CN114874291B - Artificial tiger bone polypeptide and preparation method and application thereof - Google Patents
Artificial tiger bone polypeptide and preparation method and application thereof Download PDFInfo
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Physical Education & Sports Medicine (AREA)
- Life Sciences & Earth Sciences (AREA)
- Rheumatology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an artificial tiger bone polypeptide, a preparation method and application thereof, wherein the amino acid sequence of the artificial tiger bone polypeptide is shown as SEQ ID NO.1 or SEQ ID NO.2, and the preparation process is as follows: firstly, extracting 1g of artificial tiger bone powder in 300mL of ultrapure water at 80 ℃ for 4 hours, centrifuging to obtain a supernatant to obtain an artificial tiger bone polypeptide solution, ultrafiltering the solution by a 3KD ultrafiltration membrane, collecting filtrate, and vacuum freeze-drying at-40 ℃; then adopts Superdex TM peptide 10/300GL gel filtration and separation, and finally high performance liquid chromatography purification; the artificial tiger bone polypeptide has the functions of promoting osteoblast proliferation and resisting osteoporosis, and provides a feasible research path for osteoporosis treatment.
Description
Technical Field
The invention relates to biomedical technology, in particular to an artificial tiger bone polypeptide and a preparation method and application thereof.
Background
Tiger bone is the skeleton of tiger, which comes from Ben Cao Jing Ji Zhi (materia Medica of materia Medica). Tiger bone, pungent, sweet, salty, warm, ancient writing states that tiger bone is good at dispelling pathogenic qi, arresting convulsion and epilepsy, strengthening tendons and bones, etc., and is suitable for arthralgia, arthralgia due to wind-cold-dampness, soreness of waist and backache, etc. Ancient formulas such as Huguzhuifeng pills, longhu san and Hugu Jiu are commonly used for treating osteoporosis. At present, tiger is an important protection for wild animals, so tiger bones are forbidden to be used as medicinal materials. However, due to the need of medical and health of the folk of traditional Chinese medicine, a great deal of research on tiger bone substitutes is carried over, and artificial tiger bone powder is one of them. The artificial tiger bone powder is approved by the national food and drug administration as a new medicine in 8 months and 30 days of 2003, and is also called as a gold Tiange capsule. The method adopts unprotected animal bones and natural tiger bones to carry out contrast analysis and research, and develops artificial tiger bones which are almost the same as the fingerprint spectrum of the natural tiger bones. Has similar components and safety to those of natural tiger bone powder, has the functions of strengthening tendons and bones, and is used for improving symptoms such as soreness and back pain, soreness and weakness of waist and knees, flaccidity of lower limbs, difficult walking and the like. The clinical artificial tiger bone powder is mainly used for treating bone diseases such as osteoporosis, fracture, rheumatoid arthritis and the like.
According to the published data, the total amino acid content of the artificial tiger bone powder is 16.82 g/100 g, which is quite consistent with 16.06 g/100 g of the natural tiger bone powder; analyzing the contents of peptides and proteins of the artificial tiger bone powder and the natural tiger bone powder by adopting HPLC, and uniformly dividing the contents into 5 peaks, wherein the retention time of the two peaks is basically consistent; the artificial tiger bone powder and the natural tiger bone powder have 13 inorganic elements and microelements which are the same. However, the active polypeptide in the artificial tiger bone is not researched and reported at present, and the structure, activity and extraction preparation process of the active polypeptide of the artificial tiger bone are unknown. The active polypeptide in the artificial tiger bone, the structure and the function thereof are found, which is helpful for secondary development of the artificial tiger bone.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and provides an artificial tiger bone polypeptide and a preparation method and application thereof.
The aim of the invention is realized by the following technical scheme:
an artificial tiger bone polypeptide has an amino acid sequence shown as SEQ ID NO.1 or SEQ ID NO. 2.
