CN103275181B - A kind of tuna meat mincing polypeptide class Angiostatin and its production and use - Google Patents

Abstract

The invention discloses a tuna minced meat polypeptide angiogenesis inhibitory factor and its preparation method and application. The amino acid sequence of the polypeptide angiogenesis inhibitory factor is Trp-Val-Thr-Gly-Ile (WVTGI), which is detected by ESI/MS Give the molecular ion peak <i>m/z</i>? 575.42? Da ([M+H] ⁺ ). Tuna minced peptide angiogenesis inhibitor Trp-Val-Thr-Gly-Ile (WVTGI) can effectively inhibit the angiogenesis of chicken chorioallantoic membrane (CAM) and the proliferation of prostate cancer cell DU-145. The tuna minced meat peptide angiogenesis inhibitor Trp-Val-Thr-Gly-Ile (WVTGI) can be used to prepare drugs for inhibiting angiogenesis and preventing and treating prostate cancer. <b><u /></b>

Description

A kind of tuna minced meat polypeptide angiogenesis inhibitory factor and its preparation method and application

technical field

The invention relates to a tuna minced polypeptide angiogenesis inhibitor, a preparation method of the tuna minced polypeptide angiogenesis inhibitor, and a use of the tuna minced polypeptide angiogenesis inhibitor.

Background technique

Angiogenesis plays an important role in normal physiological processes such as human development and tissue repair, and is also one of the important pathological features of tumors. Folkman, a professor at Harvard Medical School in the United States, believes that the growth of tumors depends on the formation of new blood vessels, and the purpose of treating tumors can be achieved by inhibiting tumor angiogenesis. At present, this view has been confirmed by a large number of experiments, and tumor angiogenesis inhibitors (tumor angiogenesis inhibitors, TAIs), which target blood vessels to treat tumors, have also become a research hotspot.

Tuna is one of the important operating fish species of pelagic fisheries in the world, with an annual output of more than 6 million tons, accounting for more than 70% of the total output of high seas fisheries. The leftovers such as dark meat, viscera, fish head and fish skin produced during tuna processing account for about 50% to 70% of the total weight. Studies have found that the crude protein content in tuna leftovers is relatively high, which is a good source of protein. Using the protein in tuna leftovers can produce a variety of polypeptides with physiological activities, such as blood pressure lowering peptides, antioxidant peptides, immune peptides, etc.

However, the applicant found that the research on the preparation of polypeptide angiogenesis inhibitors by enzymatic hydrolysis technology using tuna leftovers and minced meat as raw materials is still in a blank stage, while the preparation of highly active polypeptides using enzymatic hydrolyzate of tuna leftovers and minced meat Class angiogenesis inhibitory factors and their applications have not been reported.

Contents of the invention

The first technical problem to be solved by the present invention is to provide an angiogenesis inhibitory factor of tuna minced polypeptide based on the technical status quo of utilization of the above-mentioned tuna minced meat, which can effectively inhibit chicken chorioallantoic membrane (CAM) Angiogenesis, also has a good inhibitory effect on the proliferation of prostate cancer cell DU-145.

The second technical problem to be solved by the present invention is to provide a method for preparing tuna minced meat polypeptide angiogenesis inhibitory factors.

The third technical problem to be solved by the present invention is to provide the application of a tuna minced meat polypeptide angiogenesis inhibitor in the preparation of drugs for inhibiting angiogenesis and preventing and treating prostate cancer.

The technical scheme adopted by the present invention to solve the above-mentioned first technical problem is: a kind of tuna minced meat polypeptide angiogenesis inhibitory factor, characterized in that the amino acid sequence of the angiogenesis inhibitory factor is Trp-Val-Thr-Gly-Ile (WVTGI), ESI/MS detection gave the molecular ion peak m/z 575.42 Da ([M+H] ⁺ ).

