CN114409735A

CN114409735A - Clinacanthus nutans antioxidant undecapeptide and preparation method and application thereof

Info

Publication number: CN114409735A
Application number: CN202210103859.XA
Authority: CN
Inventors: 严芬; 刘文静; 张丹; 胡诗琦; 吴崟沣
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2022-01-28
Filing date: 2022-01-28
Publication date: 2022-04-29
Anticipated expiration: 2042-01-28
Also published as: CN114409735B

Abstract

The patent discloses an antioxidant undecapeptide of clinacanthus nutans and a preparation method and application thereof, wherein the antioxidant undecapeptide is DMGPPLSEKLH. The clinacanthus nutans antioxidant peptide is prepared by taking clinacanthus nutans as a raw material and adopting an ultrasonic-assisted alkali-soluble acid extraction technology, and a purification method of the clinacanthus nutans antioxidant peptide is further established by separation and purification technologies such as ion chromatography, reversed-phase high performance liquid chromatography and the like. In vitro antioxidant experiments show that the clinacanthus nutans antioxidant undecapeptide can effectively remove free radicals such as DPPH, ABTS, hydroxyl free radicals and the like, has the characteristics of simple structure, strong antioxidant activity and high safety, can be used as a good substitute of a chemically synthesized antioxidant, has certain antitumor activity, and provides theoretical basis and practical reference for research and development of novel natural additives in the industries of foods, medicines and cosmetics.

Description

Clinacanthus nutans antioxidant undecapeptide and preparation method and application thereof

Technical Field

The invention relates to clinacanthus nutans antioxidant peptide and a preparation method and application thereof, and belongs to the technical field of food and medicine.

Background

Clinacanthus nutans, also known as crocodile flower, is a plant of the genus crocodile of the family Acanthaceae. Clinacanthus nutans is widely distributed from tropical zone of south China to Malaysia, Java, Calimanda and other places in south China, Hainan, Guangdong, Guangxi and Yunnan. Clinacanthus nutans mostly grows in low-altitude sparse forest close to equator or in moist sandy soil in a bush, is a high and large herbaceous plant, is in an upright or climbing shape, and is in a light green shape when stems are cylindrical and dry. Clinacanthus nutans is generally taken as a medicine by whole plants or leaves, is sweet in taste, slightly bitter and cool in nature, and has the effects of clearing heat and promoting diuresis, inducing diuresis and reducing edema, activating blood and dredging channels, removing dampness, resisting tumors and the like.

Free radicals are atoms or radicals having unpaired electrons, which are intermediates of human metabolism. The proliferation of free radicals in the body can induce oxidative stress, and the antioxidant defense system of the body is seriously unbalanced, thereby causing cell damage. Under normal conditions, the body produces reactive oxygen radicals: (Reactive oxygen speciesROS) can be maintained at a low level under the action of own antioxidant enzyme systems (superoxide dismutase, catalase, glutathione peroxidase and the like) and endogenous antioxidants (VE, VC, carnosine, glutathione and the like), so that the body is not damaged by free radicals, and meanwhile, when endogenous or exogenous stimulation promotes abnormal metabolism of the body to generate a large amount of ROS or the balance between the antioxidant and the oxidant of the body is abnormal along with the growth of the age, oxidative stress can be caused, namely, the excessive ROS can damage the normal redox level of the bodyBalance, causing oxidative damage to biological macromolecules (such as protein, lipid, DNA and the like) in cells, thereby accelerating the aging of the organism and causing various diseases such as neurodegenerative diseases, atherosclerosis, chronic inflammation, cancer and the like.

