CN114573664B - Anti-oxidation tridecape of clindamianum as well as preparation method and application thereof - Google Patents
Anti-oxidation tridecape of clindamianum as well as preparation method and application thereof Download PDFInfo
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- CN114573664B CN114573664B CN202210288893.9A CN202210288893A CN114573664B CN 114573664 B CN114573664 B CN 114573664B CN 202210288893 A CN202210288893 A CN 202210288893A CN 114573664 B CN114573664 B CN 114573664B
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Abstract
The invention relates to an antioxidant tridecapeptide of clinopodium polycephalum, and a preparation method and application thereof, belonging to the technical field of biology. The amino acid sequence of the antioxidant peptide is LLPENDPSANHLM, the preparation method of the antioxidant peptide is to extract crude products from the clindamycin leaves serving as raw materials by adopting an alkali-soluble acid precipitation method, and then obtain the antioxidant peptide through separation, purification, identification and synthesis technology. Pharmacological experiments prove that the anti-oxidation tridecapeptide of the clindamycin provided by the invention can protect HepG-2 cells from H to different degrees 2 O 2 The poison has the characteristics of simple structure, strong antioxidation activity and high safety, and can be used as an excellent substitute for chemical synthesis antioxidants. The invention also provides theoretical basis and practical reference for developing novel natural additives in the industries of foods, medicines and cosmetics.
Description
Technical Field
The invention particularly relates to an antioxidant peptide of clinopodium polycephalum, and a preparation method and application thereof, and belongs to the technical field of biology.
Background
Free radicals are atoms or groups of atoms with unpaired electrons, and are intermediates of metabolism in the human body. The living body free radical is active oxygen free radicalReactive oxygen speciesROS) is the main component, and also comprises partial active nitrogen free radicalReactive nitrogenspeciesRNS). ROS mainly include hydrogen peroxide, hydroxyl radicals, superoxide anions, hydroperoxides, and the like; RNS includes nitric oxide, peroxynitrate, nitrogen dioxide, etc. Under normal conditions, ROS produced by the organism can be maintained at a low level under the action of an antioxidant enzyme system (superoxide dismutase, catalase, glutathione peroxidase and the like) and an endogenous antioxidant (VE, VC, carnosine, glutathione and the like) to protect the organism from free radicals. However, due to the low physiological level, when the body is greatly overused with free radicals caused by external interference, the actions of endogenous antioxidant enzymes and antioxidants are very little. Meanwhile, when endogenous or exogenous stimulation promotes abnormal metabolism of the organism to generate a large amount of ROS or the balance between antioxidant and oxidant of the organism with the increase of age is abnormal, oxidative stress is caused, and the excessive ROS can damage the normal redox balance of the organism, so that oxidation damage of intracellular biomacromolecules such as protein, lipid, DNA and the like is caused, thereby accelerating the aging of the organismCauses neurodegenerative diseases, atherosclerosis, chronic inflammation, cancer and other diseases.
Thus, there is a need to seek exogenous antioxidants to scavenge excessive free radicals in the body to maintain the health of the body. The antioxidant can effectively inhibit negative effects caused by oxidative stress in human body. Antioxidants can protect tissues and organs from ROS damage. Synthetic antioxidants such as 2, 6-di-tertiary hydroxy-p-cresol (BHT), butyl Hydroxy Anisole (BHA), tert-butyl hydroquinone (TBHQ), gallic acid (PG) and the like have a very good function of scavenging free radicals, but are harmful to the liver, spleen and lung of human bodies and have potential teratogenicity and carcinogenesis, and governments in various countries have forced to prescribe allowable daily intake values (ADI) for them and control excessive addition. The natural antioxidants have both strong antioxidant activity and high safety, so that people gradually shift research emphasis to the natural antioxidants. Bioactive peptide is a compound with molecular structure between amino acid and protein, and has effects of regulating metabolism and participating in vital activity. Bioactive peptides having antioxidant activity are also referred to as antioxidant peptides. The antioxidant active polypeptide has the characteristics of low toxicity, easy absorption, high efficiency and the like, is considered as an ideal substitute for artificially synthesized antioxidants, can effectively remove excessive ROS (such as hydroxy free radicals, superoxide anion free radicals, nitric oxide free radicals and the like) in a body, protects the normal structure and functions of cells and mitochondria, prevents lipid peroxidation and helps the body resist diseases. The research range of antioxidant peptides is wide, and the antioxidant peptides have all the functions of stable, safe and effective oxidation resistance and aging resistance. The natural reducing peptide can reduce the occurrence rate of diseases related to oxidation, aging and the like, so that the natural reducing peptide is often used for research and development of anti-aging health-care foods and cosmeceuticals.