The technical solution adopted by the present invention to solve the above-mentioned second technical problem is: a preparation method of tuna minced polypeptide angiogenesis inhibitory factor, which is characterized in that it includes the following steps:

1) Take minced tuna, add buffer solution according to the material-to-liquid ratio of 1:3~1:5, adjust the pH value to 7.5~8.0, keep warm at 35~40°C for 5-10 minutes, and homogenate;

2) According to the quality of tuna minced meat, add trypsin (trypsin), the amount of enzyme added is 2000~2500 u/g, the enzymolysis temperature is 35~40℃, and the enzymolysis time is 4~6 h; the enzymolysis solution is heated to 90~ 95°C, and keep at this temperature for 10-15 minutes, inactivate the enzyme; centrifuge the inactivated enzymatic hydrolysis solution at 9,000-10,000 r/min for 15-20 minutes, take the supernatant, freeze-dry, and obtain the enzymatic hydrolysis product.

3) The prepared tuna minced meat enzymatic hydrolyzate is subjected to ultrafiltration and chromatography in sequence to obtain the tuna minced meat polypeptide angiogenesis inhibitory factor.

Preferably, the tuna in the step 1) is skipjack ( Katsuwonus pelamis ).

Preferably, the buffer in step 1) is a K ₂ HPO ₄ -KH ₂ PO ₄ buffer with a pH of 8.0.

Preferably, the enzyme activity of trypsin in the step 2) is ≥1.0×10 ⁴ u/g.

As an improvement, the specific process of ultrafiltration and chromatography in step 3) is:

Ultrafiltration: Dissolve the enzymatic hydrolyzate of minced tuna in double-distilled water to prepare a solution with a concentration of 15-20 mg/mL. The filter membrane is subjected to ultrafiltration treatment, and the part with a molecular weight less than 1 kDa is collected and freeze-dried, which is the ultrafiltration enzymatic hydrolyzate;

Chromatography: Dissolve the above-mentioned ultrafiltration enzymatic hydrolyzate in double-distilled water to form a solution with a concentration of 10-15 mg/mL. After separation by anion exchange resin, water, 0.09-0.11 mol/L, 0.45-0.55 mol/L and 0.90- 1.10 mol/L NaCl solution was used for gradient elution, and the eluted fractions were collected according to the absorbance curve at 220 nm. Among them, the component with the highest inhibitory effect on the angiogenesis of chicken embryo chorioallantoic membrane (CAM) was ion exchange chromatography enzyme Hydrolyzate; the above-mentioned ion-exchange chromatography enzymatic hydrolyzate was prepared into a solution of 8-12 mg/mL with double distilled water, separated by gel column chromatography, and eluted with double distilled water. According to the absorbance curve at 220 nm The eluted fractions were collected, among which, the fraction with the highest inhibitory effect on CAM angiogenesis was gel chromatography enzymatic hydrolyzate, and the above gel chromatography enzymatic hydrolyzate was prepared into a solution of 45-55 μg/mL with double distilled water, Purified by reversed-phase high-performance liquid chromatography (RP-HPLC), according to the inhibitory effect on CAM angiogenesis, a polypeptide with high angiogenesis inhibitory effect Trp-Val-Thr-Gly-Ile (WVTGI) was obtained, which was detected by ESI/MS. Molecular ion peak m/z 575.42 Da ([M+H] ⁺ ).

Preferably, the anion exchange resin is DEAE-52 cellulose, and the gel is Sephadex G-25; the reversed-phase high performance liquid chromatography conditions are: injection volume 10-15 μL; chromatographic column is Zorbax C18 (250 mm × 4.6 mm, 5 μm); mobile phase is 30% acetonitrile; UV detection wavelength is 220 nm.

The technical solution adopted by the present invention to solve the above third technical problem is: the application of a peptide angiogenesis inhibitor of minced tuna meat, which is characterized in that Trp-Val-Thr-Gly-Ile (WVTGI) is present at 1 μg/ At the concentration of 1, 10 μg/mouse and 100 μg/mouse, the inhibition rates of CAM angiogenesis were 42.7%, 64.0% and 87.6%, respectively; the IC ₅₀ against prostate cancer cell DU-145 was 0.22 mg/mL; Trp -Val-Thr-Gly-Ile (WVTGI) has the advantages of safety, no side effects, strong activity, etc., and can be used to prepare drugs for inhibiting angiogenesis and preventing and treating prostate cancer.