The antioxidant can effectively inhibit negative effects caused by oxidative stress in human body, and can protect tissues and organs from being damaged by ROS. Synthetic antioxidants such as BHT, PG, etc. have a good function of scavenging free radicals, but have a certain toxic effect on the enzyme system of the human body, while natural antioxidants have both strong antioxidant activity and high safety, so people gradually turn their research into natural antioxidants. The antioxidant peptide is one of the main members of natural antioxidants, participates in the processes of antioxidation and detoxification in organism cells, and the reduction of the antioxidant capacity can induce the cells to generate oxidative stress, cause the damage and the function loss of the cells, and have close relation with the occurrence of cell aging and diseases. The antioxidant active polypeptide has the characteristics of low toxicity, high efficiency and the like, and is considered as an ideal substitute of artificially synthesized antioxidants as an antioxidant for food and organisms. The antioxidant active polypeptide can effectively remove excessive ROS (such as hydroxyl free radical, superoxide anion free radical, nitric oxide free radical, etc.) in vivo, protect normal structure and function of cells and mitochondria, prevent lipid peroxidation, and help organism resist diseases. Research has demonstrated that ROS play an important role in the development of cancer. They are required for normal physiological activities such as intracellular signaling and homeostasis, cell death, etc. at appropriate concentrations. Excessive ROS easily attack deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), cause cell mutation, inhibit the expression of cancer suppressor genes and induce cell canceration. Under oxidative stress, free radicals are continuously and excessively generated, and then DNA, protein and lipid are damaged, and the health of cells is threatened, so that diseases are caused. A large number of researches show that Keap1-Nrf2/ARE is an important oxidative stress path of a body, wherein Nrf2 is a key factor, and the key factor can prevent normal cell degeneration and promote tumor development. When the organism in the physiological state is in an oxidative stress state, Keap1 and Nrf2Uncoupling, nuclear incorporation after phosphorylation of Nrf2 and anti-oxidant reaction element(s) ((Antioxidant ResponseElementAnd ARE) to start the downstream II phase detoxification enzyme regulated and controlled by ARE, and improve the anti-oxidative stress capability of cells, thereby playing a role in preventing tumors. The cationic peptide has the characteristics of easy modification and easy combination with tumor cells, and has lower toxicity to normal cells, so that the cationic peptide has better anti-tumor potential.

At present, the chemical components and pharmacological activity of clinacanthus nutans are rarely researched at home and abroad, and particularly, the protein and polypeptide of clinacanthus nutans are rarely researched. Therefore, it is necessary to study the preparation of polypeptide and antioxidant activity of Clinacanthus nutans by Clinacanthus nutans research subjects, and important scientific basis is provided for the comprehensive development of Clinacanthus nutans and the application of Clinacanthus nutans in the fields of foods, medicines and the like.

Disclosure of Invention

The invention aims to provide clinacanthus nutans antioxidant undecapeptide and a preparation method and application thereof.

The comparison of a Uniprot database and a NCBI database shows that the amino acid sequence of the antioxidative undecapeptide is Asp-Met-Gly-Pro-Pro-Leu-Ser-Glu-Lys-Leu-His, and is represented by DMGPPLSEKLH with a single letter.

The method comprises the following steps of extracting clinacanthus nutans crude protein by an ultrasonic-assisted alkali-soluble acid precipitation method, and then carrying out anion exchange chromatography, reverse high performance liquid chromatography separation and purification, identification and synthesis to obtain the clinacanthus nutans antioxidant undecapeptide, and the specific steps comprise:

1) extracting the protein in clinacanthus nutans by adopting an alkali-soluble acid precipitation method to prepare a crude extract of the clinacanthus nutans protein;

2) separating the crude extract of clinacanthus nutans protein by using DEAE-Sepharose ion exchange chromatography, eluting with 0.2 mol/L to 1 mol/L NaCl Tris-hydrochloric acid buffer solution with pH =8.4 in a linear gradient manner at a flow rate of 1mL/min, detecting the absorbance of an effluent sample by using a chromatographic chart acquisition analyzer HD-A, collecting each peak component, measuring the antioxidant activity, and collecting the component CNPH-III with the highest activity;

3) and (3) further separating and purifying CNPH-III by using reverse phase high performance liquid chromatography (RP-HPLC), wherein the mobile phase A is acetonitrile containing 0.1% TFA, the mobile phase B is deionized water containing 0.1% TFA, and the elution gradient of the RP-HPLC is as follows: 0-5min, 5% -10% A; 5-15min, 10% -50% A; 15-35 min, 50% -80% A, flow rate of mobile phase of 1.0 mL/min, detection wavelength of 214 nm, and collecting the component with highest activity (retention time of elution peak of 3-4 min);

4) adopting liquid chromatography mass spectrometer to carry out amino acid sequence determination to the peptide section that the elution peak that retention time is 3-4min contained, carrying out Fmoc solid phase synthesis to the amino acid sequence that obtains of survey, thereby obtain the antioxidative undecapeptide of clinacanthus nutans.

The clinacanthus nutans antioxidant undecapeptide provided by the invention can be applied to preparation of anti-liver cancer drugs.

The clinacanthus nutans antioxidant undecapeptide provided by the invention can also be applied to preparation of products with antioxidant functions.

