CN104894200A - Method for preparing cartilage angiogenesis inhibiting factor for scalloped hammerhead shark - Google Patents

Abstract

The invention relates to a preparation method of scalloped hammerhead shark angiogenesis inhibitory factor. Pro-Asp-Tyr-Lys-Phe was prepared by homogenizing hammerhead shark cartilage, extracting with guanidine hydrochloride, acetone precipitation and fractionation, enzymatic hydrolysis, gel filtration chromatography, cell membrane chromatography and high performance liquid chromatography. -Lys. The angiogenesis inhibitory factor can effectively inhibit the angiogenesis of chick embryo chorioallantoic membrane, and can promote three kinds of angiogenesis: vascular endothelial cell growth factor, basic fibroblast growth factor and platelet-derived growth factor in lung cancer tissues of mice bearing Lewis lung cancer. Factor expression was significantly inhibited.

Description

Preparation method of scalloped hammerhead cartilage angiogenesis inhibitory factor

Preparation method of scalloped hammerhead cartilage angiogenesis inhibitory factor

technical field

The invention relates to a preparation method of angiogenesis inhibitory factor from aquatic organisms, in particular to a preparation method of scalloped hammerhead cartilage angiogenesis inhibitory factor.

Background technique

Angiogenesis is one of the important pathological features of vascular proliferative diseases such as tumors, diabetic retinopathy, rheumatoid arthritis and chronic inflammation. The anti-angiogenesis-based tumor biotherapy research has become a research hotspot of anti-tumor drugs in the past ten years.

Compared with chemotherapy aimed at directly killing tumor cells, tumor angiogenesis inhibitor (TAI) has the following unique advantages: (1) TAI directly acts on vascular endothelial cells, while anticancer drugs diffuse through tissues Affected by tissue fibrosis, necrosis, etc., the effective drug concentration in the tissue is often not reached; (2) Compared with genotype-unstable tumor cells, vascular endothelial cells are normal cells with stable genotypes and are not prone to drug resistance; (3) The vascular endothelial cells of the primary tumor and the secondary tumor are the same, but the biological characteristics of the tumor cells in the primary tumor and the secondary tumor are quite different, and the chemotherapy response is also different; (4) The proliferation rate of the tumor vascular endothelial cells Many times faster than normal tissue, TAI has only minor effects on normal tissue. Based on the above reasons, anti-angiogenesis tumor treatment strategies will play an important role in future tumor treatment.

The applicant's research found that the research on the preparation of angiogenesis inhibitory factors using scalloped hammerhead cartilage as raw material is in a blank stage, and the application of scalloped hammerhead cartilage angiogenesis inhibitors in the preparation of drugs for inhibiting angiogenesis and preventing and treating tumors It has not been reported.

Contents of the invention

The purpose of the present invention is still to provide a method for preparing the scalloped hammerhead cartilage angiogenesis inhibitory factor with convenient operation and high purity of the final product.

The technical scheme adopted by the present invention to solve the above-mentioned technical problems is: a preparation method of scalloped hammerhead cartilage angiogenesis inhibitory factor, which is characterized in that it comprises the following steps:

1) Preparation of active protein components of scalloped hammerhead cartilage: Add the crushed and homogenized scalloped hammerhead cartilage into 1.0 mol/L guanidine hydrochloride solution at a solid-to-liquid ratio of 1 g: 8-10 mL, at 4 °C , After shaking and extracting for 32-36 h, centrifuge at 4 °C and 10 000 r/min for 15-20 min, take the supernatant and put it into a dialysis bag with a molecular weight cut-off of 1 kDa, and use Tris-HCl (pH 7.6) to buffer The liquid was dialyzed below 4°C for 22-24 hours to obtain crude hammerhead cartilage protein extract; the crude hammerhead cartilage protein extract was placed at 4°C, and acetone pre-cooled below 4°C was slowly added to the acetone concentration After standing still for 0.5-1 h, centrifuge at 10 000 r/min below 4 °C for 15-25 min, take the supernatant and add pre-cooled acetone below 4 °C until the acetone concentration reaches 60%, and let stand for 0.5- After 1 h, centrifuge at 10,000 r/min for 15-25 min at below 4°C, take the precipitate and place it in a dialysis bag with a molecular weight cut-off of 1 kDa, dialyze with double distilled water at below 4°C for 22-24 h, and freeze-dry the dialysate , to get the cartilage active protein component of Hammerhead Shark.