Compared with the prior art, the present invention has the advantages that: the process of the present invention is scientific and reasonable, selects trypsin (trypsin) as the enzyme for enzymolysis, and simultaneously integrates membrane ultrafiltration classification, macroporous resin desalination and chromatographic purification through biological enzymolysis, The enzymatic hydrolysis process is easy to monitor, and the prepared polypeptide angiogenesis inhibitory factor has higher activity; compared with the chemically synthesized polypeptide angiogenesis inhibitory factor, the polypeptide angiogenesis inhibitory factor prepared by the present invention is safe and non-toxic The invention has the advantages of side effects, strong activity and the like, and can be used to prepare drugs for inhibiting angiogenesis and preventing and treating prostate cancer.

Description of drawings

Fig. 1 is the anion exchange resin DEAE-52 cellulose chromatogram of ultrafiltration hydrolyzate of the present invention;

Fig. 2 is the Sephadex G-25 chromatogram of the ion-exchange chromatography enzymatic hydrolyzate of the present invention;

Fig. 3 is the reverse phase high performance liquid chromatography (RP-HPLC) analytical figure of gel chromatography enzymatic hydrolyzate of the present invention;

Fig. 4 is the reverse phase high performance liquid chromatography (RP-HPLC) figure of Trp-Val-Gly-Thr-Ile (WVGTI) of the present invention;

Fig. 5 is a mass spectrum (ESI/MS) of Trp-Val-Gly-Thr-Ile (WVGTI) of the present invention.

Detailed ways

Below in conjunction with embodiment the present invention is described in further detail.

A preparation method of peptide angiogenesis inhibitors of tuna minced meat, the preparation process is as follows: Tuna minced meat "enzymolysis" enzymatic hydrolyzate "membrane ultrafiltration" ion exchange chromatography "gel filtration chromatography" high performance liquid chromatography Preparation and analysis of "tuna minced meat polypeptide angiogenesis inhibitory factor".

Example 1:

1) Take minced tuna, add K ₂ HPO ₄ - KH ₂ PO ₄ buffer solution (pH 8.0) according to the ratio of material to liquid 1:3~1:5, incubate at 38°C for 10 min, and homogenate;

2) According to the quality of tuna minced meat, add trypsin (trypsin), the amount of enzyme added is 2500 u/g, the enzymolysis temperature is 38°C, and the enzymolysis time is 5 hours; the enzymolysis solution is heated to 90°C and kept at this temperature After 10 min, the enzyme was inactivated; the inactivated enzymatic hydrolyzate was centrifuged at 10,000 r/min for 20 min, and the supernatant was taken and freeze-dried to obtain the enzymatic hydrolyzate.

3) The enzymatic hydrolysis product obtained in step 2) is subjected to ultrafiltration and chromatography in sequence to obtain the tuna minced meat polypeptide angiogenesis inhibitory factor, and its structure is determined by amino acid sequence analysis and mass spectrometry. The specific process is as follows:

①Ultrafiltration: The enzymatic hydrolyzate of minced tuna meat is subjected to ultrafiltration treatment with a 1 kDa ultrafiltration membrane at a working pressure of 0.1-0.15 MPa and a working temperature of 25 ℃, and the fraction with a molecular weight less than 1 kDa is collected and freeze-dried to obtain ultrafiltration. filter enzymatic hydrolyzate;

②Anion-exchange chromatography: Dissolve the above-mentioned ultrafiltered enzymatic hydrolyzate in double distilled water to form a solution with a concentration of 15 mg/mL, and separate it with DEAE-52 cellulose anion-exchange resin, then add water, 0.1 mol/L, 0.5 mol/L and 1.0 mol/L NaCl solution was used for elution, and the eluted fractions were collected according to the absorbance curve at 220 nm. Among them, the fraction with the highest inhibitory effect on angiogenesis of chicken embryo chorioallantoic membrane (CAM) was ion exchange chromatography enzymatic hydrolysis Object (F5) (Figure 1);

③Gel chromatography: The above-mentioned ion-exchange chromatography enzymatic hydrolyzate (F5) was made into a 10 mg/mL solution with double distilled water, separated by Sephadex G-25 column chromatography, and then washed with double distilled water. Carry out elution, and collect the eluted fractions according to the absorbance curve at 220 nm. Among them, the fraction with the highest inhibitory effect on angiogenesis of chicken embryo chorioallantoic membrane (CAM) is gel chromatography hydrolyzate (F52) (Fig. 2 ).

