Phyllanthus emblica polypeptide with effects of protecting liver and reducing blood fat and application of phyllanthus emblica polypeptide

Abstract

The invention discloses phyllanthus emblica polypeptide with effects of protecting liver and reducing blood fat and application thereof, and relates to the technical field of biology. The phyllanthus emblica polypeptide with the effects of protecting the liver and reducing the blood fat is obtained from phyllanthus emblica through enzymolysis separation for the first time, the phyllanthus emblica polypeptide is prepared by adopting a composite enzymolysis process and combining with an ultrasonic-assisted means, and the phyllanthus emblica polypeptide has excellent activity of protecting the liver and reducing the blood fat and has a relatively great application prospect and market.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a Phyllanthus emblica polypeptide having liver-protecting and blood-lipid-lowering effects and an application thereof. Background Art

[0002] Phyllanthus emblica is a plant belonging to the genus Phyllanthus in the family Euphorbiaceae. Its fruit is sweet and sour when eaten fresh, crisp and slightly astringent, with a sweet aftertaste, hence the name Phyllanthus emblica, also known as throat emblica, amla fruit, ox emblica fruit, etc. This product is spherical or oblate, with a diameter of 1.2-2cm. The surface is brown to dark green, with light yellow granular protrusions, wrinkles and inconspicuous 6 edges, and the fruit stalk is about 1mm. Phyllanthus emblica trees are drought-resistant and barren-resistant, with very strong adaptability, and like light and warmth. The growth period is 1-3 years. It is sweet, sour, astringent, and cool. It enters the lung and stomach meridians. It has the effects of clearing heat and cooling blood, promoting digestion and strengthening the stomach, and promoting body fluid and relieving cough.

[0003] Hyperlipidemia, also known as high blood lipids or dyslipidemia, usually refers to elevated triglycerides and (or) total cholesterol, elevated low-density lipoprotein cholesterol and decreased high-density lipoprotein cholesterol in plasma. The disease can occur in people of different ages and genders, especially in people aged 50 to 69, with a clear genetic tendency. The causes of the disease include gene mutations, a variety of environmental factors, such as poor eating habits, lack of physical activity, obesity, and other diseases such as diabetes, nephrotic syndrome, liver disease, etc. Existing treatments for hyperlipidemia cannot effectively achieve the purpose of protecting the liver and lowering blood lipids, and there is an urgent need to provide a new treatment method. Summary of the invention

[0004] In view of the technical problems existing in the prior art, the present invention provides a Phyllanthus emblica polypeptide with liver protection and blood lipid lowering effects and its application. The present invention obtains a Phyllanthus emblica polypeptide with liver protection and blood lipid lowering effects by enzymatic separation from Phyllanthus emblica for the first time. The preparation method adopts a composite enzymatic hydrolysis process combined with ultrasound-assisted means to prepare the Phyllanthus emblica polypeptide. The Phyllanthus emblica polypeptide has excellent liver protection and blood lipid lowering activities and has great application prospects and markets.

[0005] Specifically, the present invention first provides a method for preparing a polypeptide of Phyllanthus emblica, characterized in that the method comprises the following steps: 1) Wash the emblica buds, cut them into small pieces, and dry them with air at room temperature for later use; 2) putting the air-dried pieces of Phyllanthus emblica in step 1) into a wall-breaking machine for crushing to obtain granules; 3) Step 2) the mass ratio of the emblica granules to deionized water is (1-3):(5-8) and the slurry is beaten to form a homogenate, and the pH is adjusted to 8.5-9.5 for later use; 4) Prepare a 150-250 IU / mL complex enzyme aqueous solution by mixing pectinase and alkaline protease in a mass ratio of (2-5):(3-7) for later use; 5) adding the complex enzyme aqueous solution of step 4) to the homogenate of step 3), the volume ratio of the homogenate to the complex enzyme aqueous solution being (5-9):(1-5), maintaining the temperature at 55-65°C, and performing ultrasound-assisted enzymolysis for 5 hours, and heating to 95°C for 10 minutes after enzymolysis; 6) The enzymatic hydrolysate of step 5) is filtered by ultrafiltration membrane, and the polypeptide molecules with a molecular weight cutoff of 1500-2500Da are retained. The retained enzymatic hydrolysate is subjected to LC-MS / MS analysis to analyze its sequence characteristics; and the collected Phyllanthus emblica polypeptides are concentrated by low temperature vacuum; 7) The vacuum concentrated solution in step 6) is transferred to a spray dryer to collect dry powder.

[0006] Preferably, the mass ratio in step 3) is 1:5, 2:7 or 3:8.

[0007] Preferably, the pH in step 3) is 8.5, 9.0 or 9.5.

[0008] Preferably, the mass ratio in step 4) is 2:3, 3:5 or 5:7.

[0009] Preferably, the enzyme activity unit in step 4) is 150 IU / mL, 200 IU / mL or 250 IU / mL.