The invention has the following remarkable advantages:

the clinacanthus hypochondriacus antioxidant undecapeptide DMGPPLSEKLH provided by the invention has strong DPPH, OH and ABTS free radical scavenging capacity, and shows that the clinacanthus hypochondriacus antioxidant undecapeptide has important value in the aspects of development and application of antioxidant activity. When the concentration of the peptide reaches 2 mg/mL, the DPPH, ABTS and hydroxyl free radical clearance rates are 37.858 +/-1.001%, 16.569 +/-0.554% and 65.983 +/-1.191%, respectively. IC of antioxidant peptide DMGPPLSEKLH for DPPH free radical clearance rate₅₀IC of value 19.064 + -1.569 mg/mL for ABTS free radical clearance₅₀IC of value 19.771 + -5.003 mg/mL, for hydroxyl radical clearance₅₀The value was 1.013. + -. 0.050 mg/mL. When the concentration of the antioxidant peptide DMGPPLSEKLH is 10 mg/mL, the inhibition rate of HepG2 cells reaches 56.213 +/-1.739%. The antioxidant undecapeptide has a certain anti-liver cancer effect.

Drawings

FIG. 1 is a chromatogram of DEAE sepharose anion of a crude extract of clinacanthus nutans.

FIG. 2 is a reverse phase high performance liquid chromatogram of anion chromatography elution peak III.

Fig. 3 is a mass spectrum of antioxidant peptide DMGPPLSEKLH.

Fig. 4 shows the effect of natural antioxidant peptide DMGPPLSEKLH on free radical scavenging. A: ABTS free radical scavenging activity; b: DPPH free radical scavenging activity; c: hydroxyl radical scavenging activity.

FIG. 5 shows the inhibitory effect of natural antioxidant peptide DMGPPLSEKLH on HepG-2 cells.

Detailed Description

The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and process are given, but the scope of the present invention is not limited to the following implementation examples.

Example 1 separation and purification of crude extract of Clinacanthus nutans

Clinacanthus nutans pretreatment: the surface of clinacanthus nutans is cleaned, blackened and rotten clinacanthus nutans leaves are removed, and then the clinacanthus nutans are ground by liquid nitrogen for standby.

Extracting clinacanthus nutans crude protein and polypeptide: the ground clinacanthus nutans powder is prepared by mixing the following raw materials in a liquid-to-solid ratio of 1: 10 (m/v) distilled water was added, the ultrasonic power was set at 135W, and ultrasonic was performed at 25 ℃ for 35 min. Then filtering out residues, adjusting the pH value of the filtrate to 8.0 by using 1 mol/L NaOH, stirring the filtrate at room temperature for 30 min, centrifuging the filtrate at 5000 r/min for 20 min, adjusting the pH value of the centrifuged supernatant to 3 by using 1 mol/L HCl, centrifuging the supernatant at 5000 r/min for 20 min, dissolving the centrifuged precipitate in 10-30 mL of Tris-hydrochloric acid buffer solution with the pH value of 8.4, then filling the solution into a dialysis bag with the molecular weight cutoff of 3500D, dialyzing the solution in deionized water overnight, and freeze-drying to obtain the crude Clinacanthus nutans protein.

And (3) anion chromatography separation and purification: preparing the clinacanthus nutans crude protein lyophilized powder into 6 mg/ml solution with 0.02M Tris-hydrochloric acid buffer solution with pH of 8.4, completely dissolving, filtering with 0.22 μ M pore diameter microfiltration membrane, then separating and purifying by DEAE-Sepharose ion exchange column chromatography, balancing with 0.02M Tris-hydrochloric acid buffer solution with pH value of 8.4, loading the filtered sample on the column with flow rate of 1mL/min, eluting with 0.2 mol/L to 1 mol/L NaCl Tris-hydrochloric acid buffer solution with pH =8.4 in linear gradient, detecting the absorbance of the effluent sample by a chromatogram collection analyzer HD-A, collecting 3 peak components according to elution peak (figure 1), comparing hydroxyl free radical, DPPH, ABTS free radical scavenging ability, collecting highest active component (CNPH-III), vacuum freeze drying, and storing at-20 deg.C.