The Clinopodium polycephalum, called crocodile flower, is a plant belonging to the genus crocodile flower of the family Acanthaceae. The plant of Clinopodium polycephalum is widely distributed in tropical areas of south China to malaysia, java, calimanthes, hainan, guangdong, guangxi and Yunnan. The clindamycin is a tall herb plant which is upright or climbed, and has a cylindrical stem and light green color when dried. The clinopodium polycephalum is generally used as a medicine in whole plant or leaf, has sweet and slightly bitter taste and cool nature, and has the effects of clearing heat and promoting diuresis, promoting urination and detumescence, promoting blood circulation and dredging channels, dehumidifying, resisting tumor and the like. Through the reference to related documents, the research reports on the chemical components and the pharmacological activity of the compound are less at home and abroad. The plant extract contains rich chemical components, such as triterpenes, flavone carboglycosides, sulfur-containing glycosides, phytosterols, pheophytin and the like, most of which have been proved by modern pharmacological researches to have a certain inhibition effect on tumors, and few researches on proteins and polypeptides of clinopodium are carried out. The separation and purification of the polypeptide and the functional research have important roles in the comprehensive development of plants, scientific research, food, medicine and other fields. But also the development of natural antioxidant health food and natural antioxidant at present becomes a scientific hot spot of modern life. Therefore, it is necessary to use the devil's grass study object to conduct the research of polypeptide preparation and antioxidant activity, and provide important scientific basis for comprehensive development and utilization of devil's grass. The molecular weight 1450.2 Da of the product can remove free radicals, and is a safe antioxidant active peptide of clinopodium polycephalum, which can be used as an antioxidant.
Disclosure of Invention
The invention aims to provide an antioxidant tridecapeptide of clinopodium polycephalum and a preparation method thereof, and the antioxidant tridecapeptide of clinopodium polycephalum can be applied to health-care products, food additives or cosmetics.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention firstly provides an antioxidant tridecape of clinopodium polycephalum, which contains more hydrophobic amino acids, has the molecular weight of 1450.2 Da, and has the amino acid sequence of Leu-Leu-Pro-Glu-Asn-Asp-Pro-Ser-Ala-Asn-His-Leu-Met, and is represented by LLPENDPSANHLM by single letter.
The invention also provides a preparation method of the antioxidant tridecapeptide of the clinopodium polycephalum, which comprises the following steps:
1) Extracting protein from clinopodium polycephalum by alkali-dissolution and acid-precipitation to prepare clinopodium polycephalum protein crude extract;
2) Separating the crude extract of the clindamycin by using DEAE-Sepharose ion exchange chromatography, linearly gradient eluting with 0.2 mol/L to 1 mol/L NaCl (Tris-hydrochloric acid buffer solution) with pH=8.4 at a flow rate of 1 mL/min, detecting absorbance of the effluent sample by using a chromatographic profile acquisition analyzer HD-A, collecting each peak-out component according to the elution peak, measuring antioxidant activity, and collecting the component CNPH-III with the highest activity;
3) The component CNPH-III is further separated and purified by reverse phase high performance liquid chromatography RP-HPLC, wherein the mobile phase A is acetonitrile containing 0.1 percent TFA, the mobile phase B is deionized water containing 0.1 percent TFA, and the elution gradient is as follows: 0-5min,5% -10% A;5-15min,10% -50% of A;15-35 min,50% -80% A, the flow rate of the mobile phase is 1.0 mL/min, the detection wavelength is 214 nm, and the elution peak with the retention time of 3-4 min is collected;
4) And (3) carrying out amino acid sequence determination on peptide fragments contained in an elution peak with the retention time of 3-4 min by adopting a liquid chromatography-mass spectrometer, and carrying out Fmoc solid-phase synthesis on the determined amino acid sequence, thereby obtaining the antioxidant tridecapeptide of the clinopodium polycephalum.
