CN104894200B - Preparation method of cartilage angiogenesis inhibiting factor of Sphyrna lewini - Google Patents

Abstract

The invention relates to a preparation method of a Sphyrna lewini angiogenesis inhibiting factor. The preparation method comprises homogenizing cartilage of Sphyrna lewini, extracting with guanidine hydrochloride, precipitating with acetone, performing enzymolysis, performing gel filtration chromatography, purifying with cell membrane chromatography, and purifying with high performance liquid chromatography to obtain Pro-Asp-Tyr-Lys-Phe-Lys. The angiogenesis inhibiting factor can effectively inhibit the angiogenesis of chick chorioallantoic membrane, and has obvious inhibiting effect on the expression of three angiogenesis promoting factors, namely vascular endothelial cell growth factor, basic fibroblast growth factor and platelet-derived growth factor of lung cancer tissues of Lewis lung cancer-bearing mice.

Description

Preparation method of cartilage angiogenesis inhibitor of hammerhead shark

Preparation method of cartilage angiogenesis inhibitor of hammerhead shark

technical field

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

Background technique

Angiogenesis is one of the important pathological features of angioproliferative 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 decade.

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 anti-cancer drugs are affected by tissue diffusion. Due to the influence of tissue fibrosis and necrosis, 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 genotype and are not easy to develop 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 response to chemotherapy is also different; (4) The proliferation rate of tumor vascular endothelial cells is higher than that of the secondary tumor. Normal tissue is many times faster and TAI has little effect on normal tissue. Based on the above reasons, anti-angiogenic tumor therapy strategies will play an important role in tumor therapy in the future.

The applicant's research found that the research on the preparation of angiogenesis inhibitory factors using hammerhead shark cartilage as a raw material is at a blank stage, and the application of hammerhead shark cartilage angiogenesis inhibitory factors in the preparation of drugs for inhibiting angiogenesis and preventing and treating tumors There are no reports.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a preparation method of hammerhead shark cartilage angiogenesis inhibitory factor with convenient operation and high final product purity.

The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a preparation method of hammerhead shark cartilage angiogenesis inhibitory factor, which is characterized by comprising the following steps:

1) Preparation of active protein components of hammerhead shark cartilage: add the crushed and homogenized hammerhead cartilage to 1.0 mol/L guanidine hydrochloride solution at a solid-liquid ratio of 1 g: 8-10 mL, at 4 ℃ After 32-36 h of vibration extraction, 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) as a buffer. The solution was dialyzed below 4 °C for 22-24 h to obtain the crude extract of hammerhead shark cartilage protein; 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 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 below 4 °C for 15 to 25 min, take the precipitate and place it in a dialysis bag with a molecular weight cut-off of 1 kDa and dialyze it with double distilled water at below 4 °C for 22 to 24 h. The dialysate is freeze-dried. , the cartilage active protein component of hammerhead shark.

2) Enzymatic hydrolysis of the active protein component of hammerhead shark cartilage: add the active protein component of hammerhead shark cartilage to Gly-NaOH buffer (0.05 mol/L, pH 9-10) to obtain a mixed solution; raise the temperature of the mixed solution to 45-55 °C, preheat for 5-10 min, and add alkaline protease (enzyme activity ≥2.0×10 ⁵ U/g), the enzymatic hydrolysis temperature is 45-55 ℃, after enzymatic hydrolysis for 5-6 h, the solution is heated to 90-95 ℃, and after maintaining this temperature for 10-15 min, 10 000 g Centrifuge for 20-25 min, and take the supernatant, which is the enzymatic hydrolysis product of cartilage active protein components;

3) Preparation of cartilage angiogenesis inhibitory factor of hammerhead shark: The enzymatic hydrolysis product 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 ultrafiltration enzymatic hydrolysate. , and then the enzymatic hydrolysate was purified by gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) successively to obtain angiogenesis inhibitory factor of hammerhead shark cartilage.

