CN109748951B

CN109748951B - Angelica sinensis antioxidant polypeptide and preparation method and application thereof

Info

Publication number: CN109748951B
Application number: CN201910018454.4A
Authority: CN
Inventors: 龙莉莉; 宋延民
Original assignee: Xiangya Hospital of Central South University
Current assignee: Xiangya Hospital of Central South University
Priority date: 2019-01-09
Filing date: 2019-01-09
Publication date: 2021-12-03
Anticipated expiration: 2039-01-09
Also published as: CN109748951A

Abstract

The invention discloses an angelica antioxidant polypeptide and a preparation method and application thereof, wherein the amino acid sequence of the antioxidant polypeptide Asao-1 is shown as SEQ ID NO. 1; the invention takes the medicine angelica as raw material, firstly obtains short peptide segment by the enzymolysis of neutral protease and trypsinase, and then obtains the medicine angelica by separation and purification. The polypeptide has good antioxidant activity, is a source of traditional Chinese medicines, and has good safety.

Description

Angelica sinensis antioxidant polypeptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to angelica sinensis antioxidant polypeptide and a preparation method and application thereof.

Background

The oxidation process of biomolecules is a free radical mediated process that has many adverse effects on food and biological systems. Free radicals are inevitably produced during aerobic metabolism in aerobic organisms. The radical mainly includes superoxide anion radical, hydroxyl radical, hydrogen peroxide, singlet oxygen molecule, hypochlorous acid, and the like, which are very unstable in natural environment and are very easily reacted with other substances. Free radicals can effectively kill pathogenic macrophages and other phagocytic cells of the immune system when the cells are under physiological and pathological conditions. But if free radicals are produced in large quantities without control, they can cause severe damage to biological systems by destroying the cell membrane phospholipid molecule layer, proteins and DNA. These negative effects of free radicals are thought to be associated with aging and many health disorders in humans, such as diabetes, cancer, neurodegenerative and inflammatory diseases.

The existence of natural antioxidants in human body can eliminate the harm of free radicals to human body, such as vitamin C and vitamin E. Some synthetic antioxidants (butylated hydroxyanisole and butylated hydroxytoluene) can also act to scavenge free radicals, but studies have shown that these synthetic antioxidants may cause damage to the liver of the human body and even cause carcinogenesis. The short peptides mostly contain 2-20 amino acid residues, and obtain peptides with certain biological characteristics which are not possessed by the protein per se, such as the activity inhibition of certain enzymes, the antioxidant capacity, the immunoregulation, the antihypertensive effect, the antithrombotic effect and the like.

China has rich Chinese herbal medicine resources, but the research on the effective components of the Chinese herbal medicine is less at present. Dang Gui is sweet in flavor and tonifying pungent in property, bitter in flavor and purging heat and unblocking in property, it can tonify blood, activate blood and move qi to alleviate pain. When the roots of the angelica are developed and contain a large amount of proteins, a large amount of active polypeptides can be generated after the proteins are subjected to enzymolysis by different proteases, so that the polypeptides with antioxidant activity can be screened, and the utilization of biological resources is enhanced.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides angelica antioxidant polypeptide and a preparation method and application thereof.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

the amino acid sequence of the angelica antioxidant polypeptide is shown as SEQ ID NO. 1: Gln-Pro-His-Val-His-Tyr (QPHVHY) (SEQ ID NO. 1).

The preparation method of the antioxidant polypeptide comprises the following steps:

(1) obtaining a peptide segment by enzymolysis of neutral protease and trypsin;

(2) separating and purifying the peptide segment obtained in the step (1) by Sephadex G-25 and RP-HPLC two steps to obtain three components (G-1, G-2 and G-3), and then carrying out DPPH free radical scavenging experiment to obtain antioxidant activity of the three components G-1, G-2 and G-3;

(3) and (3) obtaining the amino acid sequence information of the polypeptide component G-3 with the strongest antioxidant activity obtained in the step (2) by an Edman degradation method.

The angelica antioxidant polypeptide can be used for preparing antioxidants.

The invention takes the medicine angelica as raw material, firstly obtains short peptide segment by the enzymolysis of neutral protease and trypsinase, and then obtains the medicine angelica by separation and purification. The polypeptide has good antioxidant activity, is a source of traditional Chinese medicines, and has good safety.

Drawings

FIG. 1 is a Sephadex G-25 separation and purification map;

FIG. 2 shows DPPH.radical activity of each fraction after Sephadex G-25 separation and purification;

FIG. 3 is a diagram of the Sephadex G-25 component G-3 separated and purified by RP-HPLC; wherein an arrow indicates a target peak containing antioxidant polypeptide AsAo-1;

FIG. 4 is a concentration-inhibition curve of DPPH.radical activity of the antioxidant polypeptide AsAo-1.

Detailed Description

Other various experimental procedures involved in the present invention are all conventional procedures in the art, and those not specifically described herein can be implemented by those skilled in the art with reference to various common tool books, scientific documents, or related specifications, manuals, etc. before the filing date of the present application.

EXAMPLE 1 sample preparation of active Polypeptides

The root tissue of angelica sinensis is washed by deionized water, water is sucked dry, 10 g of the root tissue is weighed and cut into small blocks by a knife, 10 mL of PBS buffer solution (pH 7.5) is added, then the small blocks are evenly ground by a mortar and placed in a desk type centrifuge with the temperature of 4 ℃ and centrifuged for 30min at 10000 g/min. Collecting supernatant, adding neutral protease and trypsin (at a ratio of 1: 1), reacting in 45 deg.C constant temperature water bath for 6 hr, and inactivating in 100 deg.C water bath for 10 min. After the sample is cooled, the sample is placed in a desktop centrifuge at 4 ℃ and centrifuged at 10000 g/min for 30 min.