The invention also provides application of the clindamycin antioxidant tridecapeptide in preparation of antioxidant functional products.
The invention has the beneficial effects that:
the novel clindamycin anti-oxidation active peptide LLPENDPSANHLM provided by the invention has a strong DPPH, OH, ABTS free radical scavenging capability, and has important value in the aspects of anti-oxidation activity development and application. When the peptide concentration reached 2 mg/mL, the DPPH, ABTS and hydroxyl radical clearance were 41.117 + -0.924%, 18.934 + -0.828% and 14.102 + -0.179% respectively. IC of antioxidant peptide LLPENDPSANHLM on DPPH free radical clearance 50 The value of the compound is 7.104 +/-1.875 mg/mL, and the compound has IC (integrated circuit) on the clearance rate of ABTS free radicals 50 IC with value of 12.854 + -2.032 mg/mL and its clearance to hydroxyl radical 50 The value is 14.357 +/-1.827 mg/mL.
The invention establishes HepG-2 cell H 2 O 2 The damage model, through research and selection, induces cells to generate 50% survival rate, and the final concentration is 400 mu M H 2 O 2 As a model. The concentration of the Clinopodium polycephalum antioxidant peptide LLPENDPSANHLM is less than 2.0mg/mLHas no toxic effect on HepG-2 cells in the range of degree, and has the effect of protecting HepG-2 from oxidative damage of hydrogen peroxide. After the HepG-2 cells are treated by 0.5, 1.0, 1.5 and 2.0mg/mL of antioxidant peptide LLPENDPSANHLM for 24 hours, the normal cell proportion is not obviously changed compared with the polypeptide treatment group, which fully shows that the multi-antioxidant peptide LLPENDPSANHLM has no toxic effect on the HepG-2 cells at the concentration of 0-2.0 mg/mL. HepG-2 cells were used at a final concentration of 400. Mu. M H 2 O 2 After the induction of the injury, the cell viability decreased to 49.053 ±0.638%. If the HepG-2 cells are pretreated with 0-2.0 mg/mL of antioxidant peptide LLPENDPSANHLM before the oxidative damage is induced by hydrogen peroxide, the apoptosis rate of the cells is reduced (the cell survival rate reaches 54.668 +/-1.079% -71.154 +/-0.791%), which proves that the natural antioxidant peptide LLPENDPSANHLM can effectively protect the oxidative damage of the hydrogen peroxide of the HepG-2 cells.
The invention effectively prepares the antioxidant peptide from the clinopodium polycephalum, and provides scientific basis for realizing modernization of the clinopodium polycephalum traditional Chinese medicine and developing novel, efficient and safe health food.
Drawings
FIG. 1 is a DEAE sepharose anion chromatogram of a crude extract of Clinopodium polycephalum.
FIG. 2 is a reverse high performance liquid chromatogram of anion chromatography elution peak three.
Fig. 3 is a mass spectrum of the Clinopodium polycephalum antioxidant peptide LLPENDPSANHLM.
FIG. 4 shows the in vitro antioxidant activity of native antioxidant peptide LLPENDPSANHLM. A: ABTS radical scavenging activity; b: DPPH radical scavenging activity; c: hydroxyl radical scavenging activity.