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

Gel filtration chromatography: The above ultrafiltration enzymatic hydrolysis solution was prepared into a solution of 15-25 mg/mL with pH 6.5-7.5 phosphate buffer, and subjected to Sephadex G-15 column chromatography (2.6 × 80 cm ) was separated, 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 digest.

Purification by Cell Membrane Chromatography: The above gel chromatography enzymatic hydrolysate was prepared into a solution of 40-50 μg/mL with three-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 the angiogenesis of chick chorioallantoic membrane (CAM) is the cell membrane chromatography-purified enzymatic hydrolysate.

RP-HPLC purification: The above-mentioned cell membrane chromatography-purified enzymatic hydrolysate was made into a solution of 80-100 μg/mL with three-distilled water, and purified by RP-HPLC. According to the inhibitory effect on chicken embryo chorioallantoic membrane (CAM) angiogenesis A highly active polypeptide Pro-Asp-Tyr-Lys-Phe-Lys (PDYKFK) was obtained, and the molecular weight detected by ESI-MS was 796.90 Da.

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

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

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

Compared with the prior art, the present invention has the advantages that the extraction and purification processes are more advanced, 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 chromatography chromatogram of the ultrafiltration enzymatic hydrolysate (≤1 kDa component) of the present invention;

Figure 2 is the cell membrane chromatogram of the preparation of the enzymatic hydrolyzate (Fr.C) of Sephadex G-15 of the present invention;

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

Figure 4 is a flow chart of the preparation steps of the present invention.

Detailed ways

The present invention will be further described in detail below with reference to the embodiments of the accompanying drawings.

Example:

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

1) Preparation of active protein components of hammerhead shark cartilage: The crushed and homogenized hammerhead shark ( Sphyrna lewini ) cartilage was added to 1.0 mol/L guanidine hydrochloride solution at a solid-liquid ratio of 1 g: 10 mL. After 36 h of shaking and extraction at 4 °C, centrifuge at 4 °C and 10 000 r/min for 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) buffer in the dialysis bag. Dialyzed at below 4 °C for 24 h to obtain crude hammerhead shark cartilage protein extract; put the crude hammerhead shark cartilage protein extract at 4 °C, slowly add pre-cooled acetone below 4 °C until the acetone concentration is 30%, After standing 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 acetone concentration reaches 60%. 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 and dialyze it with double distilled water at below 4 °C for 24 h.

2) Enzymatic hydrolysis of the active protein component of hammerhead shark cartilage: add the active protein component of hammerhead shark cartilage to Gly-NaOH buffer (0.05 mol/L, pH 9 in a solid-liquid ratio of 1 g: 25 mL) ~10) to obtain a mixed solution; the temperature of the mixed solution was raised to 45-55 °C, preheated for 5 min, and alkaline protease (enzyme activity ≥ 2.0×10 ⁵ ) was added according to 1.8% of the mass of the active protein component of hammerhead shark cartilage. U/g), the enzymatic hydrolysis temperature was 50 °C, after the enzymatic hydrolysis for 5 h, the solution was heated to 95 °C, kept at this temperature for 12 min, centrifuged at 10 000 g for 25 min, and the supernatant was taken, which is the cartilage active protein Component enzymatic hydrolysis products;

3) Preparation of cartilage angiogenesis inhibitory factor of hammerhead shark: The enzymatic hydrolysis product 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 ultrafiltration enzymatic hydrolysate. , and then the enzymatic hydrolysate was purified by gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) successively to obtain angiogenesis inhibitory factor of hammerhead shark cartilage.

①Gel filtration chromatography: The above ultrafiltration enzymatic hydrolysis solution 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). Elution was performed with pH 7.0 phosphate buffer, and the eluted fractions were collected according to the absorbance curve at 215 nm. Among them, the strongest inhibitory effect on the angiogenesis of chick chorioallantoic membrane (CAM) was gel chromatography enzyme The lysate Fr.C (Fig. 1).

