WO2013023443A1

WO2013023443A1 - Pharmaceutical application of peptide of soft-shell turtle

Info

Publication number: WO2013023443A1
Application number: PCT/CN2012/001097
Authority: WO
Inventors: 钟虹光; 卢建中; 易敏之; 马莉; 刘根云
Original assignee: 江中药业股份有限公司
Priority date: 2011-08-18
Filing date: 2012-08-17
Publication date: 2013-02-21
Also published as: CN102319420A; CN102319420B

Abstract

The present invention aims at providing an application of a peptide of a soft-shell turtle in preparing a medicament having a protective feature against radiation hazards. The oligopeptide of the soft-shell turtle of the present invention uses the soft-shell turtle as a main ingredient, with small peptides having a distribution of molecular weights of less than 10,000 daltons, where molecular weights ranging between 140 and 1,000 daltons accounts for over 50%.

Description

Application of turtle peptide in pharmaceutical industry

[0001] The present invention relates to the use of turtle peptides, and more particularly to the use in the field of medicaments having protective properties against radiation hazards. Background technique

[0002] Application No. 201010103157. 9 Patent document discloses a preparation method of soft-shelled peptides of soft-shelled turtles; Chinese Journal of Food Science 2008, 02: Xu Huaide et al. "In vitro ACE inhibition and antioxidant activity of proteinase products of turtles" discloses in vitro experiments The effects of ACE inhibition and antioxidant activity of the soft-shelled proteolytics were confirmed; neither the prior art nor the patents reported the use of soft-shelled peptides for assisting the inhibition of tumor cell growth or metastasis, nor on the soft-shelled turtles having a molecular weight below 1000. Polypeptides have been reported to protect against radiation hazards. Summary of the invention

[0003] The object of the present invention is to provide a new use of turtle peptide, that is, a new application in pharmacy; another object of the present invention is to provide a protective function against radiation hazards, which has high nutritional value and absorption rate. High, no harm to the body's health drugs or health foods.

[0004] The turtle peptide of the present invention is a small molecule peptide having a molecular weight distribution of 1000OODalton as a main raw material, wherein the molecular weight range of 140-1000 accounts for 50% or more.

The present invention also relates to the use of turtle peptide as a medicament for the preparation of a protective function against radiation hazards.

[0006] The use of a turtle peptide having a molecular weight distribution within 100OODalton is useful as a medicament for preparing a protective function against radiation hazards.

[0007] The use of a turtle peptide having a molecular weight distribution within 2000 Dalt _O n is useful as a medicament for preparing a protective function against radiation hazards.

[0008] The use of a turtle peptide having a molecular weight distribution within the range of 140 to 100 Odalton is useful as a medicament for preparing a protective function against radiation.

[0009] The use of a turtle peptide having a molecular weight distribution in the range of 300 to 700 Dalton is useful as a medicament for preparing a protective function against radiation.

[0010] The turtle peptide of the present invention is prepared from the whole of the genus Trionyx sinensis Wiegmann.

[0011] In order to better understand the essence of the present invention, the pharmacological experiments and results of the turtle peptide having a molecular weight of 140 to 1000 Dalton will be described below to illustrate its new use in the pharmaceutical field.

C0012] The effect of a turtle peptide with a molecular weight of less than 100OODalton on the protective effect against radiation damage.

[0013] 1. Materials and methods

1. 1 Sample source and treatment: The test drug is the turtle liquid oral powder dry powder, lg turtle peptide / dry powder, provided by Jiangzhong Pharmaceutical Co., Ltd., the clinical dose is 4g turtle peptide / d, 100mL / do

[0014] 1. 2 Experimental animals and environment: clean-grade healthy Kunming mice, male, weighing 18-22 g, provided by Experimental Animal Center of Jiangxi College of Traditional Chinese Medicine, license number: SCXK (赣) 2006-0001. The animals are kept in the animal room of Jiangxi College of Traditional Chinese Medicine. Environmental permit: SYXK (赣) 2004-0001, feeding environment temperature 21~ 23 ° C, relative humidity 50~ 60%.

[0015] 1. 3 experimental methods

1. 3. 1 Animal grouping: According to body weight, they were randomly divided into 5 groups, 12 in each group. A blank control group, a model control group, and a low, medium and high dose group of the turtle liquid oral solution were established.

[0016] 1. 3. 2 dose design: the recommended daily intake of the turtle liquid oral liquid is: 4. 0g / 60kg body weight. From this, it is estimated that the daily intake of mice is: low dose group, 0. 333g crude drug / kg body weight; medium dose group, 0. 667g crude drug / kg body weight; high dose group, 1. 334g crude drug / kg body weight, respectively It is equivalent to 5, 10 and 20 times the daily intake of people. The sample was prepared in distilled water to a corresponding concentration (0. 333 g dry powder / 20 mL, 0. 667 g dry powder / 20 mL, 1. 334 g dry powder / 20 mL).

