimental animals is carried out according to the method of the embodiment 12, only the mulberry leaf flavone extract in the compound puer tea functional composition is replaced by the mulberry twig flavone extract to obtain the compound puer tea functional composition 2, and simultaneously, the mulberry leaf flavone extract is replaced by the mulberry twig flavone extract alone to be used as a contrast. The compound group (compound Pu' er tea functional composition 2) comprises the following components in percentage by weight: pu' er tea extract group (250mg/kg), dendrobe polysaccharide (100mg/kg) and mulberry twig flavone (100 mg/kg); 3 single-use groups: the single group of Pu' er tea extract (250mg/kg) and the single group of dendrobium polysaccharide extract (100 mg/kg); ramulus Mori flavone extract is used singly (100 mg/kg); the positive control was metformin (200mg/kg) and gavage was performed for 10 weeks. The body weight of each group of mice was measured, and the change in body weight was calculated. The total cholesterol content in blood samples is detected, and the result shows that compared with a single group of Pu ' er tea extract, dendrobium polysaccharide extract and mulberry twig flavone extract, the compound Pu ' er tea functional composition 2 group can remarkably reduce the weight and is superior to a positive control (figure 4A), which shows that the compound Pu ' er tea functional composition has better weight-reducing and lipid-lowering effects. Meanwhile, the compound Pu 'er tea functional composition 2 can also obviously reduce the content of total cholesterol (T-CHO) (figure 4B), which shows that the compound Pu' er tea functional composition enhances the effect of reducing blood fat by reducing weight and the content of total cholesterol.
Example 14 lipid-lowering action of Compound Puer tea functional composition 2
The adipose tissues of each group of examples 12 to 13 of the animal experiment were sectioned and observed for morphology and size. As shown in FIG. 5, the fat cells in the normal adipose tissues were small and no cell hypertrophy occurred; and the model group tissues have severe fat cell hypertrophy and fat accumulation. Although the single group of the Pu ' er tea extract, the single group of the dendrobium polysaccharide extract and the single group of the mulberry leaf/mulberry twig flavone extract have certain effect of improving fat cell hypertrophy, the effect is weaker, after the compound Pu ' er tea functional compositions 1 and 2 are administrated, the fat tissue cell hypertrophy phenomenon is obviously weakened, the lipid accumulation condition is obviously improved, and the compound Pu ' er tea functional compositions are close to the normal group. Therefore, the compound Pu' er tea functional composition enhances the lipid-lowering effect by weakening fat cell hypertrophy and improving lipid accumulation.
Example 15 Effect of the Compound Puer tea functional composition on improving non-alcoholic fatty liver disease
Liver tissues of each group of examples 12 to 13 of the animal experiment were sectioned to observe the lesion and improvement. The results are shown in FIG. 6, in which the liver cells of the liver tissue of the normal group were uniform in size and free from abnormalities and lesions; the liver of the model group has serious pathological changes, is a typical symptom of the nonalcoholic fatty liver, and is represented by the conditions of cell swelling, serious adiposity, even large-area cell necrosis, fat vacuole and the like. Although the single group of Pu' er tea extract, the single group of dendrobium polysaccharide extract or the single group of mulberry leaf/mulberry twig flavone extract can improve symptoms, the effects are weaker. After the compound Pu' er tea functional compositions 1 and 2 are administered, the degrees of hepatocyte cell enlargement and hepatocyte necrosis are obviously weakened, and the fat vacuoles basically disappear and are close to the normal group. Therefore, the Pu' er tea compound composition enhances the improvement effect on the non-alcoholic fatty liver by preventing hepatocyte enlargement and hepatocyte necrosis, reducing fat vacuole and the like.