④ Purification by high-performance liquid chromatography: The gel chromatography hydrolyzate (F52) prepared above was made into a 50 μg/mL solution with double distilled water, and purified by reverse-phase high-performance liquid chromatography (RP-HPLC) (conditions : Injection volume 15 μg; chromatographic column is Zorbax C18 (250 mm × 4.6 mm, 5 μm); mobile phase: 30 % acetonitrile; UV detection wavelength 220 nm), according to the angiogenesis of chicken embryo chorioallantoic membrane (CAM) Inhibition of a high angiogenesis inhibitory polypeptide (F52-II). (See Figure 3).

⑤ Structure determination: RP-HPLC detects that the collected high-angiogenesis inhibitory polypeptide is a single peak (see Figure 4), and the amino acid sequence is determined as Trp-Val-Thr-Gly-Ile (WVTGI) by protein/peptide sequence analyzer, ESI /MS detection gave a molecular ion peak m/z 575.42 Da ([M+H] ⁺ ).

The anti-tumor polypeptide Trp-Val-Thr-Gly-Ile (WVTGI) prepared above was subjected to the proliferation inhibition experiment of prostate cancer cell line DU-145. The experimental results showed that the peptide had an IC ₅₀ of 0.22 mg/mL against prostate cancer cell line DU-145.

The prepared polypeptide Trp-Val-Thr-Gly-Ile (WVTGI) was used to inhibit the angiogenesis of chicken embryo chorioallantoic membrane (CAM). Encapsulation technology-incubation method without air chamber" [recorded in "Journal of Sun Yat-Sen University (Natural Science Edition)", Volume 2 (Volume 42) in 2003: Pages 126-128], the size of the filter paper was 3 mm when measured ×3 mm; the incubation time before adding the sample is 4 days; the sample volume: Trp-Val-Thr-Gly-Ile (WVTGI) is 1 μg/piece, 10 μg/piece, 100 μg/piece; 0.01mol/L Phosphate buffered saline (PBS) at pH 7.6 was used as negative control; 5 μg chondroitin sulfate (CS) was used as positive control. After adding the sample, continue to incubate for 24 hours, divide the CAM into 3 regions with a radius interval of 5 mm centered on the administration point, count the number of blood vessels in each region, and calculate the angiogenesis inhibition rate according to the following formula:

Angiogenesis inhibition rate = (the number of vascular branch points in the negative control group - the number of vascular branch points in the treatment group) / the number of vascular branch points in the negative control group × 100%

The results showed that Trp-Val-Thr-Gly-Ile (WVTGI) could significantly inhibit the angiogenesis of CAM in a dose-dependent manner. The resulting inhibition rates were 42.7%, 64.0%, and 87.6%, respectively (Table 1).

Table 1 Inhibitory effect of tuna minced meat polypeptide angiogenesis inhibitor Trp-Val-Thr-Gly-Ile (WVTGI) on CAM angiogenesis

group number of embryos Dosage (μg/piece) Vessel branch points ( ±s) Inhibition rate(%) control 5 -- 72.3±5.2 -- Chondroitin Sulfate 5 5 29.3±1.9 59.5 WVTGI 5 1 41.4±1.9 42.7 WVTGI 5 10 26.0±2.8 64.0 WVTGI 5 100 8.97±0.5 87.6

Finally, it should be noted that what is listed above is only a specific embodiment of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

<110> Zhejiang Ocean University

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<120> A peptide angiogenesis inhibitory factor of minced tuna and its preparation method and application

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Claims (4)

1. a tuna meat mincing polypeptide class Angiostatin, is characterized in that the aminoacid sequence of this tuna meat mincing polypeptide class Angiostatin is that Trp-Val-Thr-Gly-Ile SEQ ID NO:1, ESI/MS detection provides molecular ion peak m/z575.42 Da([M+H] ⁺).