[0010] Preferably, the volume ratio in step 5) is 5:1, 7:3 or 9:5.

[0011] Preferably, the maintenance temperature in step 5) is 55°C, 60°C or 65°C.

[0012] Preferably, the enzymatic hydrolysis in step 5) is carried out for 5 h, 7 h or 9 h.

[0013] Preferably, the polypeptide sequences of Phyllanthus emblica isolated in step 6) are shown in SEQ ID NO.1-2.

[0014] Another aspect of the present invention is to provide the use of Phyllanthus emblica polypeptide in the preparation of medicines with liver-protecting and blood lipid-lowering effects.

[0015] Preferably, the polypeptide sequence of the Emblica officinalis is shown in SEQ ID NO.1-2.

[0016] Another object of the present invention is to provide a medicine with liver protection and lipid-lowering effects, wherein the medicine uses the Phyllanthus emblica polypeptide as a main active ingredient, and the amino acid sequence of the Phyllanthus emblica polypeptide is shown in any one of SEQ ID NO.1-2.

[0017] The advantages of the present invention are as follows: the present invention obtains the Phyllanthus emblica polypeptide with liver protection and blood lipid lowering effects by enzymatic separation from Phyllanthus emblica for the first time, and the preparation method adopts a composite enzymatic hydrolysis process combined with ultrasound-assisted means to prepare the Phyllanthus emblica polypeptide, and the Phyllanthus emblica polypeptide has excellent liver protection and blood lipid lowering activities, and has great application prospects and market. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0019] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0020] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0021] Example 1 The preparation method of emblica polypeptide comprises the following steps: 1) Wash the emblica buds, cut them into small pieces, and dry them with air at room temperature for later use; 2) Put the air-dried pieces of Phyllanthus emblica in step 1) into a wall-breaking machine and grind them into fine particles; 3) adding deionized water in a mass ratio of 1:5 to the fine particles of Phyllanthus emblica in step 2), beating the fine particles to form a homogenate, adjusting the pH value to 8.5, and setting aside; 4) Pectinase and alkaline protease are prepared into a 150 IU / mL complex enzyme aqueous solution in a mass ratio of 2:3 for later use; 5) adding the aqueous solution of the complex enzyme of step 4) to the homogenate of step 3) at a volume ratio of 5:1, maintaining the temperature at 60°C, and performing ultrasound-assisted enzymolysis for 5 hours, and then rapidly heating to 95°C for 10 minutes after enzymolysis; 6) The enzymatic hydrolysate of step 5) is filtered by ultrafiltration membrane, and the polypeptide molecules with a molecular weight cutoff of 1500-2500Da are retained. The retained enzymatic hydrolysate is subjected to LC-MS / MS analysis to analyze its sequence characteristics; and the collected Phyllanthus emblica polypeptides are concentrated by low temperature vacuum; 7) The vacuum concentrated solution in step 6) is transferred to a spray dryer to collect dry powder.

[0022] The results showed that two kinds of Phyllanthus emblica polypeptides were obtained through sequencing and identification analysis, and the sequences were shown in SEQ ID NO.1-2. After purity determination, the purity of the obtained Phyllanthus emblica polypeptides could reach 92.6% and 95.2% respectively.

[0023] Example 2 2.1 Analysis of the lipid-lowering effect of Phyllanthus emblica polypeptide A total of 40 healthy male SD rats weighing 150-170g were selected. 30 rats were selected to be fed with high-fat diet for 3 consecutive weeks to construct a hyperlipidemia rat model, and 10 rats were selected as the blank control group, which were fed with ordinary diet for 3 consecutive weeks. Subsequently, the model group was divided into three groups, one of which was the model control group, which was fed with high-fat diet; the other two groups were fed with high-fat diet supplemented with 15% mass ratio of emblica polypeptide 1-2, and the blank control group was still fed with ordinary diet. After 3 weeks, the serum total cholesterol (TC), total triglyceride (TG), low-density lipoprotein (LDL-C), and high-density lipoprotein (HDL-C) levels of rats in each group were measured. The test results are shown in Table 1.

[0024] Table 1 Analysis of TC, TG, LDL-C and HDL-C contents in rat serum The results are shown in Table 1: Compared with the blank group, the total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein (LDL-C) of the rats in the model control group were significantly increased, confirming that the model of the present invention was successfully constructed. Compared with the model control group, the total cholesterol (TC), total triglycerides (TG), and low-density lipoprotein (LDL-C) of the rats in the treatment groups 1-2 of the present invention were significantly reduced and returned to normal levels, further confirming that the Phyllanthus emblica polypeptides 1-2 of the present invention have better lipid-lowering activity, especially the Phyllanthus emblica polypeptide 2 has the best lipid-lowering effect.