FIG. 5 shows the toxic effect of natural antioxidant peptide LLPENDPSANHLM on HepG-2 cells.
FIG. 6 is H 2 O 2 Model of induced HepG-2 cell damage.
FIG. 7 is a diagram of the natural antioxidant peptide pair H 2 O 2 Protection of induced HepG-2 cells.
Detailed Description
The embodiment is implemented on the premise of the technical scheme of the invention, and detailed implementation modes and processes are given, but the protection scope of the invention is not limited to the following implementation examples.
EXAMPLE 1 isolation and purification of crude extract of Clinopodium polycephalum
Pretreatment of Clintonia sessilifolia: the surfaces of the clinkery grass leaves are cleaned, the black and rotten clinkery grass leaves are removed, and then the clinkery grass is ground by utilizing liquid nitrogen for standby.
Extracting crude protein and polypeptide of clinopodium polycephalum: grinding the clindamia powder according to a feed liquid ratio of 1:10 (m/v) distilled water was added thereto, the ultrasonic power was set at 135W, and the ultrasonic treatment was carried out at 25℃for 35 minutes. Filtering out residues, regulating the pH value of filtrate to 8.0 by adopting 1 mol/L NaOH, stirring at room temperature for 30 min, centrifuging at 5000 r/min for 20 min, regulating the pH value of the supernatant after centrifugation to 3.0 by adopting 1 mol/L HCl, centrifuging at 5000 r/min for 20 min, dissolving the precipitate after centrifugation in 10-30mL of Tris-hydrochloric acid buffer solution with the pH value of 8.4, loading into a dialysis bag with the molecular weight cutoff of 3500D, dialyzing overnight in deionized water, and freeze-drying the dialysate to obtain the crude protein of the clinopodium polycephalum.
Anion chromatographic separation and purification: the method comprises preparing the coarse protein lyophilized powder of Clinopodium polycephalum into 6 mg/mL solution with 0.02M Tris-hydrochloric acid buffer solution with pH value of 8.4, completely dissolving, filtering with 0.22 μm aperture microfiltration membrane, separating and purifying with DEAE-Sepharose ion exchange column chromatography, balancing with 0.02M Tris-hydrochloric acid buffer solution with pH value of 8.4, and loading the filtered sample onto column. The flow rate was set at 1 mL/min, the elution was linearly carried out with 0.2 mol/L to 1 mol/L NaCl Tris-hydrochloric acid buffer solution at pH=8.4, the absorbance of the effluent sample was detected by a chromatographic collection analyzer HD-A, 3 out-peak components were collected according to the elution peak (FIG. 1), the hydroxyl radical, DPPH, ABTS radical scavenging ability were compared, the highest active component (CNPH-III) was collected, and the mixture was lyophilized in vacuo and stored at a low temperature of-20℃for use.
Reversed phase high performance liquid chromatography separation and purification: dissolving the component CNPH-III dry powder with deionized water, filtering with a micro-filtration membrane with a pore size of 0.22 mu m, separating and purifying by a Thermo C18 reverse high performance liquid chromatographic column, wherein the sample concentration is 10mg/mL, the sample injection amount is 20 mu L, and the mobile phase A: acetonitrile containing 0.1% tfa by mass, mobile phase B: deionized water containing 0.1% TFA by mass fraction, flow rate of mobile phase was 1.0. 1.0 mL/min. The gradient elution mode is adopted, and the elution conditions are as follows: 0-5min,5% -10% mobile phase A;5-15min,10% -50% of mobile phase A;15-35 min,50% -80% of mobile phase A. The detection wavelength was 214 nm. 3 off-peak fractions were collected from the elution peaks (FIG. 2), the hydroxyl radical, DPPH, and ABTS radical scavenging ability of each off-peak fraction was measured, and the fraction with the highest activity (elution peak with retention time of 3-4 min, CNPH-III-2) was collected and lyophilized under vacuum.