The preparation method of the artificial tiger bone polypeptide comprises the following steps:
(1) Adding 1g of artificial tiger bone powder into 200ml of pure water, dissolving in water bath at 80deg.C for 4 hr, and vacuum filtering to obtain solution. Filtering with 3KD ultrafilter membrane, collecting polypeptide solution smaller than 3kDa, and vacuum freeze-drying at-40deg.C to obtain polypeptide lyophilized powder;
(2) By Superdex TM Separating peptide 10/300GL by gel filtration, and subjecting the polypeptide lyophilized powder obtained in the step 1 to Superdex TM And (3) separating and purifying the peptide by using a peptide 10/300GL gel column. The mobile phase is aqueous solution containing 0.1% trifluoroacetic acid and 30% acetonitrile in volume ratio, the flow rate is 1mL/min, the detection wavelength of protein chromatograph is 280nm, and the elution peak is collected and freeze-dried in vacuum at-40 ℃.
(3) Purifying by high performance liquid chromatography, passing the polypeptide obtained in the step (2) through a reversed phase high performance liquid column, wherein the chromatographic column is Zorbax SB C 18 (250 mm. Times.9.4 mm,5 μm); the column temperature is room temperature; the mobile phase is acetonitrile and water, and gradient elution: the acetonitrile concentration is changed from 0% to 95% within 0-30 min, the elution speed is 2.5-3.0 mL/min, the ultraviolet detection wavelength is 220nm, and the components are collected according to peaks to obtain the artificial tiger bone polypeptide.
The application of the artificial tiger bone polypeptide in preparing the medicines for promoting osteoblast proliferation and resisting osteoporosis.
Compared with the prior art, the invention has the beneficial effects that: the invention extracts the artificial tiger bone polypeptide, has obvious effects on resisting osteoporosis and promoting osteogenesis, provides a feasible research path for osteoporosis treatment, has simple whole process steps and low material consumption, and is suitable for mass popularization and application.
Drawings
FIG. 1 is a graph showing the separation effect of the artificial tiger bone after 3KD of the components are separated by a gel column;
FIG. 2 is a reverse HPLC separation and purification diagram of artificial tiger bone polypeptide 1;
FIG. 3 is a reverse HPLC separation and purification diagram of artificial tiger bone polypeptide 2;
FIG. 4 is an effect of artificial tiger bone polypeptides 1 and 2 on osteoblast proliferation, wherein (a) the effect of artificial tiger bone polypeptide 1 on osteoblast proliferation, (b) the effect of artificial tiger bone polypeptide 2 on osteoblast proliferation;
FIG. 5 is an effect of artificial tiger bone polypeptides 1 and 2 on osteoblast alkaline phosphatase activity, wherein (a) the effect of artificial tiger bone polypeptide 1 on osteoblast alkaline phosphatase activity and (b) the effect of artificial tiger bone polypeptide 2 on osteoblast alkaline phosphatase activity.
Detailed Description
Example 1:
1 Material
1.1 medicinal materials
Artificial tiger bone powder, crude drug, golden flower pharmaceutical factory, production lot number: 210311-1; white solid powder.
1.2 laboratory apparatus
High speed refrigerated centrifuges (Thermo Fisher Scientific company, usa); mini PROTEAN Tetra Cell Tris-glycine SDS polyacrylamide gel electrophoresis apparatus (BIO-RAD Co., USA); AR2130 type electronic precision balance (Ohaus Corp, usa); LC-10N/12N/18N freeze dryer (Shanghai Libang West instruments and technology Co., ltd., china); DHG-9080A type electrothermal blowing dry box (Shanghai Ha Wei, china); vivaflow 50 3000MWCO PES Ultrafiltration apparatus (Sartorius Stedim Lab Ltd.GL10 3UT, UK); sep-Pak Vac 1cc (50 mg) tC18 column (Waters, USA); RE-52AA rotary evaporator (Shanghai Qinghai Shanghai, china); nano LCU3000 system (Thermo Fisher Scientific, USA); accaim PepMap 100C18 chromatographic column (Thermo Fisher Co., U.S.A.); liquid chromatograph-mass spectrometer (Thermo Fisher Scientific company, U.S.A.)
2 method
2.1 Single factor experiment of extraction conditions of Artificial tiger bone
15 parts of artificial tiger bone powder are taken as a group of 1g each and 5 parts.
