Composition containing cyclocarya paliurus extract and application thereof

Abstract

The invention discloses a composition containing a cyclocarya paliurus extract and application of the composition. The invention provides a composition (NGI extract) containing a cyclocarya paliurus extract. The flavone content of the composition is greater than 50%. The composition containing the cyclocarya paliurus extract has the application of reducing blood sugar and blood fat.

Description

Technical Field

[0001] The invention relates to the application field of traditional Chinese medicine, and in particular to a composition containing a Cyclocarya paliurus extract and application thereof. Background Art

[0002] Cyclocarya paliurus is a plant of the genus Cyclocarya paliurus in the Juglandaceae family of the dicotyledon class. It is also known as the money tree and sweet tea tree because its fruits resemble copper coins and its leaves have sweet taste. It is widely distributed in areas south of the Yangtze River, such as Hunan, Jiangxi, Zhejiang, Anhui, and Guangdong. A large number of wild ancient Cyclocarya paliurus trees grow in the primeval forests in southwestern Hunan, which is one of its important places of origin. So far, more than 210 secondary metabolites have been extracted and isolated from Cyclocarya paliurus.

[0003] This type of compound has also been reported to have a variety of pharmacological activities, and has potential efficacy in anti-cancer, hypoglycemic and anti-inflammatory pharmacological activities. However, some extracts of Cyclocarya paliurus have not yet been discovered. As a unique medicinal and edible plant in my country, Cyclocarya paliurus is rich in active ingredients such as triterpenes, flavonoids, and polysaccharides in its leaves. Modern pharmacological studies have confirmed that it has antioxidant and immunomodulatory effects.

[0004] However, the research on Cyclocarya paliurus extract by researchers in this field is not comprehensive enough, so further research on the uses of Cyclocarya paliurus still has important economic value. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the application of Cyclocarya paliurus extract is not comprehensive enough. To this end, the present invention provides a composition containing Cyclocarya paliurus extract and its application. The composition containing Cyclocarya paliurus extract. The composition containing Cyclocarya paliurus extract has the therapeutic use of lowering blood sugar and blood lipids.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The present invention provides a composition of a Cyclocarya paliurus extract, which is prepared by the following preparation method: (1) crushing Cyclocarya paliurus fallen leaves and mixing them with water, stirring at 40-80°C (for example, 50°C, 60°C, 70°C or 80°C) for 2-12 hours (for example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours), filtering to remove the Cyclocarya paliurus fallen leaf residue to obtain a filtrate, concentrating the filtrate and freeze-drying to obtain an extract A.

[0008] (2) Extract A is dissolved in water, filtered through a needle filter to remove insoluble matter, and extracted with a chloroform-alcohol solvent system to remove protein; then, the extract is dialyzed using a dialysis membrane to obtain a dialysate, and the dialysate is concentrated and freeze-dried to obtain Extract B.

[0009] (3) The extract B is purified and separated by a chromatographic column, the components containing polysaccharides are identified by a sulfuric acid-phenol method, and the components containing non-polysaccharides are combined, concentrated, and freeze-dried to obtain the composition of the Cyclocarya paliurus extract.

[0010] In the present invention, the content of flavonoids in the composition is greater than 50%, preferably greater than 56.8%.

[0011] In the present invention, in step 1, the weight ratio of the Cyclocarya paliurus fallen leaves to the water is 1:(2-10); preferably 1:(3-8).

[0012] In the present invention, in step 1, the weight ratio of the Cyclocarya paliurus fallen leaves to the water is 1:4.

[0013] In the present invention, in step 2, the weight ratio of the extract A to the water is 1:(10-50); for example, 1:40.

[0014] In the present invention, in step 2, the alcohol solvent is n-pentanol or n-butanol.

[0015] In the present invention, in step 2, the alcohol solvent is n-butanol.

[0016] In the present invention, in step 2, in the chloroform-alcohol solvent system, the volume ratio of the alcohol solvent to the chloroform is 1:(2-4); for example, 1:3.

[0017] In the present invention, in step 3, the chromatographic column is a DEAE Sephacel gel chromatographic column.

[0018] The present invention also provides a method for preparing a composition of Cyclocarya paliurus extract, and the preparation method is as described above.

[0019] The present invention provides a composition comprising the Cyclocarya paliurus extract.

[0020] In the present invention, the composition further comprises a plant extract, and the plant extract is extracted from one or more of Malus hupehensis, mulberry leaves, cinnamon, honeysuckle, Momordica grosvenori, licorice and mulberry branches.

[0021] The present invention also provides a use of the above Cyclocarya paliurus extract composition in the preparation of a drug for treating and / or preventing lowering blood sugar levels.

[0022] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0023] The reagents and raw materials used in the present invention are commercially available.

[0024] The positive and progressive effect of the present invention is that the present invention provides a composition containing the Cyclocarya paliurus extract. The composition containing the Cyclocarya paliurus extract has the purpose of lowering blood sugar and blood lipid. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The activation effect of Cyclocarya paliurus extract 1 on β-galactosidase. DETAILED DESCRIPTION

[0026] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional conditions, methods and conditions, or selected according to the product instructions.