After freeze-drying the collected antioxidant components, determining the amino acid sequence by using a liquid chromatography and mass spectrometry (LC-MS/MS) method (figure 3), and comparing the Uniprot database with the NCBI database to find a new peptide, wherein the amino acid sequence of the antioxidant peptide is as follows: LLPENDPSANHLM. The amino acid sequence was sent to Shanghai Jier Biochemical Co.Ltd for Fmoc solid phase synthesis.
Example 2 in vitro antioxidant Activity assay of Natural antioxidant peptides
Weighing synthetic polypeptide sample, and preparing into sample solutions with different concentrations by distilled water.
DPPH radical clearance: accurately weighing 3.94 mg of DPPH, dissolving by adopting an ethanol solution with the volume fraction of 95 percent, and fixing the volume to 100 mL to prepare a DPPH solution with the volume of 0.1 mmo 1/L. Fully and uniformly mixing the 1 mL sample solution with 1 mL of DPPH solution, standing at room temperature in a dark place for 30 min, and measuring the light absorption value at 517 nm; using 1 mL volume fraction of 95% ethanol solution instead of DPPH solution as a control group; 1 mL distilled water was used as a blank instead of the sample. The DPPH radical scavenging rate of the sample was calculated using the following formula:
wherein: a is that 1 -absorbance of the sample set; a is that 2 -absorbance of control group; a is that 3 Light absorbance value of blank control group
ABTS radical scavenging activity: ABTS stock mother liquor of 7 mM and potassium persulfate solution of 2.45 mM were prepared immediately before use at 1:1, and placing the mixture in a dark place at room temperature of 16 h, and diluting the mixture with 5 mM phosphate buffer solution with pH of 7.4 until the absorbance at 734 nm is 0.70+/-0.02, thus obtaining the ABTS free radical solution. 1 mL of ABTS free radical solution was mixed with 1 mL sample solutions of different concentrations in equal volumes, reacted at room temperature for 10 min, and absorbance was measured at 734 nm. Distilled water was used as a blank instead of the sample solution. ABTS radical scavenging activity of the samples was calculated as follows:
wherein: a is that 1 -absorbance of the sample set; a is that 2 Blank absorbance values
Hydroxyl radical scavenging activity: 8mmol/L ferrous sulfate solution, 3 mmol/L salicylic acid solution and 20mmol/L hydrogen peroxide solution are prepared for later use. 200. Mu.L of the sample was thoroughly mixed with 60. Mu.L of 8mmol/L ferrous sulfate, 200. Mu.L of 20mmol/L salicylic acid, 50. Mu.L of 20mmol/L hydrogen peroxide, incubated in a 37℃water bath for 30 min, cooled to room temperature, added with 90. Mu.L deionized water, and centrifuged at 4000 rpm for 30 min. The supernatant was taken and the absorbance of the reaction was measured at 510 and nm. Distilled water was used as a blank. The scavenging activity of the hydroxyl radical was calculated according to the following formula:
wherein: a is that 0 Blank absorbance; a is that 1 Sample set absorbance
As shown in FIG. 4, the antioxidant peptides LLPENDPSANHLM of the clindamycin have better scavenging rates on DPPH free radicals, ABTS free radicals and hydroxyl free radicals.
EXAMPLE 3 toxicity of Natural antioxidant tridecapeptide on HepG-2 cells and study of HepG-2 cytoprotective Effect
Cell culture: hepG-2 cells were cultured in DMEM medium containing 10% FBS by volume fraction at 37℃and 5% CO 2 And culturing and passaging in an incubator. Observing under an inverted microscope, pumping the culture solution in a sterile operation table until the cell growth is fused to 80%, and firstly using PBS phosphate buffer solution with pH of 7.3(from Zhongwuxiaonong Ruidong Biotechnology Co., ltd., 23.48, g/2L), the cell surface layer was washed, 1 mL pancreatin digest was added, and the mixture was placed in an incubator at 37℃for 5 minutes, and then the effect of pancreatin digest was stopped by adding 1 mL DMEM complete medium containing 10% fbs by volume. All the liquid was then transferred into a 1.5 mL centrifuge tube, centrifuged at 2000 rpm for 10 min, the supernatant discarded after centrifugation, fresh broth was added to the pellet and the cells were evenly dispersed by pipetting several times. Cells were evenly distributed in fixed proportions into various sizes of dishes according to the experimental design.