The first group measures the effect of different water bath temperatures on the percent dissolution of artificial tiger bone powder. Extracting 5 parts of artificial tiger bone powder in 200ml of ultrapure water at 50 ℃,60 ℃,70 ℃,80 ℃ and 90 ℃ respectively under water bath stirring for 2 hours, suction filtering, drying undissolved artificial tiger bone powder, weighing, and calculating the dissolution gram number/total weight, thus obtaining the extraction rate.
The second set measures the effect of different solvent volumes on the percent dissolution of artificial tiger bone powder. Taking 5 parts of artificial tiger bone powder, respectively extracting in water bath at 80 ℃ in ultrapure water of different volumes (100 ml,150ml,200ml,250ml,300 ml) for 2 hours, suction filtering, drying undissolved artificial tiger bone powder, weighing, calculating the number of dissolved grams/total weight, and calculating the extraction rate.
The third group measures the effect of different water bath times on the percent dissolution of artificial tiger bone powder. Extracting 5 parts of artificial tiger bone powder in 200ml of ultrapure water at 80 ℃ for different times (1 h,2h,3h,4h and 5 h) by stirring in a water bath, filtering, drying undissolved artificial tiger bone powder, weighing, calculating the number of dissolved grams/total weight, and calculating the extraction rate.
2.2 Ultrafiltration of anti-osteoporosis active peptides
And (3) carrying out ultrafiltration separation on the extracted artificial tiger bone polypeptide by using an ultrafiltration membrane with a cutoff of 3kDa on an ultrafiltration instrument, sequentially collecting filtrate to obtain polypeptide smaller than 3kDa, and carrying out vacuum freeze drying at-40 ℃ to obtain unfiltered polypeptide larger than 3 kDa. Obtaining polypeptide freeze-dried powder. Osteoblast proliferation experiments prove that the part smaller than 3KD has the effect of promoting the proliferation of the osteoblast.
2.3 Superdex is used TM peptide 10/300GL gel column filtration separation
The step 1 is performed to obtain a product smaller than3KD polypeptide Superdex TM And (3) separating and purifying the peptide by using a peptide 10/300GL gel column. The mobile phase is aqueous solution containing 0.1% trifluoroacetic acid and 30% acetonitrile in volume ratio, the flow rate is 1mL/min, the detection wavelength of protein chromatograph is 280nm, and the elution peak is collected and freeze-dried in vacuum at-40 ℃.
2.4 high Performance liquid chromatography purification
Passing the obtained polypeptide through reversed phase high performance liquid column, wherein the chromatographic column is Zorbax SB C 18 (250 mm. Times.9.4 mm,5 μm); the column temperature is room temperature; the mobile phase is acetonitrile and water, and gradient elution: the acetonitrile concentration is changed from 0% to 95% within 0-30 min, the elution speed is 2.5-3.0 mL/min, the ultraviolet detection wavelength is 220nm, and the components are collected according to peaks to obtain the artificial tiger bone polypeptide 1 and the artificial tiger bone polypeptide 2.
2.5 cell experiments
2.5.1 Effect of polypeptide in Artificial tiger bone powder on MC3T3-E1 cell proliferation
Mouse MC3T3-E1 preosteoblasts were cultured in alpha-MEM complete medium. MC3T3 osteoblast cell line with good growth state is prepared by 1×10 4 Density of each well was seeded in 96-well plates at 37℃with 5% CO 2 After 24h adaptation of the incubator, the well plates were divided into a blank group, which was given alpha-MEM complete medium, and an experimental group, which was given different concentrations: 1. Mu.g/mL, 2. Mu.g/mL and 5. Mu.g/mL of artificial tiger bone polypeptide 1 or artificial tiger bone polypeptide 2, 100. Mu.L per well, 6 duplicate wells per group, and after 48h of intervention, 10. Mu.L of CCK-8 detection solution per well, 100. Mu.L of serum-free alpha-MEM medium were added, incubated for 1h at 37℃in a 5% CO2 incubator, and the absorbance at 450nm was measured with an enzyme-labeled instrument.