Claims (9)
1. The preparation method of the compound composition is characterized by mainly comprising the following steps:
(1) adding water into Pu 'er tea, refluxing for 1-5h at 50-90 deg.C, filtering, repeating the above operation for more than two times, mixing filtrates, concentrating, and drying to obtain Pu' er tea extract;
(2) adding water into dried dendrobe according to the mass ratio of material liquid of 1 (10-60), stirring at the speed of 1000rpm for 1-4h, carrying out 100-500W ultrasonic crushing for 5-30min, adding trichloroacetic acid precipitated protein into the obtained extracting solution, adjusting the pH to 5-8 by using alkali liquor, centrifuging at 5000r/min of 3000-;
(3) adding the active ingredients of the dried mulberry plants into the dried mulberry plant active ingredients according to the mass ratio of 1: (3-15) adding an ethanol water solution, leaching at room temperature for 12-48h, filtering to remove residues, evaporating to remove the solvent, adding petroleum ether for extraction, removing oil, adding an ethyl acetate-water solution with a volume ratio of (1:2) - (2:1) into the obtained suspension for extraction for more than two times, combining the obtained ethyl acetate phases, and evaporating to remove the solvent to obtain the effective part flavone extract of the Morus plant;
(4) and (3) mixing the Pu' er tea extract obtained in the step (1), the dendrobium polysaccharide extract obtained in the step (2) and the effective part flavone extract of the mulberry plant obtained in the step (3) to obtain a compound composition.
2. The preparation method according to claim 1, wherein the mass ratio of the material liquid in the step (1) of extracting and drying the Pu' er tea is 1 (1-6).
3. The method according to claim 1, wherein the dendrobium in step (2) is selected from dendrobium officinale, dendrobium nobile, dendrobium chrysotoxum, dendrobium fimbriatum, dendrobium devonianum, dendrobium candidum and dendrobium loddigesii; the final concentration of ethanol is 60-80% (v/v) when the dendrobium polysaccharides are precipitated.
4. The method according to claim 1, wherein the effective parts of Morus plants in step (3) comprise ramulus Mori, folium Mori and cortex Mori; the ethanol water solution concentration is 60-90% (v/v) when extracting effective components of Morus plant.
5. A compounded composition is characterized by being prepared by the preparation method of any one of claims 1 to 4, wherein the mass part ratio of the Pu' er tea extract, the dendrobium polysaccharide extract and the effective part flavone extract of the mulberry is (0.5-10) to (0.5-10) 1.
6. A functional food comprising the formulated composition of claim 5.
7. The use of the formulated composition of claim 5 for the preparation of a food and/or a pharmaceutical product for ameliorating high-fat high-sugar induced diseases.
8. The use according to claim 7, wherein the food and/or pharmaceutical product is capable of ameliorating high-fat high-sugar diet-induced diseases including hyperglycemia, hyperlipidemia and non-alcoholic fatty liver disease.
9. The use according to claim 7 or 8, wherein the food and/or pharmaceutical product enhances the hypoglycemic effect by inhibiting α -glucosidase activity, increasing insulin sensitivity; the food and/or medicine can enhance lipid-lowering effect by increasing adsorption of oil and fat, reducing weight, preventing fat cell hypertrophy and lowering total cholesterol content; the food and/or medicine can improve the effect of non-alcoholic fatty liver by preventing hepatomegaly and hepatonecrosis and reducing fatty vacuole.
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| CN1706837A (en) * | 2005-04-30 | 2005-12-14 | 苏州大学 | Extraction process of total mulberry twig flavone |
| CN102524464A (en) * | 2012-02-22 | 2012-07-04 | 红云红河烟草(集团)有限责任公司 | Preparation method of dendrobe health-care Pu'er tea cream |
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| CN1706837A (en) * | 2005-04-30 | 2005-12-14 | 苏州大学 | Extraction process of total mulberry twig flavone |
| CN102524464A (en) * | 2012-02-22 | 2012-07-04 | 红云红河烟草(集团)有限责任公司 | Preparation method of dendrobe health-care Pu'er tea cream |
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