Cytotoxicity: and determining the toxic effect of the antioxidant peptide LLPENDPSANHLM on HepG-2 cells by adopting a CCK-8 method, sucking the old culture medium (without fully growing the cells, the cell density is too high to influence the cell growth state) when the density of the HepG-2 cells is increased to 70% -90%, cleaning by using PBS buffer with the pH of 7.3 preheated at 37 ℃ in advance to remove residual serum on the surface, adding 1 mL pancreatin digestive juice, and digesting for 5min in a constant-temperature incubator at 37 ℃. Digestion was stopped by adding 1 mL volume fraction 10% fbs in DMEM complete medium to the dishes after the cells had floated. At 1x10 5 cell/mL density was seeded in 96-well plates at 90. Mu.L per well, with 4 replicate wells per concentration gradient in parallel. 37 ℃,5% concentration CO 2 Culture 24 h in incubator followed by addition of 10. Mu.L of DMEM medium containing antioxidant peptide LLPENDPSANHLM per well at concentration gradients of 0, 0.5, 1.0, 1.5 and 2.0mg/mL, culture 24 h. After pipetting the medium, 100. Mu.L of CCK-8 (prepared in DMEM medium) containing a volume fraction of 10% was added, and after incubation in an incubator of 1 h the absorbance was determined with an enzyme-labeled instrument at a wavelength of 450 nm (FIG. 4). Glutathione (GSH) was used as a control and DMEM medium was used as a blank. The viability of each group of drugs on cells was calculated according to the following formula:
wherein: a is that 1 Blank absorbance; a is that 2 -absorbance of control group; a is that 3 Sample set absorbance
HepG-2 cell peroxidationEstablishing a hydrogen oxidation damage model: the density was 1.0X10 5 cells/mL of HepG-2 cells were seeded in 96-well plates. A blank (90. Mu.L of LDMEM medium), a control (90. Mu.L of cell suspension and an experimental group (90. Mu.L of cell suspension) were set up at 37℃with 5% CO 2 After 24. 24H incubation in incubator, the blank and control groups were each added with 10. Mu.L of LDMEM medium, and the experimental groups were added with 10. Mu.L of H at final concentrations of 100, 200, 400, 600, 800. Mu. Mol/L 2 O 2 After further incubation for 2, 4, 8 hours, cell viability was measured. Selecting H with cell viability around 50% 2 O 2 The concentration was used as a model.
HepG-2 cytoprotective action: the density was 1.0X10 5 HepG-2 cells of cells/mL were seeded in 96-well plates, and a blank, a control, a lesion and a protection group were set, each group was added with 80. Mu.L of cell suspension, and the blank was added with DEME medium. At 37 ℃,5% CO 2 After incubation in the incubator at 24 h, the blank, control and lesion groups were added with 10 μl of medium and the protective groups were added with 10 μl of antioxidant peptide LLPENDPSANHLM or Glutathione (GSH) solution at final concentrations of 0.5, 1.0, 1.5 and 2.0mg/mL for continued incubation at 24 h. Then, 10. Mu.L of medium was added to each of the blank group and the control group, and 10. Mu. L H was added to each of the injured group and the protected group 2 O 2 After further incubation of the solution (final concentration 400. Mu. Mol/L) with 4 h, the cell viability was examined.