2.5.2 Effect of Polypeptides in Artificial tiger bone powder on osteoblast alkaline phosphatase Activity
Taking MC3T3-E1 cells which are in good condition and can be passaged, adding 0.25% trypsin for digestion, blowing with alpha-MEM culture solution containing 10% FBS to form suspended cell solution, and using 5×10 per well 4 Individual cells were seeded in 96-well culture plates at 100 μl per well volume. Placing the culture plate into a constant temperature incubator at 37deg.C with 5% CO 2 Culturing for 24h under saturated humidity condition, and changing the induced differentiation culture medium: containing 10nM dexamethasone, 10mM beta-glycylSodium oil phosphate and 50. Mu.g/mL ascorbic acid and 10% FBS alpha-MEM medium. The well plates were divided into a blank group, which was given induction differentiation medium, and an experimental group, which was given different concentrations: 1. Mu.g/mL, 2. Mu.g/mL, and 5. Mu.g/mL of artificial tiger bone polypeptide 1 or artificial tiger bone polypeptide 2. Culturing was continued for 7 days, with liquid changes every three days. After the original culture solution is sucked and removed, the culture solution is washed by precooled PBS for 2 to 3 times, 50 mu L of cell lysate is added into each hole for cracking for 30 minutes, the mixture is collected in an EP tube, the mixture is centrifuged at 12000rpm at 4 ℃ for 15 minutes to obtain the supernatant, and the alkaline phosphatase activity is measured by an alkaline phosphatase kit, and the specific operation is carried out according to the specification.
2.5.3 Effect of polypeptide in Artificial tiger bone powder on bone mineralization nodule formation of osteoblasts (alizarin red ARS staining)
Taking cells in good condition and passaging, adding 0.25% trypsin for digestion, blowing with alpha-MEM culture solution containing 10% FBS to form suspended cell solution, and allowing each well to be 5×10 4 Individual cells were inoculated in 24-well plates and after 24h, the induced differentiation medium was changed: alpha-MEM medium containing 10nM dexamethasone, 10mM sodium beta-glycerophosphate and 50. Mu.g/mL ascorbic acid and 10% FBS. The well plates were divided into a blank group, which was given induction differentiation medium, and an experimental group, which was given different concentrations: 1. Mu.g/mL, 2. Mu.g/mL, and 5. Mu.g/mL of artificial tiger bone polypeptide 1 or artificial tiger bone polypeptide 2. Changing the culture solution every 3 days, continuously culturing for 21 days, sucking the original culture solution, washing with precooled PBS for 2-3 times, fixing in 4% paraformaldehyde for 30min, washing with PBS for 2-3 times, adding 500 μl of 0.1% alizarin red-Tris-HCl dye solution (pH=8.3), placing in a 37 ℃ incubator for dyeing for 30min, taking out a 24-pore plate, sucking the dye solution, washing with distilled water for 3 times, drying, observing under an inverted phase contrast microscope, and photographing.
2.6 liquid chromatography-tandem mass spectrometry analysis
A Nano LCU3000 system was used to assemble Accaim PepMap Nano C18 chromatographic column and Acclaim PepMap 100C18 chromatographic column as separation columns, and the flow rate was set at 400p/min. A 120min linear gradient is as follows: 10min 2% B/98% A-5% B,70min 5% B-19% B,15min 19% B-30% B,10min 30% B-98%B,5min 100%B,2min to 2% B. Acquisition of mass spectral data, from a full scan covering m/z 350 to 1800, the first 20 precursors are fragmented by higher energy collisions in order of intensity. The normalized collision energy was set to 27% and the dynamic banking time was 30s. The automatic gain control of MS1 and MS2 is set to 3e6 and 1e5, and the resolution of MS1 and MS2 is set to 120 and 15K.
2.7 alignment with database
All MS original files of polypeptide analysis were retrieved by nonspecific cleavage of amino acid sequences of pig, goat and red deer bones using Uniprot system. Results were screened by setting peptide levels of 1% fdr with methionine and proline oxidation as variable modifications.
3 results
3.1 Single factor experiment
As shown in the experimental results of Table 3-1, the dissolution weight of the artificial tiger bone powder was maximum and the extraction rate was 9.82% at the water bath temperature of 80℃under the same solvent volume (200 ml) and water bath time (2 h).