② Purification by cell membrane chromatography: The above gel chromatography enzymatic hydrolysate Fr.C was prepared into a solution of 50 μg/mL with three distilled water, and added to the cell membrane chromatography column for purification (conditions: injection volume 50 μL; human umbilical vein blood vessels) Cell membrane column of endothelial cell line ECV304 (150 mm × 4.6 mm, 5 μm); column temperature is 37 °C; mobile phase: triple distilled water; UV detection wavelength: 215 nm; flow rate: 0.2 mL/min). The component with the strongest inhibitory effect on angiogenesis in the chorioallantoic membrane (CAM) was the enzymatic hydrolyzate 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 prepared into a solution of 80-100 μg/mL with three-distilled water, and purified by RP-HPLC (conditions: injection volume 15 μL; chromatographic column). Zorbax C18 (250 mm × 4.6 mm, 5 μm); mobile phase is 20 % acetonitrile; UV detection wavelength is 215 nm), and a highly active polypeptide was obtained according to its inhibitory effect on the angiogenesis of chick chorioallantoic membrane (CAM). .

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

The obtained Pro-Asp-Tyr-Lys-Phe-Lys (PDYKFK) was tested for the angiogenesis inhibitory activity of chicken embryo chorioallantoic membrane (CAM). Allantoic Membrane Technology - Airless Chamber Incubation" (recorded in "Journal of Sun Yat-Sen University (Natural Science Edition)", 2003, Volume 2 (Volume 42): pp. 126-128). The results showed that the angiogenesis inhibitor PDYKFK of hammerhead shark significantly inhibited the angiogenesis of chick chorioallantoic membrane (CAM) in a dose-dependent manner (Table 1).

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

The obtained PDYKFK was tested for the expression of pro-angiogenic factors, and the determination method was based on "Scorpion Venom Polypeptide Extract Combined with Chemotherapy Inhibits Lewis Lung Cancer Angiogenesis Experimental Study" (recorded in "Chinese Journal of Traditional Chinese Medicine", No. 12, 2011 (Vol. 36: pp. 1644-1649). The angiogenesis inhibitor PDYKFK (20.0 mg/kg·d×14) was intraperitoneally injected into Lewis lung cancer-bearing mice, and lung cancer tissues were taken for immunohistochemical examination. The factor PDYKFK has a significant inhibitory effect on the expression of three pro-angiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) (Table 2).

Table 2 Inhibitory effect of hammerhead shark angiogenesis inhibitory factor on the expression of pro-angiogenic factor

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

SEQUENCE LISTING

<110> Zhejiang Ocean University

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

<130> zjou-2015-wb0508

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 6

<212> PRT

<213> Synthetic

<400> 1

Pro Asp Tyr Lys Phe Lys

1 5

Claims (1)