2) Enzymolysis of active protein components of hammerhead shark cartilage: Add Gly-NaOH buffer (0.05 mol/L, pH 9-10) to obtain a mixed solution; the temperature of the mixed solution was raised to 45-55 ℃ and preheated for 5-10 min, and alkaline protease (enzyme activity ≥ 2.0×10 ⁵ U/g), the enzymatic hydrolysis temperature is 45-55 ℃, after 5-6 hours of enzymatic hydrolysis, the solution is heated to 90-95 ℃, and kept at this temperature for 10-15 minutes, 10 000g Centrifuge for 20-25 min, and take the supernatant, which is the enzymatic hydrolysis product of cartilage active protein components;

3) Preparation of inhibitory factor for cartilage angiogenesis of hammerhead shark: the enzymatic hydrolyzate of the prepared cartilage active protein component was subjected to ultrafiltration treatment with a 1 kDa ultrafiltration membrane, and the fraction with a molecular weight of less than 1 kDa was collected to obtain an ultrafiltered enzymatic hydrolyzate , and then the enzymolysis solution was purified by gel filtration chromatography, cell membrane chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) in sequence to obtain cartilage angiogenesis inhibitory factor of hammerhead shark.

Preferably, the scalloped hammerhead shark in the step 1) is Sphyrna lewini . As an improvement, the specific process of gel filtration chromatography, cell membrane chromatography and RP-HPLC purification in step 3) is:

Gel Filtration Chromatography: The above ultrafiltration enzymatic hydrolyzate was made into a solution of 15-25 mg/mL with pH 6.5-7.5 phosphate buffer solution, and it was subjected to Sephadex G-15 column chromatography (2.6 × 80 cm ) separation, and eluted with pH 6.5-7.5 phosphate buffer, and the eluted fractions were collected according to the absorbance curve at 215 nm. Gel chromatography enzymatic hydrolyzate.

Cell membrane chromatography purification: the above gel chromatography enzymatic hydrolyzate was prepared into a solution of 40-50 μg/mL with triple distilled water, added to a cell membrane chromatography column for purification, and the eluted fractions were collected according to the absorbance curve at 215 nm, among which , the highest inhibitory effect on chick embryo chorioallantoic membrane (CAM) angiogenesis is cell membrane chromatography purified enzymatic hydrolyzate.

RP-HPLC purification: The above cell membrane chromatography purified enzymatic hydrolyzate was made into a solution of 80-100 μg/mL with triple distilled water, and purified by RP-HPLC. A highly active polypeptide Pro-Asp-Tyr-Lys-Phe-Lys (PDYKFK) was obtained with a molecular weight of 796.90 Da by ESI-MS.

Preferably, the cell membrane chromatography conditions are: injection volume 5-10 μL; cell membrane chromatography column (150 mm × 4.6mm, 5 μm); column temperature is 37°C; mobile phase: triple distilled water; ultraviolet detection wavelength 215 nm; Flow rate: 0.2 mL/min.

Preferably, the RP-HPLC conditions are: injection volume 15-20 μL; chromatographic column is Zorbax C18 (250 mm × 4.6 mm, 5 μm); mobile phase is 20% acetonitrile; ultraviolet detection wavelength is 215 nm.

More preferably, the cell membrane used in the cell membrane chromatography column is the cell membrane of human umbilical vein endothelial cell strain ECV304.

Compared with the prior art, the present invention has the advantages of advanced extraction and purification techniques, and the prepared angiogenesis inhibitory factor has significant activity, and can be used for the prevention and treatment of tumor diseases.

Description of drawings

Fig. 1 is the Sephadex G-15 column chromatogram of the ultrafiltration enzymatic hydrolyzate (≤1 kDa component) of the present invention;

Fig. 2 is the cell membrane chromatogram of the hydrolyzate (Fr.C) prepared from Sephadex G-15 of the present invention;

Fig. 3 is the RP-HPLC chromatogram of the cell membrane chromatography purified enzymatic hydrolyzate (Fr.C-II) of the present invention;

Fig. 4 is a flowchart of the preparation steps of the present invention.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

Example:

A method for preparing angiogenesis inhibitory factor of scalloped hammerhead shark, the preparation process is as follows: cartilage of scalloped hammerhead shark → tissue crushing → guanidine hydrochloride extraction → fractional precipitation with acetone → purification by gel filtration chromatography → purification by cell membrane chromatography → high-efficiency Liquid chromatography purification → angiogenesis inhibitory factor.