Table 3-1: experimental results of influence of different water bath temperatures on extraction rate of polypeptide of artificial tiger bone powder
As shown in the experimental results of Table 3-2, the dissolution weight was maximum and the extraction rate was 9.82% at the same solvent volume of 200ml under the conditions of the water bath temperature (80 ℃) and the water bath time (2 hours).
Table 3-2: experimental results of influence of different solvent volumes on extraction rate of polypeptide of artificial tiger bone powder
As shown in the experimental results of tables 3 to 3, the dissolution weight was maximum and the extraction rate was 14.61% at the same water bath temperature (80 ℃) and solvent volume (200 ml) for 4 hours.
Table 3-3: influence of different water bath time on extraction rate of polypeptide of artificial tiger bone powder
In conclusion, the extraction rate of the artificial tiger bone powder is highest when the water bath time is 4 hours in 200ml of ultrapure water at 80 ℃.
3.2 Superdex is used TM peptide 10/300GL gel column filtration separation results
As shown in FIG. 1, the highest collection peak type of polypeptide fraction 1, polypeptide fraction 2, polypeptide fraction 3 and polypeptide fraction 4 was selected in combination with the absorbance at 280 nm. Repeating the sample collection for multiple times according to the method, and performing vacuum freeze drying at-40 ℃. Osteoblast proliferation experiments show that the polypeptide component 1 and the polypeptide component 2 have obvious bone proliferation promoting activity, so that the polypeptide component 1 and the polypeptide component 2 are selected for further separation and purification.
3.3 high Performance liquid chromatography purification results
The polypeptide component 1 and the polypeptide component 2 are purified by high performance liquid chromatography. Fig. 2 and 3 show the results of high performance liquid chromatography purification of the artificial tiger bone polypeptide component 1 and the polypeptide component 2, respectively, and the results show that the obtained polypeptide has good purity. And (3) respectively collecting the purified artificial tiger bone polypeptides according to peaks to obtain an artificial tiger bone polypeptide 1 and an artificial tiger bone polypeptide 2, repeatedly loading and collecting, and performing vacuum freeze drying at-40 ℃ to perform subsequent experiments.
3.4 Effect of target peptides on osteoblast proliferation
As shown in fig. 4, proliferation of MC3T3-E1 cells was significantly increased (P < 0.01) in both the artificial tiger bone polypeptide 1 and polypeptide 2 groups after 48h of intervention compared to the blank group. Wherein P <0.05, P <0.01, P <0.001 compared to the blank.
3.5 Effect of peptides of interest on osteoblast alkaline phosphatase (ALP) Activity
As shown in fig. 5, osteoblast alkaline phosphatase activity represents osteoblast differentiation activity. The alkaline phosphatase activity of group 1 of artificial tiger bone polypeptides was significantly increased (P < 0.01) compared to the blank group and increased with increasing concentration. The activity of alkaline phosphatase in group 2 of artificial tiger bone polypeptides was significantly increased (P < 0.01) and increased with increasing concentration. Wherein (a) the effect of artificial tiger bone polypeptide 1 on osteoblast alkaline phosphatase activity, (b) the effect of artificial tiger bone polypeptide 2 on osteoblast alkaline phosphatase activity; compared with the blank group, the artificial tiger bone polypeptide 1 and the artificial tiger bone polypeptide 2 have obvious promotion effect on the ALP activity of osteoblasts (P < 0.01). Wherein P <0.05, P <0.01, P <0.001 compared to the blank.
3.6 Effect of target peptides on the formation of bone mineralization nodules in osteoblasts
After alizarin red staining, MC3T3-E1 cells form multiple layers of cells and develop into small colonies, collagen and calcium salt are secreted simultaneously so that the small colonies are mineralized to form opaque nodules, massive precipitates are formed at the nodules, and the quantity and the size of the nodules represent the mineralization degree of osteoblasts. Compared with a blank group (Ctrl), the number of calcified nodules of the artificial tiger bone polypeptide 1 group increases with the increase of the concentration, and the number of calcified nodules of the artificial tiger bone polypeptide 2 group increases with the increase of the concentration, which indicates that both the artificial tiger bone polypeptides 1 and 2 have the effect of promoting the calcification of osteoblasts.