[0027] Example 1: Step 1: Take 500 g of dried Cyclocarya paliurus fallen leaves, grind them and mix them with 2000 g of water, stir them at 70°C for 8 hours, remove the Cyclocarya paliurus fallen leaf residue to obtain a filtrate, reduce the pressure and concentrate the filtrate to about 100 mL, and then freeze-dry the concentrate at -78°C (dry ice ethanol bath) to obtain 25.6 g of extract A.

[0028] Step 2: Take 5 g of the above extract A, dissolve it in 200 mL of water, filter it through a needle filter (Φ25 μm) to remove insoluble matter, then extract it three times with a mixture of 60 mL of chloroform and 20 mL of an alcohol solvent (such as n-butanol or n-pentanol) to remove protein, and then dialyze it with a dialysis membrane with a molecular weight cutoff of 2000-10000 for 36 hours. After that, the liquid in the dialysis bag is concentrated to about 10 mL under reduced pressure, and it is freeze-dried at -78°C (dry ice ethanol bath) to obtain 1.2 g of extract B.

[0029] Step 3: The extract B is purified and separated by a DEAE Sephacel gel chromatography column, and the polysaccharide-rich components are identified by the sulfuric acid phenol method, which are combined, concentrated, and freeze-dried to obtain a polysaccharide-rich extract C; and then the other main peak substances are combined (combined with non-polysaccharide components) to obtain a flavonoid-rich Cyclocarya paliurus extract 1.

[0030] Example 2: Referring to the general method of Example 1, the only difference is that the stirring temperature in step 1 is 50°C, and Cyclocarya paliurus extract 2 is obtained.

[0031] Example 3: Referring to the general method of Example 1, the only difference is that the stirring temperature in step 1 is 60°C, and Cyclocarya paliurus extract 3 is obtained.

[0032] Example 4: Referring to the general method of Example 1, the only difference is that the stirring temperature in step 1 is 80°C, and Cyclocarya paliurus extract 4 is obtained.

[0033] Example 5: Referring to the general method of Example 1, the only difference is that the stirring temperature in step 1 is 90°C, and Cyclocarya paliurus extract 5 is obtained.

[0034] Example 6: Referring to the general method of Example 1, the only difference is that the stirring temperature in step 1 is 100°C, to obtain Cyclocarya paliurus extract 6.

[0035] According to the UV-Vis spectrophotometry method described in General Chapter 0401 of the 2015 edition of the Chinese Pharmacopoeia, the weight content of flavonoid compounds in the sample was measured with rutin as a reference; the weight content of polysaccharide compounds was measured with D-glucose as a control. The test results of the samples of Examples 1-5 are specifically shown in Table 1 below.

[0036] Table 1:

[0037] Note: “ / ” in the table means not tested.

[0038] From the above experimental results, it can be seen that within the range of 90-100°C, the content of flavonoids decreases significantly; within the range of 50-80°C, the content of flavonoids in the Cyclocarya paliurus extract is above 50%.

[0039] Effect Example 1: β-galactosidase assay method (ONPG).

[0040] This method is based on the enzymatic hydrolysis of o-nitrophenyl-β-D-galactoside (ONPG) to generate o-nitrophenol (ONP), and the activity of β-galactosidase is determined by measuring the absorbance change of ONP at 420 nm.

[0041] Experimental materials: Substrate: o-nitrophenyl-β-D-galactoside (ONPG).

[0042] Buffer: ; Enzyme solution: β-galactosidase sample to be tested.

[0043] Spectrophotometer: used to measure the absorbance at 420 nm.

[0044] Constant temperature water bath: to control the reaction temperature (37°C).

[0045] Test tubes, pipettes, and cuvettes: used for reactions and measurements.

[0046] Experimental steps: Reagent preparation: ONPG substrate solution: Dissolve ONPG in buffer to prepare a 4 mg / mL solution; then adjust the pH to 7.0 by adding the above buffer.

[0047] The solution of Cyclocarya paliurus extract 1 was prepared into a 1 mg / mL aqueous solution.

[0048] Detection of absorbance change rate: Add the following reagents into the cuvette: 0.7 mL buffer, 0.2 mL ONPG substrate solution, 0.1 mL enzyme solution (adjust the amount according to the enzyme activity). Then gently mix, immediately put into the spectrophotometer, and detect the absorbance change value for 1 minute.

[0049] Blank control: Add 0.8 mL of buffer and 0.2 mL of ONPG substrate solution to the blank control tube; then measure the absorbance of the blank control.

[0050] Enzyme activity calculation: Net absorbance: .

[0051] Definition of enzyme activity unit: One unit of enzyme activity (U) is defined as the amount of enzyme required to hydrolyze 1 μmol ONPG to produce o-nitrophenol (ONP) per minute at 37°C and pH 7.0.