As shown in FIGS. 5, 6 and 7, after the HepG-2 cells are treated by LLPENDPSANHLM of 0-2 mg/mL for 24 hours, the normal cell proportion is not significantly changed compared with the polypeptide treatment group (GSH and LLPENDPSANHLM treatment group), which fully shows that GSH and polypeptide LLPENDPSANHLM have no toxic effect on the HepG-2 cells at the concentration of 0-2 mg/mL. HepG-2 cells were treated with LLPENDPSANHLM at 0.5, 1.0, 1.5 and 2.0mg/mL for 24 hours and then with H at a final concentration of 400. Mu.M 2 O 2 The cell survival rate can be improved by inducing oxidative damage, which indicates that the natural antioxidant peptide LLPENDPSANHLM can effectively protect HepG-2 cells from oxidative damage of hydrogen peroxide.
In the foregoing, only the preferred embodiments of the present invention are described, and all equivalent changes and modifications made according to the claims of the present invention should be covered in the protection scope of the present invention.
SEQUENCE LISTING
<110> university of Fuzhou
<120> an antioxidant tridecapeptide from Clinopodium polycephalum, and its preparation method and application
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 13
<212> PRT
<213> artificial sequence
<400> 1
Leu Leu Pro Glu Asn Asp Pro Ser Ala Asn His Leu Met
1 5 10
Claims (2)
1. An antioxidant tridecapeptide of clindamycin, which is characterized in that: the amino acid sequence of the anti-oxidation tridecapeptide of the clindamycin is Leu-Leu-Pro-Glu-Asn-Asp-Pro-Ser-Ala-Asn-His-Leu-Met.
2. A method for preparing the antioxidant tridecapeptide of clindamycin according to claim 1, which is characterized in that: the method comprises the following steps:
1) Extracting protein in clindamycin by adopting an alkali-dissolution acid precipitation method, and preparing a clindamycin crude extract: grinding the clindamia powder according to a feed liquid ratio of 1:10 Adding distilled water m/v, setting ultrasonic power to 135W, performing ultrasonic treatment at 25 ℃ for 35 min, filtering out residues, regulating the pH value of filtrate to 8.0 by using NaOH of 1 mol/L, stirring at room temperature for 30 min, centrifuging at 5000 r/min for 20 min, regulating the pH value of the supernatant after centrifugation to 3.0 by using HCl of 1 mol/L, centrifuging at 5000 r/min for 20 min, dissolving the precipitate after centrifugation in Tris-hydrochloric acid buffer solution of which the pH value is 8.4 of 10-30mL, filling into dialysis bags of which the cut-off molecular weight is 3500D, dialyzing overnight in deionized water, and freeze-drying the dialysate to obtain a crude extract of the clinkering grass protein;
2) Separating the crude extract of the clindamycin by using DEAE-Sepharose ion exchange chromatography, linearly gradient eluting with 0.2-1 mol/L NaCl Tris-hydrochloric acid buffer solution with pH=8.4 at a flow rate of 1 mL/min, detecting absorbance of the effluent sample by using a chromatographic acquisition analyzer HD-A, collecting each peak component according to the elution peak, measuring antioxidant activity, and collecting the component CNPH-III with the highest antioxidant activity;
3) Separating and purifying the component CNPH-III by reverse phase high performance liquid chromatography RP-HPLC, wherein the chromatographic column is a Thermo C18 reverse phase high performance liquid chromatography column, 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 is: 0-5min,5% -10% A;5-15min,10% -50% of A;15-35 min,50% -80% A, the flow rate of the mobile phase is 1.0 mL/min, the detection wavelength is 214 nm, and the elution peak with the retention time of 3-4 min is collected;
4) And (3) carrying out amino acid sequence determination on peptide fragments contained in an elution peak with the retention time of 3-4 min by adopting a liquid chromatography-mass spectrometer, and carrying out Fmoc solid-phase synthesis on the determined amino acid sequence, thereby obtaining the antioxidant tridecapeptide of the clinopodium polycephalum.
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| 大孔吸附树脂纯化美洲大蠊多肽的工艺研究;廖芳 等;中草药;第47卷(第19期);3420-3425 * |
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