1) Preparation of active protein components of hammerhead shark cartilage: crushed and homogenized cartilage of Sphyrna lewini was added to 1.0 mol/L guanidine hydrochloride solution at a solid-to-liquid ratio of 1 g: 10 mL. After extraction for 36 h at 4 °C with shaking, centrifuge at 10 000 r/min for 20 min at 4 °C, take the supernatant and put it into a dialysis bag with a molecular weight cut-off of 1 kDa, and use Tris-HCl (pH 7.6) buffer in the Dialyze at a temperature below 4°C for 24 hours to obtain the crude scalloped hammerhead cartilage protein extract; place the crude scalloped hammerhead cartilage protein extract at 4°C, slowly add acetone pre-cooled below 4°C until the acetone concentration is 30%, After standing still for 1 h, centrifuge at 10 000 r/min below 4 °C for 25 min, take the supernatant and add pre-cooled acetone below 4 °C until the concentration of acetone reaches 60%, after standing for 1 h, centrifuge at 10 000 r/min below 4 °C Centrifuge at r/min for 25 min, take the precipitate and place it in a dialysis bag with a molecular weight cut-off of 1 kDa, dialyze with double-distilled water below 4 °C for 24 h, freeze-dry the dialysate, and obtain active protein fraction of hammerhead shark cartilage

2) Enzymolysis of active protein components of hammerhead shark cartilage: Add Gly-NaOH buffer (0.05 mol/L, pH 9 ~10), to obtain a mixed solution; the temperature of the mixed solution was raised to 45-55 ℃ and preheated for 5 min, and alkaline protease (enzyme activity ≥ 2.0×10 ⁵ U/g), the enzymolysis temperature was 50°C, after 5 hours of enzymolysis, the solution was heated to 95°C, and kept at this temperature for 12 minutes, centrifuged at 10 000g for 25 minutes, and the supernatant was taken, which was cartilage active protein Component enzymatic hydrolysis products;

3) Preparation of inhibitory factor for cartilage angiogenesis of hammerhead shark: the enzymatic hydrolyzate of the prepared cartilage active protein component was subjected to ultrafiltration treatment with a 1 kDa ultrafiltration membrane, and the fraction with a molecular weight of less than 1 kDa was collected to obtain an ultrafiltered enzymatic hydrolyzate , and then the enzymolysis solution was purified by gel filtration chromatography, cell membrane chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) in sequence to obtain cartilage angiogenesis inhibitory factor of hammerhead shark.

①Gel filtration chromatography: The above-mentioned ultrafiltration enzymatic hydrolyzate was prepared into a 25 mg/mL solution with pH 7.0 phosphate buffer, and separated by Sephadex G-15 column chromatography (2.6 × 80 cm). Elute with pH 7.0 phosphate buffer, and collect the eluted fractions according to the absorbance curve at 215 nm. Among them, the most potent inhibitory effect on chick embryo chorioallantoic membrane (CAM) angiogenesis is gel chromatography enzyme Solution Fr.C (Figure 1).

② Purification by cell membrane chromatography: the above gel chromatography enzymatic hydrolyzate Fr.C was prepared into a 50 μg/mL solution with triple distilled water, and added to a cell membrane chromatography column for purification (conditions: injection volume 50 μL; human umbilical vein blood Cell membrane column of endothelial cell line ECV304 (150 mm × 4.6 mm, 5 μm); column temperature: 37 ℃; mobile phase: triple distilled water; UV detection wavelength: 215 nm; flow rate: 0.2 mL/min), among which, for chicken embryo The component with the strongest inhibitory effect on angiogenesis of chorioallantoic membrane (CAM) was Fr.C-II, purified by cell membrane chromatography (Figure 2).