[0052] Calculation formula: Enzyme activity (U / mL) = ΔA420 × Vtotal t × ε × d × Venzyme Enzyme activity (U / mL) = t × ε × d × Venzyme ΔA420 × Vtotal; where: ΔA420: net absorbance.

[0053] Vtotal: total reaction volume (mL).

[0054] t: reaction time (minutes).

[0055] ε: molar extinction coefficient of ONP (approx. ).

[0056] d: cuvette light path (usually 1 cm).

[0057] Venzyme: enzyme solution volume (mL).

[0058] Add the reagents in the cuvette according to the reagent dosage in Table 2, then gently mix, and immediately put it into the spectrophotometer to measure the absorbance change rate of the reaction solution in the cuvette at 37°C. The test results are as follows: Figure 1 shown.

[0059] Table 2:

[0060] The above experimental results show that the extract can significantly activate the activity of galactosidase, reaching a maximum of 287U / ml, an increase of about 130%.

[0061] Effect Example 2: Blood glucose experiment: 6-month-old male zebrafish were divided into a blank group (feeding water), a model group (1% glucose), a low-dose group 1 (1% glucose + 10 mg extract 1), a high-dose group 1 (1% glucose + 100 mg), a low-dose group 2 (1% glucose + 10 mg extract 2), a low-dose group 3 (1% glucose + 10 mg extract 3), a low-dose group 4 (1% glucose + 10 mg extract 4), a low-dose group 5 (1% glucose + 10 mg extract 5), a low-dose group 6 (1% glucose + 10 mg extract 6), and a positive control group (1% glucose + 10 mg metformin), with 10 fish in each group. First, the fish were cultured with 1% glucose (feeding water for the blank group) for 7 days, and the water was changed every day. The extract was added to the feeding water of the drug administration group from the 8th day, and metformin was added to the positive control group for 7 consecutive days, and the water was changed every day. After 7 days, the fish were taken out and placed in ice water. After the fish became rigid, they were taken out and dried with absorbent paper to remove the residual glucose on the surface of the fish. The fish tail was cut off and blood was sucked up by a test strip. The blood glucose level was tested with a blood glucose meter.

[0062] The test results are shown in Table 3 below: Table 3:

[0063] As shown in the table above, compared with the blank group, the blood glucose level in the model group was 1.9 times that of the blank group. In addition, compared with the positive control group, the blood glucose level in the model group was reduced by about 45%. This indicates that the zebrafish blood glucose model has been successfully established.

[0064] In addition, compared with the model group, the blood glucose values ​​of the low-dose group 1 and the high-dose group 1 were 2.83 mmol / L and 2.71 mmol / L, respectively. In addition, the effect of the low-dose group 1 was equivalent to that of the positive control group, and it can be seen that the blood glucose concentration was significantly reduced. It can be seen that the above experimental results fully demonstrate that the Cyclocarya paliurus extract of the present invention has an excellent hypoglycemic effect.

Claims

1. A composition of Cyclocarya paliurus extract, characterized in that: The extract is prepared by the following method: (1) crushing the fallen leaves of Cyclocarya paliurus and mixing them with water, stirring at 40-70°C for 2-12 hours, filtering to remove the residue of the fallen leaves of Cyclocarya paliurus to obtain a filtrate, concentrating and freeze-drying the filtrate to obtain extract A; (2) dissolving extract A in water, filtering through a needle filter to remove insoluble matter, extracting and removing protein with a chloroform-alcohol solvent system; then dialyzing with a dialysis membrane to obtain a dialysate, concentrating and freeze-drying the dialysate to obtain extract B; (3) purifying and separating extract B through a chromatographic column, identifying the components containing polysaccharides through a sulfuric acid-phenol method, combining, concentrating and freeze-drying the components of non-polysaccharides to obtain the composition of the Cyclocarya paliurus extract.

2. The composition of Cyclocarya paliurus extract according to claim 1, characterized in that In step 1, the weight ratio of the Cyclocarya paliurus fallen leaves to the water is 1:(2-10).

3. The composition of Cyclocarya paliurus extract according to claim 1, characterized in that In step 1, the weight ratio of the Cyclocarya paliurus fallen leaves to the water is 1:

4.

4. The composition of Cyclocarya paliurus extract according to claim 1, characterized in that In step 2, the weight ratio of the extract A to the water is 1:(10-50).

5. The composition of Cyclocarya paliurus extract according to claim 1, characterized in that In step 2, the alcohol solvent is n-pentanol or n-butanol.

6. The composition of Cyclocarya paliurus extract according to claim 5, characterized in that In step 2, the alcohol solvent is n-butanol.

7. The composition of Cyclocarya paliurus extract according to claim 1, characterized in that In step 2, in the chloroform-alcohol solvent system, the volume ratio of the alcohol solvent to the chloroform is 1:(2-4).

8. Use of the Cyclocarya paliurus extract composition according to any one of claims 1 to 7 in the preparation of a drug for lowering blood sugar levels.

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