Separating and purifying by reversed-phase high performance liquid chromatography: dissolving the CNPH-III freeze-dried powder with deionized water, filtering with a 0.22 mu m pore diameter microfiltration membrane, and separating and purifying by using a Thermo C18 chromatographic column, wherein the sample concentration is 10 mg/mL, the sample injection amount is 20 mu L, and the mobile phase A: acetonitrile with mass fraction of 0.1% TFA, mobile phase B: deionized water containing 0.1% TFA by mass fraction, and the flow rate of the mobile phase was 1.0 mL/min. Adopting a gradient elution mode, wherein the elution conditions are as follows: 0-5min, 5% -10% of mobile phase A; 5-15min, 10% -50% of mobile phase A; 15-35 min, 50% -80% mobile phase A. The detection wavelength was 214 nm. Measuring scavenging ability of hydroxyl free radical, DPPH, and ABTS free radical of the eluate corresponding to each absorption peak, collecting eluate (CNPH-III-2 with highest antioxidant activity, FIG. 2) with retention time of 3-4min, and vacuum freeze drying.

Freeze-drying the collected antioxidant components, determining the amino acid sequence by a liquid chromatography-mass spectrometry (LC-MS/MS) method (figure 3), and finding a new peptide by comparing a Uniprot database with an NCBI database to obtain the amino acid sequence of the antioxidant peptide: DMGPPLSEKLH are provided. The amino acid sequence was sent to Gill Biochemical Co., Ltd, Shanghai for Fmoc solid phase synthesis.

Example 2 in vitro antioxidant Activity assay of Natural antioxidant peptides

Weighing the synthetic polypeptide freeze-dried sample, and preparing sample solutions with different concentrations.

DPPH radical clearance: 3.94mg of DPPH is accurately weighed, dissolved by ethanol with volume fraction of 95 percent, and prepared into 0.1 mmo1/L DPPH solution after the volume is adjusted to 100 mL. Mixing 1mL sample solution and 1mL DPPH solution, standing at room temperature in dark place for 30 min, and measuring light absorption value at 517 nm; 1mL of ethanol solution with 95% volume fraction is used as a control group instead of DPPH solution; a blank was prepared by replacing the sample with 1mL of distilled water. The DPPH radical clearance of the samples was calculated using the following formula:

in the formula: a. the₁-absorbance of the sample set; a. the₂-control absorbance values; a. the₃Blank control groupAbsorbance value

ABTS free radical scavenging activity: a 7 mM stock solution of ABTS and a 2.45 mM solution of potassium persulfate were prepared and mixed in a 1: 1, placing the mixture in a dark place for 16 hours at room temperature, and then diluting the mixture by using 5mM phosphate buffer solution with pH7.4 until the light absorption value at 734 nm is 0.70 +/-0.02 to obtain the ABTS free radical solution. 1mL of ABTS free radical solution and 1mL of sample solution with different concentrations are mixed in equal volume, reacted at room temperature for 10 min, and then the absorbance is measured at 734 nm. Distilled water was used as a blank instead of the sample. The ABTS free radical scavenging activity of the samples was calculated according to the following formula:

in the formula: a. the₁-absorbance of the sample set; a. the₂Blank set absorbance value

Hydroxyl radical scavenging activity: 8mmol/L ferrous sulfate solution, 3 mmol/L salicylic acid solution and 20mmol/L hydrogen peroxide solution are prepared for standby. And (3) fully mixing 200 mu L of sample solution with 60 mu L of 8mmol/L ferrous sulfate, 200 mu L of 20mmol/L salicylic acid and 50 mu L of 20mmol/L hydrogen peroxide, incubating in a water bath at 37 ℃ for 30 min, cooling to room temperature, adding 90 mu L deionized water, and centrifuging at 4000 rpm for 30 min. The supernatant was taken to measure the absorbance of the reaction at 510 nm. Distilled water was used as a blank control. The scavenging activity of hydroxyl radicals is calculated according to the following formula:

in the formula: a. the₀-blank set absorbance values; a. the₁Absorbance of sample set

As measured by the example, the clinacanthus nutans antioxidant peptide DMGPPLSEKLH has a better clearance rate for DPPH radical, ABTS radical and hydroxyl radical as shown in fig. 4.

EXAMPLE 3 HepG-2 cytotoxicity and anticancer Studies of Natural antioxidant undecapeptide

Cell culture: HepG-2 cells inDMEM medium with 10% FBS by volume at 37 ℃ in 5% CO₂Culturing and subculturing in an incubator. Observing under an inverted microscope until the cells grow and fuse to 80%, draining the culture solution in a sterile operating platform, cleaning the cell surface layer by PBS phosphate buffer solution with pH of 7.3 (purchased from 23.48 g/2L of Hippo east China Biotech Co., Ltd.) and adding 1mL of pancreatin digestive juice, placing the cell surface layer in an incubator at 37 ℃ for 5 minutes, and then adding 1mL of DMEM culture medium containing 10% FBS by volume fraction to stop the action of the pancreatin digestive juice. All the liquid was then transferred into a 1.5 mL centrifuge tube and centrifuged at 2000 rpm for 10 min. After centrifugation, the supernatant was discarded, and fresh DMEM medium was added to the pellet, and the cells were uniformly dispersed by several times of pipetting. Cells were evenly distributed in fixed proportions into various sizes of petri dishes according to the experimental design.