Example 2 Sephadex G-25 gel chromatography column separation of antioxidant polypeptide fractions

Sephadex G-25 was loaded onto a 2.5 cm by 60 cm glass column, equilibrated overnight with distilled water, the sample from example 1 was filtered through a 0.45 μm microporous membrane, loaded onto the column, eluted with distilled water at a flow rate of 1 mL/min, detected at a wavelength of 215 nm, three absorption peaks were collected in total (see FIG. 1), lyophilized, and the DPPH.clear rate was determined (see FIG. 2).

EXAMPLE 3 measurement of DPPH radical scavenging Activity

DPPH was dissolved in absolute ethanol to prepare a solution with a concentration of 0.2 mmol/L. Adding 2 mL of sample solution into 2 mL of DPPH solution with concentration of 0.2 mmol/L, shaking, mixing, standing at room temperature in dark place for 30min, then placing in a 4 ℃ bench centrifuge, centrifuging at 3000 g/min for 30min, collecting supernatant, measuring light absorption value at 517 nm, and recording as A_iIn the control group, 2 mL of absolute ethyl alcohol is added into 2 mL of DPPH solution, and the light absorption value is recorded as A₀(ii) a 2 mL of sample solution with certain concentration is added with 2 mL of absolute ethyl alcohol and evenly mixed, and the measured light absorption value is recorded as A_j(ii) a The mixture was zeroed with 2 mL of distilled water plus 2 mL of absolute ethanol.

The clearance rate (K) of the zymohydrolysis product to DPPH & is calculated by the following formula: k = [1- (A)_i - A_j)/A₀]×100%。

Example 4 RP-HPLC separation of antioxidant Polypeptides

The DPPH & clearance of the Sephadex G-25 gel fraction G-3 is detected to be highest, the Sephadex G-25 gel fraction G-3 is freeze-dried and then dissolved in ultrapure water to prepare a polypeptide solution, the polypeptide solution is filtered by a 0.45 mu m microporous membrane, and the polypeptide solution is further separated and purified by a WATERS high performance liquid chromatograph 2695. The separation and purification conditions were as follows, with respect to a chromatographic column, Asahi Ultimate XB-C18 (4.6 mm. times.250 mm, 5 μm), a loading amount of 100 μ L, a flow rate of 1 mL/min, a column temperature of 30 ℃, a detection wavelength of 215 nm, and a linear gradient elution of 5% to 30% acetonitrile (containing 0.1% TFA). The peak of each fraction was collected and the RP-HPLC profile is shown in FIG. 3. After freeze-drying each peak, the DPPH radical scavenging rate was determined, and it was identified that the objective peak shown by the arrow in FIG. 3 has the strongest DPPH radical scavenging ability, and the DPPH radical scavenging activity is shown in FIG. 4.

Example 5 identification of amino acid sequences of antioxidant Polypeptides by Edman degradation

The amino acid sequence of the antioxidant peptide is obtained by determination by an N-terminal Edman degradation method and is completed on an ABI 491-A type gas phase sequencer. The set cycle number is 8, the obtained PTH-amino acid is detected by on-line HPLC, the determination of the whole amino acid sequence of the antioxidant peptide is completed at one time, and the amino acid is identified as shown in SEQ ID NO. 1.

SEQUENCE LISTING

<110> Hunan ya Hospital of Zhongnan university

<120> Chinese angelica antioxidant polypeptide and preparation method and application thereof

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 6

<212> PRT

<213> Artificial Synthesis

<400> 1

Gln Pro His Val His Tyr

1 5

Claims

1. The angelica sinensis antioxidant polypeptide is characterized in that the amino acid sequence of the angelica sinensis antioxidant polypeptide is shown as SEQ ID No. 1.

2. The method for producing the antioxidant polypeptide of claim 1, comprising the steps of:

(1) taking root tissues of angelica sinensis, cleaning the root tissues with deionized water, sucking water, weighing 10 g, cutting into small pieces with a knife, adding 10 mL of PBS (phosphate buffer solution) with the pH of 7.5, uniformly grinding with a mortar, placing in a table centrifuge at 4 ℃, and centrifuging for 30min at 10000 g/min; collecting supernatant, adding neutral protease and trypsin, reacting in 45 deg.C constant temperature water bath for 6 hr, and inactivating in 100 deg.C water bath for 10 min; after the sample is cooled, placing the sample in a table centrifuge at 4 ℃, and centrifuging for 30min at 10000 g/min; obtaining a peptide segment;

(2) loading Sephadex G-25 into a 2.5 cm × 60 cm glass column, balancing with distilled water overnight, filtering the peptide segment obtained in the step (1) by a 0.45 μm microporous membrane, loading the peptide segment onto the column, eluting with distilled water at the flow rate of 1 mL/min, detecting at the wavelength of 215 nm, collecting three absorption peaks, G-1, G-2 and G-3 in total, and then obtaining the antioxidant activity of three components, namely G-1, G-2 and G-3 through a DPPH & free radical scavenging experiment; freeze drying the component with the highest antioxidant activity, dissolving with ultrapure water, preparing polypeptide solution, filtering with 0.45 μm microporous membrane, and further separating and purifying with WATERS high performance liquid chromatograph 2695; the separation and purification conditions were as follows, with respect to a chromatographic column, Asahi Ultimate XB-C18 (4.6 mm. times.250 mm, 5 μm), a loading amount of 100 μ L, a flow rate of 1 mL/min, a column temperature of 30 ℃, a detection wavelength of 215 nm, and a linear gradient elution of 5% to 30% acetonitrile containing 0.1% TFA.

3. The use of the antioxidant polypeptide of claim 1 in the preparation of an antioxidant.