2. a preparation method for a kind of tuna meat mincing polypeptide class Angiostatin according to claim 1, is characterized in that comprising the following steps:

1) get tuna meat mincing, add damping fluid by solid-liquid ratio 1:3 ~ 1:5, adjust ph to 7.5 ~ 8.0, in 35 ~ 40 DEG C of insulation 5 ~ 10 min, homogenate;

2) according to tuna meat mincing quality, trypsin trypsin is added), enzyme concentration is 2000 ~ 2500 u/g, and hydrolysis temperature is 35 ~ 40 DEG C, enzymolysis time 4 ~ 6 h; Enzymolysis solution is warming up to 90 ~ 95 DEG C, and after this temperature keeps 10 ~ 15min, makes enzyme-deactivating; Centrifugal 15 ~ 20 min of enzymolysis solution 9000 ~ 10000 r/min, get supernatant liquor, freeze-drying, obtain enzymolysis product.

3) by preparation tuna meat mincing enzymolysis product successively through ultrafiltration and chromatography, obtain tuna meat mincing polypeptide class Angiostatin;

Tuna in described step 1) is stripped tuna;

The ultrafiltration of described step 3) and the detailed process of chromatography are:

Ultrafiltration: tuna meat mincing enzymolysis product is dissolved in distilled water, be made into the solution that concentration is 15 ~ 20 mg/mL, under the working temperature of the operating pressure of 0.1 ~ 0.15 MPa and 20 ~ 25 DEG C, adopt 1 kDa ultra-filtration membrane to carry out uf processing, collect molecular weight and be less than 1 kDa part, freeze-drying, obtains ultrafiltration zymolyte;

Chromatography: above-mentioned ultrafiltration zymolyte is dissolved in distilled water and is made into the solution that concentration is 10 ~ 15 mg/mL, through Anion exchange resin separation, gradient elution is carried out by water, 0.09 ~ 0.11 mol/L, 0.45 ~ 0.55 mol/L and 0.90 ~ 1.10 mol/L NaCl solution, elution fraction is collected according to the absorbance curve under 220 nm, wherein, be ion exchange chromatography zymolyte to the highest component of chick chorioallantoic membrane (CAM) angiogenesis suppression action, above-mentioned ion exchange chromatography zymolyte distilled water is made into the solution of 8 ~ 12 mg/mL, be separated through gel filtration chromatography, wash-out is carried out with distilled water, elution fraction is collected according to the absorbance curve under 220 nm, wherein, be gel chromatography zymolyte to the highest component of CAM angiogenesis suppression action, above-mentioned gel chromatography zymolyte distilled water is made into the solution of 45 ~ 55 μ g/mL, RPLC (RP-HPLC) is utilized to carry out purifying, according to the restraining effect of CAM vasculogenesis being obtained to 1 high angiogenesis suppression action peptide T rp-Val-Thr-Gly-Ile SEQ ID NO:1, ESI/MS detects and provides molecular ion peak m/z575.42 Da([M+H] ⁺),

Described anionite-exchange resin is DEAE-52 Mierocrystalline cellulose, and described gel is sephadex G-25; Described RPLC condition is: sample size 10 ~ 15 μ L; Chromatographic column is Zorbax C18; Moving phase is 30 % acetonitriles; Ultraviolet detection wavelength is 220 nm.

3. preparation method according to claim 2, the damping fluid that it is characterized in that in described step 1) to be pH be 8.0 K ₂hPO ₄-KH ₂pO ₄damping fluid.

4. a kind of tuna meat mincing polypeptide class Angiostatin according to claim 1 is for the preparation of inhibiting angiogenesis, the purposes of preventing and treating prostate cancer medicine.