[0025] 2.2 Analysis of the liver-protective effect of Phyllanthus emblica polypeptide Thirty SPF Wistar rats were selected, half male and half female, with a body weight of 180-220g. They were randomly divided into 3 groups, of which group 1 was a blank group and fed with ordinary feed; group 2 was a positive control group and fed with ordinary feed; group 3 was treatment group 1, fed with ordinary feed supplemented with 15% by mass ratio of Phyllanthus emblica polypeptide 1; group 4 was treatment group 2, fed with ordinary feed supplemented with 15% by mass ratio of Phyllanthus emblica polypeptide 2; the above groups were fed for 1 week, and then the positive control group, treatment group 1 and treatment group 2 were given 0.5% (V / V) carbon tetrachloride (CCl4) treated oil solution 0.1ml / 10g BW by intraperitoneal injection once, and the blank group was given an equal amount of prepared oil solution (salad oil 5%, distilled monoglyceride 1.5%, distilled water 93.5%). After 24h of drug treatment, the rats in each group were killed to detect liver alanine aminotransferase (ALT) and aspartate aminotransferase (AST) indicators. The test results are shown in Table 2.

[0026] Table 2 Analysis of ALT and AST content in rat serum The results are shown in Table 2: ALT and AST in the serum of rats in the positive control group were significantly increased compared with the blank group, confirming the successful construction of the chemical liver injury model of the present invention. Further, by comparing the treatment group, it can be found that the Phyllanthus emblica polypeptide in ordinary feed can improve the liver performance of rats and effectively resist the liver damage caused by CCl4, further confirming that the Phyllanthus emblica polypeptide of the present invention has a liver-protecting effect.

[0027] It is necessary to point out that the above embodiments are limited to further elaboration and explanation of the technical solution of the present invention, and are not further limitations of the technical solution of the present invention. The method of the present invention is only a preferred implementation scheme, and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a polypeptide of Phyllanthus emblica, characterized in that: The method comprises the following steps: 1) Wash the emblica buds, cut them into small pieces, and dry them with air at room temperature for later use; 2) putting the air-dried pieces of Phyllanthus emblica in step 1) into a wall-breaking machine for crushing to obtain particles; 3) Beat the particles to deionized water in a mass ratio of (1-3):(5-8) to form a homogenate, adjust the pH to 8.5-9.5, and set aside; 4) Prepare a 150-250 IU / mL complex enzyme aqueous solution of pectinase and alkaline protease in a mass ratio of (2-5):(3-7) for later use; 5) adding the complex enzyme aqueous solution of step 4) to the homogenate of step 3), the volume ratio of the homogenate to the complex enzyme aqueous solution being (5-9):(1-5), maintaining the temperature at 55-65°C, and performing ultrasound-assisted enzymolysis for 5 hours, and heating to 95°C for 10 minutes after enzymolysis; 6) filtering the enzymatic hydrolysate in step 5) by ultrafiltration membrane, retaining polypeptide molecules with a molecular weight of 1500-2500Da, and performing LC-MS / MS analysis on the retained enzymatic hydrolysate to analyze its sequence characteristics; and vacuum concentrating the collected Phyllanthus emblica polypeptides; 7) The vacuum concentrated solution in step 6) is transferred to a spray dryer to collect dry powder.

2. The method according to claim 1, characterized in that In step 3), the mass ratio of particles to deionized water is 1:5, 2:7 or 3:8; and / or, the pH is 8.5, 9.0 or 9.

5.

3. The method according to claim 1, characterized in that The mass ratio of pectinase to alkaline protease in step 4) is 2:3, 3:5 or 5:7; and / or the parameter of the complex enzyme aqueous solution is 150 IU / mL, 200 IU / mL or 250 IU / mL.

4. The method according to claim 1, characterized in that In step 5), the volume ratio of the homogenate to the complex enzyme aqueous solution is 5:1, 7:3 or 9:

5.

5. The method according to claim 1, characterized in that The maintenance temperature in step 5) is 55°C, 60°C or 65°C.

6. The method according to claim 1, characterized in that The enzymatic hydrolysis in step 5) is performed for 5 h, 7 h or 9 h.

7. The method according to claim 1, characterized in that The polypeptide sequences of Phyllanthus emblica isolated in step 6) are shown in SEQ ID NO. 1-2.

8. A polypeptide of Phyllanthus emblica having liver protection and blood lipid lowering effects, characterized in that: The polypeptide sequences of the Emblica officinalis L. are shown in SEQ ID NO. 1-2.

9. The use of Phyllanthus emblica polypeptide in the preparation of a medicine having liver protection and blood lipid lowering effects, characterized in that: The polypeptide sequences of the emblica fruit are shown in SEQ ID NO.1-2.

10. A medicine having liver protection and blood lipid lowering effects, characterized in that: The medicine uses the Phyllanthus emblica polypeptide as an active ingredient, and the amino acid sequence of the Phyllanthus emblica polypeptide is shown in any one of SEQ ID NO.1-2.

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