3.7 Mass Spectrometry method for identifying the Components of Artificial tiger bone powder polypeptide
The molecular weight and the amino acid sequence of the obtained artificial tiger bone polypeptide 1 and the artificial tiger bone polypeptide 2 with bone promoting effect are determined by combining high-resolution mass spectrum and liquid chromatography technology: artificial tiger bone polypeptide 1, molecular weight, 1147.23g/mol, amino acid sequence (SEQ ID NO. 1), H-Gly-Leu-Pro-Gly-Pro-Pro-Gly-Glu-Arg-Gly-Gly-Pro-Gly-OH, (GLPGPPGERGGPG). Artificial tiger bone polypeptide 2, molecular weight 1341.55g/mol, amino acid sequence (SEQ ID NO. 2), H-Gly-Ala-Pro-Gly-Leu-Pro-Gly-Pro-Arg-Gly-Ile-Pro-Gly-Pro-Val-OH, (GAPGLPGPRGIPGPV).
Sequence listing
<110> Zhejiang university of Chinese medicine
<120> an artificial tiger bone polypeptide, its preparation method and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213> Artificial sequence (Unknown)
<400> 1
Gly Leu Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly
1 5 10
<210> 2
<211> 15
<212> PRT
<213> Artificial sequence (Unknown)
<400> 2
Gly Ala Pro Gly Leu Pro Gly Pro Arg Gly Ile Pro Gly Pro Val
1 5 10 15
Claims (3)
1. An artificial tiger bone polypeptide is characterized in that the amino acid sequence is shown as SEQ ID NO.1 or SEQ ID NO. 2.
2. A method of producing an artificial tiger bone polypeptide of claim 1 including the steps of:
(1) Adding 1g of artificial tiger bone powder into 200ml of pure water, dissolving in water bath at 80deg.C for 4 hr, filtering, collecting the solution, filtering with 3KD ultrafiltration membrane, collecting polypeptide solution less than 3kDa, and vacuum freeze drying at-40deg.C to obtain polypeptide lyophilized powder;
(2) By Superdex TM Gel filtration separation of peptide 10/300GL, and Superdex treatment of the polypeptide lyophilized powder obtained in step 1 TM Separating and purifying peptide 10/300GL gel column with mobile phase of water solution containing trifluoroacetic acid 0.1% and acetonitrile 30% at volume ratio of 1mL/min, detecting wavelength 280nm with protein chromatograph, collecting eluting peak, and vacuum freeze drying at-40deg.C;
(3) Purifying by high performance liquid chromatography, passing the polypeptide obtained in the step (2) through a reversed phase high performance liquid column, wherein the chromatographic column is Zorbax SB C 18 The method comprises the steps of carrying out a first treatment on the surface of the The column temperature is room temperature; the mobile phase is acetonitrile and water, and the gradient elution is carried out: the acetonitrile concentration is changed from 0% to 95% within 0-30 min, the elution speed is 2.5-3.0 mL/min, the ultraviolet detection wavelength is 220nm, and the components are collected according to peaks to obtain the artificial tiger bone polypeptide.
3. Use of an artificial tiger bone polypeptide of claim 1 in the manufacture of an anti-osteoporosis medicament.
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| CN108586604A (en) * | 2018-06-19 | 2018-09-28 | 大连工业大学 | Facilitate bone bioactivity peptide and its screening technique |
| CN111307990A (en) * | 2020-03-31 | 2020-06-19 | 金花企业(集团)股份有限公司西安金花制药厂 | Method for simultaneously determining content of cystine and methionine in artificial tiger bone powder |
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| CN108586604A (en) * | 2018-06-19 | 2018-09-28 | 大连工业大学 | Facilitate bone bioactivity peptide and its screening technique |
| CN111307990A (en) * | 2020-03-31 | 2020-06-19 | 金花企业(集团)股份有限公司西安金花制药厂 | Method for simultaneously determining content of cystine and methionine in artificial tiger bone powder |
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| 人工虎骨粉治疗绝经后骨质疏松症的临床效果及作用机制探讨;徐迎锋等;北方药学;第15卷(第11期);第98-99页 * |
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