③ RP-HPLC purification: The above-mentioned cell membrane chromatography purified enzymatic hydrolyzate Fr.C-II was made into a solution of 80-100 μg/mL with triple-distilled water, and purified by RP-HPLC (conditions: injection volume 15 μL; chromatographic column It is Zorbax C18 (250 mm × 4.6 mm, 5 μm); the mobile phase is 20 % acetonitrile; the UV detection wavelength is 215 nm), and a highly active polypeptide is obtained according to the inhibition of angiogenesis in chicken embryo chorioallantoic membrane (CAM) .

④Structural detection : The polypeptide with the strongest inhibitory effect on chick chorioallantoic membrane (CAM) angiogenesis was collected and detected as a single peak, and the amino acid sequence was determined to be Pro-Asp-Tyr-Lys-Phe using a protein/peptide sequence analyzer -Lys (PDYKFK), the molecular weight detected by ESI-MS is 796.90 Da.

The prepared Pro-Asp-Tyr-Lys-Phe-Lys (PDYKFK) was tested for the angiogenesis inhibitory activity of chicken embryo chorioallantoic membrane (CAM). Allantoic Membrane Technology - Incubation Method without Air Chamber" (recorded in "Journal of Sun Yat-Sen University (Natural Science Edition)", Volume 2 (Volume 42), 2003: Pages 126-128). The results showed that the angiogenesis inhibitor PDYKFK of scalloped hammerhead significantly inhibited the angiogenesis of chick chorioallantoic membrane (CAM) in a dose-dependent manner (Table 1).

Table 1 Inhibitory effect of scalloped hammerhead angiogenesis inhibitor on angiogenesis in chick chorioallantoic membrane (CAM)

The prepared PDYKFK was tested for its effect on the expression of pro-angiogenic factors. For the determination method, refer to Sun Xiaojia, Zhang Yueying, Jia Qing et al. "Experimental research on the inhibition of angiogenesis in Lewis lung cancer by scorpion venom polypeptide extract combined with chemotherapy" (recorded in "Chinese Journal of Traditional Chinese Medicine", 2011 No. 12 (Vol. 36): pp. 1644-1649). The angiogenesis inhibitor PDYKFK (20.0mg/kg·d×14) was injected intraperitoneally into mice bearing Lewis lung cancer, and the lung cancer tissues were taken for immunohistochemical examination. Factor PDYKFK significantly inhibited the expression of three pro-angiogenic factors including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) (Table 2).

Table 2 Inhibitory effect of angiogenesis inhibitor in scalloped hammerhead shark on the expression of pro-angiogenic factors

Although the invention has been described in conjunction with preferred embodiments, it is not intended to limit the invention, and any person skilled in the art can implement various embodiments on the subject matter set forth herein without departing from the spirit and scope of the invention. Changes, replacements and modifications of equivalents, so the protection scope of the present invention should be determined by the scope defined by the proposed claims.

SEQUENCE LISTING

the

<110> Zhejiang Ocean University

the

<120> Preparation method of scalloped hammerhead cartilage angiogenesis inhibitory factor

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<130> zjou-2015-wb0508

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<160> 1

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<170> PatentIn version 3.5

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<210> 1

<211> 6

<212> PRT

<213> Synthetic

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<400> 1

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Pro Asp Tyr Lys Phe Lys

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Claims (6)

1. the preparation method of Lu Shi hammerhead Angiostatin, is characterized in that comprising the following steps:

1) preparation of Lu Shi hammerhead cartilage activated protein component:the Lu Shi hammerhead cartilage of broken homogenate is joined in 1.0 mol/L guanidine hydrochloride solutions by solid-to-liquid ratio 1 g:8 ~ 10 mL, 4 DEG C, after vibration extracting 32 ~ 36 h, in 4 DEG C, centrifugal 15 ~ 20 min of 10 000 r/min, getting supernatant liquor loading molecular weight cut-off is in the dialysis tubing of 1 kDa, utilize the Tris-HCl damping fluid of pH 7.6 in less than 4 DEG C dialysis 22 ~ 24 h, obtain Lu Shi hammerhead chondroprotein crude extract; Lu Shi hammerhead chondroprotein crude extract is placed in 4 DEG C, acetone to the acetone concentration slowly adding less than 4 DEG C precoolings is 30%, after leaving standstill 0.5 ~ 1 h, in less than 4 DEG C centrifugal 15 ~ 25 min of 10 000 r/min, get the acetone that supernatant liquor adds less than 4 DEG C precoolings and reach 60% to acetone concentration, after leaving standstill 0.5 ~ 1 h, less than 4 DEG C, centrifugal 15 ~ 25 min of 10 000 r/min, get precipitation be placed in molecular weight cut-off be 1 kDa interior the distilled water of dialysis tubing in less than 4 DEG C dialyse 22 ~ 24 h, dialyzate lyophilize, obtains Lu Shi hammerhead cartilage activated protein component;