Determination of cell growth viability: the CCK-8 method was used to determine the effect of clinacanthus hypochondriacus antioxidant undecapeptide DMGPPLSEKLH on the growth viability of cells HepG2, when the cells were fused to 80% -90%, the cells were washed with PBS buffer (23.48 g/2L from AoRuiyuan Biotech Co., Ltd., Mitsui) of pH 7.3 preheated at 37 ℃ in advance to remove surface residual serum, 1mL of pancreatic digestion solution was added, digestion was performed in a 37 ℃ incubator for 5min, after the cells were floated, 1mL of DMEM medium containing 10% FBS by volume was added to the dish to terminate the digestion, and then the cells HepG-2 were treated with 1X 10 FBS⁵cells/mL were seeded in 96-well plates at a density of 90. mu.L per well, with 4 replicate wells per concentration gradient in parallel. 37 ℃ and 5% CO₂Culturing in an incubator for 24 h, adding 10 μ L of DMEM medium containing Clinacanthin antioxidant undecapeptide DMGPPLSEKLH to each well, wherein the concentration gradient of Clinacanthin antioxidant undecapeptide is 0, 2, 4, 6, 8, and 10 mg/mL, and culturing for 24 h. After the culture solution was aspirated, 100. mu.L of CCK-8 (prepared in DMEM medium) containing 10% by volume fraction was added, and after incubation for 1 hour in an incubator, the absorbance was measured at a wavelength of 450 nm using a microplate reader. The inhibition rate of each group of drugs on cells was calculated according to the following formula:

in the formula: a. the₁-blank set absorbance values; a. the₂-control absorbance values; a. the₃Absorbance of sample set

The clinacanthus nutans antioxidant peptide DMGPPLSEKLH measured by the example has a high inhibition rate on HepG-2, which indicates that it has a good effect on resisting liver cancer (FIG. 5).

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in the claims of the present invention should be covered in the protection scope of the present invention.

SEQUENCE LISTING

<110> Fuzhou university

<120> clinacanthus nutans antioxidant undecapeptide and preparation method and application thereof

<130>

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 11

<212> PRT

<213> Artificial sequence

<400> 1

Asp Met Gly Pro Pro Leu Ser Glu Lys Leu His

1 5 10

Claims

1. The clinacanthus nutans antioxidant undecapeptide is characterized in that: the amino acid sequence of the clinacanthus nutans antioxidant undecapeptide is Asp-Met-Gly-Pro-Pro-Leu-Ser-Glu-Lys-Leu-His.

2. A method for preparing the clinacanthus nutans antioxidant undecapeptide as claimed in claim 1, wherein the method comprises the following steps: the method comprises the following steps:

1) precipitating and extracting proteins in clinacanthus nutans by adopting an alkali-soluble acid precipitation method to prepare a crude extract of the clinacanthus nutans proteins;

2) separating the crude extract of clinacanthus nutans protein by using DEAE-Sepharose ion exchange chromatography, eluting with 0.2-1 mol/L NaCl Tris-hydrochloric acid buffer solution with pH =8.4 in linear gradient at the flow rate of 1mL/min, detecting the absorbance of an effluent sample by using a chromatographic chart acquisition analyzer HD-A, collecting each peak component, measuring the antioxidant activity, and collecting the component CNPH-III with the highest antioxidant activity;

3) and (3) further separating and purifying CNPH-III by using reverse phase high performance liquid chromatography (RP-HPLC), wherein the mobile phase A is acetonitrile containing 0.1% TFA, the mobile phase B is deionized water containing 0.1% TFA, and the elution gradient of the RP-HPLC is as follows: 0-5min, 5% -10% A; 5-15min, 10% -50% A; 15-35 min, 50% -80% A, the flow rate of mobile phase is 1.0 mL/min, the detection wavelength is 214 nm, and the elution peak with retention time of 3-4min is collected;

3. The use of the clinacanthus hypochondriacus antioxidant undecapeptide as claimed in claim 1 in the preparation of an anti-hepatoma drug.

4. The use of the clinacanthus hypochondriacus antioxidant undecapeptide as claimed in claim 1 in preparing a product with an antioxidant function.