1. The preparation method of hammerhead shark angiogenesis inhibitory factor, the amino acid sequence of described hammerhead shark angiogenesis inhibitory factor is Pro-Asp-Tyr-Lys-Phe-Lys, and its preparation method comprises the following steps: 1) Preparation of active protein components of hammerhead cartilage: Add the crushed and homogenized hammerhead cartilage to a 1.0mol/L guanidine hydrochloride solution at a solid-liquid ratio of 1g: 8-10mL, and shake at 4°C. After 32-36 hours of extraction, centrifuge at 4°C and 10000r/min for 15-20min, take the supernatant and put it into a dialysis bag with a molecular weight cut-off of 1kDa, and dialyze it with Tris-HCl buffer of pH 7.6 below 4°C for 22-20 minutes. 24h, get the crude extract of hammerhead shark cartilage protein; place the crude extract of hammerhead shark cartilage protein at 4°C, slowly add pre-cooled acetone below 4°C until the acetone concentration is 30%, and let stand for 0.5-1h Then, centrifuge at 10,000 r/min below 4°C for 15-25min, take the supernatant and add pre-cooled acetone below 4°C until the acetone concentration reaches 60%. 25min, take the precipitate and place it in a dialysis bag with a molecular weight cut-off of 1kDa and dialyze it with double distilled water below 4°C for 22-24h, and freeze-dry the dialysate to obtain the active protein component of hammerhead cartilage; 2) Enzymatic hydrolysis of the active protein component of hammerhead shark cartilage: add the active protein component of hammerhead shark cartilage with a concentration of 0.05mol/L and pH 9 to 10 at a solid-liquid ratio of 1g: 20~25mL. NaOH buffer solution to obtain a mixed solution; the temperature of the mixed solution was raised to 45-55 °C and preheated for 5-10 min, and the enzyme activity ≥ 2.0×10 ⁵ U was added according to 1.5-2.5% of the mass of the active protein components of hammerhead shark cartilage. /g of alkaline protease, the enzymatic hydrolysis temperature is 45-55 ℃, after 5-6 hours of enzymatic hydrolysis, the solution is heated to 90-95 ℃, and the temperature is kept at this temperature for 10-15 min, centrifuged at 10000g for 20-25 min, and the Serum is the enzymatic hydrolysis product of cartilage active protein components; 3) Preparation of cartilage angiogenesis inhibitory factor of hammerhead shark: The enzymatic hydrolysis product of the prepared cartilage active protein component was subjected to ultrafiltration treatment with a 1kDa ultrafiltration membrane, and the fraction with a molecular weight less than 1kDa was collected to obtain an ultrafiltration enzymatic hydrolysate, and then the enzyme hydrolyzed solution was obtained. The enzymatic hydrolysate was purified by gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography successively to obtain the cartilage angiogenesis inhibitor of hammerhead shark; Wherein, the specific process of the gel filtration chromatography, cell membrane chromatography and reversed-phase high performance liquid chromatography purification in the step 3) is as follows: gel filtration chromatography: the above-mentioned ultrafiltration enzymatic hydrolysis solution is buffered with pH 6.5-7.5 phosphate The solution was made into a solution of 15-25 mg/mL, separated by Sephadex G-15 column chromatography, eluted with pH 6.5-7.5 phosphate buffer, and the eluted fractions were collected according to the absorbance curve at 215 nm, Among them, the component with the strongest inhibitory effect on the angiogenesis of the chick embryo chorioallantoic membrane is the gel chromatography enzymatic hydrolysate; Cell Membrane Chromatography Purification: The above gel chromatography enzymatic hydrolysate was made into a solution of 40-50 μg/mL with three-distilled water, added to the cell membrane chromatography column for purification, and the eluted fractions were collected according to the absorbance curve at 215 nm. Chick embryo chorioallantoic membrane (CAM) with the strongest inhibitory effect on angiogenesis is the purified enzymatic hydrolysate by cell membrane chromatography; the cell membrane used in the cell membrane chromatography column is the cell membrane of human umbilical vein endothelial cell line ECV304; the cell membrane chromatography conditions It is: injection volume 5～10μL; cell membrane column size is 150mm×4.6mm, 5μm; column temperature is 37℃; mobile phase: triple distilled water; UV detection wavelength: 215nm; flow rate: 0.2mL/min; Reversed-phase high-performance liquid chromatography purification: The above-mentioned cell membrane chromatography-purified enzymatic hydrolysate was made into a solution of 80-100 μg/mL with three-distilled water, and purified by RP-HPLC. The polypeptide with the strongest inhibitory effect was detected as a single peak. The amino acid sequence was determined to be Pro-Asp-Tyr-Lys-Phe-Lys (PDYKFK) by a protein/peptide sequence analyzer, and the molecular weight detected by ESI-MS was 796.90 Da; The RP-HPLC conditions were as follows: the injection volume was 15-20 μL; the chromatographic column was Zorbax C18 with a size of 250 mm×4.6 mm and 5 μm; the mobile phase was 20% acetonitrile; and the ultraviolet detection wavelength was 215 nm.

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