2) enzymolysis of Lu Shi hammerhead cartilage activated protein component:by the Gly-NaOH damping fluid that Lu Shi hammerhead cartilage activated protein component adds concentration 0.05 mol/L, pH 9 ~ 10 by solid-to-liquid ratio 1 g: 20 ~ 25 mL, obtain mixed solution; Mixeding liquid temperature is risen to 45 ~ 55 DEG C of preheating 5 ~ 10 min, add enzyme activity>=2.0 × 10 according to 1.5 ~ 2.5% of Lu Shi hammerhead cartilage activated protein constituent mass ⁵the Sumizyme MP of U/g, hydrolysis temperature is 45 ~ 55 DEG C, and after enzymolysis 5 ~ 6 h, by solution warms to 90 ~ 95 DEG C, and after this temperature keeps 10 ~ 15 min, centrifugal 20 ~ 25 min of 10 000g, get supernatant liquor, are cartilage activated protein component enzymes hydrolysis products;

3) preparation of Lu Shi hammerhead cartilage Angiostatin:1 kDa ultra-filtration membrane is adopted to carry out uf processing the cartilage activated protein component enzymes hydrolysis products of preparation, collect molecular weight and be less than 1 kDa part, obtain ultrafiltration enzymolysis solution, again by enzymolysis solution successively through gel permeation chromatography, membrane flexibility and RPLC purifying, obtain Lu Shi hammerhead cartilage Angiostatin.

2. preparation method according to claim 1, is characterized in that the Lu Shi hammerhead in described step 1) is Lu Shi hammerhead sphyrna lewini.

3. preparation method according to claim 1, is characterized in that the detailed process of the gel permeation chromatography of described step 3), membrane flexibility and RPLC purifying is:

gel permeation chromatography:above-mentioned ultrafiltration enzymolysis solution pH 6.5 ~ 7.5 phosphate buffered saline buffer is made into the solution of 15 ~ 25 mg/mL, through sephadex G-15 column chromatography for separation, wash-out is carried out with pH 6.5 ~ 7.5 phosphate buffered saline buffer, elution fraction is collected according to the absorbance curve under 215 nm, wherein, be gel chromatography zymolyte to the strongest component of chick chorioallantoic membrane angiogenesis suppression action;

membrane flexibility purifying:above-mentioned gel chromatography zymolyte tri-distilled water is made into the solution of 40 ~ 50 μ g/mL, join membrane flexibility post and carry out purifying, elution fraction is collected according to the absorbance curve under 215 nm, wherein, be membrane flexibility purifying zymolyte to the strongest component of chick chorioallantoic membrane (CAM) angiogenesis suppression action;

rP-HPLC purifying:above-mentioned membrane flexibility purifying zymolyte tri-distilled water is made into the solution of 80 ~ 100 μ g/mL, RP-HPLC is utilized to carry out purifying, according to obtaining 1 high reactivity polypeptide Pro-Asp-Tyr-Lys-Phe-Lys to chick chorioallantoic membrane angiogenesis suppression action, ESI-MS detection molecules amount is 796.90 Da.

4. preparation method according to claim 3, is characterized in that described membrane flexibility condition is: sample size 5 ~ 10 μ L; Membrane flexibility post specification is 150 mm × 4.6mm, 5 μm; Column temperature is 37 DEG C; Moving phase: tri-distilled water; Ultraviolet detection wavelength 215 nm; Flow velocity: 0.2 mL/min.

5. preparation method according to claim 3, is characterized in that described RP-HPLC condition is: sample size 15 ~ 20 μ L; Chromatographic column to be specification be 250 mm × 4.6 mm, the Zorbax C18 of 5 μm; Moving phase is 20 % acetonitriles; Ultraviolet detection wavelength is 215 nm.

6. preparation method according to claim 3, is characterized in that the cytolemma adopted in described membrane flexibility post is the cytolemma of human umbilical vein endothelial cell strain ECV304.