[0017] 1. 3. 3 The effect of thyrapin oral solution on leukopenia caused by radiation: 60 mice were randomly divided into 5 groups according to body weight, blank control group, model control group, and turtle liquid oral solution was low, medium and high. Dosage group, 12 in each group. Before the experiment, 20 μL of blood was taken from the eye of each group, and the number of white blood cells in each mouse was calculated by a blood analyzer. The blank group and the model group were given distilled water, and the animals in each group were added with the corresponding test solution 0. 2mL/10g body weight, once a day for 30 days. All animals were irradiated at ⁶ °C on day 16 with an irradiation dose of 5 Gy except for the blank control group. Each group of mice took 20 μL of blood from the eyelids on the 3rd and 14th day after irradiation, and the blood analyzer calculated the number of white blood cells per mouse.

[0018] 1. 3. 4 The effect of the turtle liquid oral solution on the radiation-induced decrease of bone marrow nucleated cells and the decrease of bone marrow cell DNA content: 60 mice were randomly divided into 5 groups according to body weight, blank control group and model control group. , the soft, medium and high dose groups of the turtle liquid oral solution, 12 in each group. The blank group and the model group were given the steamed sputum water, and the animals of the squid peptide oral liquid were intragastrically administered with the corresponding test solution 0. 2mL/10 _g body weight, once a day for 30 days. All animals were irradiated with Co on day 27 except for the blank control group, and the irradiation dose was 5 Gy. The mice in each group were sacrificed from the cervical vertebra on the 3rd day after irradiation, and the femur was removed. A certain volume of Hank's solution was aspirated with a lmL syringe (6.5 needle), and all the bone marrow cells in the femur were washed out. The cell suspension was passed through a syringe of 4 gauge needle, the cells were well dispersed in the suspension, and the number of nucleated cells per mL of the bone marrow cell suspension was counted under the microscope. Another bone marrow cell solution was measured at 260 nm with an ultraviolet spectrophotometer to calculate the DM content.

C0019] 1. 3. 5 Effect of thyroid peptide oral solution on the decrease of superoxide dismutase (SOD) activity in serum and liver tissue caused by radiation: 60 mice were randomly divided into 5 groups according to body weight, blank control group and model control Group, the soft, medium and high dose groups of the turtle liquid oral solution, 12 in each group. The blank group and the model group were intragastrically digested with distilled water, and the animals in each group were added with the corresponding test solution 0. 2 mL/10 g body weight once a day for 30 days. All animals were irradiated with ^Co on day 23 except for the blank control group at an irradiation dose of 7 Gy. The mice in each group were serum-separated on the 7th day after irradiation, and then the animals were sacrificed by cervical vertebrae. The liver was taken and SOD kit was used to detect the S0D activity of red blood cells and liver tissues of each group.

[0020] 1. 3. 6 The effect of the turtle liquid oral solution on the immune function of irradiated mice: 60 mice were randomly divided into 5 groups according to body weight, blank control group, model control group, soft-shelled turtle liquid, medium, High dose group, 12 in each group. The blank group and the model group were given distilled water, and the animals in each group of the turtle liquid oral solution were intragastrically administered with the corresponding test solution 0. 2 mL/10 g body weight once a day for 30 days. All animals were exposed to Co on day 15 except for the blank control group at an irradiation dose of 2 Gy. Serum hemolysin was measured on the 15th day after irradiation, and serum hemolysin was measured by hemagglutination method to calculate the antibody volume.

C0021 ] 1 . 4 statistical method : experimental data

3⁄4 indicates that the difference between the blank group, the model control group and the turtle peptide group was compared by one-way analysis of variance, and Ρ<0.05 was judged to be significant.

[0022] 2 results 2. 1 Effect of thyrapin oral solution on leukopenia caused by radiation

The test results are shown in Table 1. Compared with the blank control group, the WBC value of the model control group decreased significantly on the 3rd and 14th day after irradiation, indicating that the model was successful. The BC value of the mice in the squid peptide oral solution and the sputum dose group decreased on the third day after irradiation, but the WBC value was significantly higher than that in the model control group, suggesting that the methicillin oral solution was inhibited. The effect of radiation-induced reduction in WBC. Compared with the model control group, the WBC values of the mice in the medium and high dose groups of the turtle liquid oral solution were significantly increased and statistically significant on the 14th day after irradiation, and the WBC values of the mice in the low dose group were increased. The trend, but not statistically significant, suggests that the administration of the squid peptide oral solution has an effect on the reduction of WBC caused by radiation.

Effect of thyrapin oral solution on leukopenia caused by radiation ( _§ )

Compared with the blank control group, *P<0.05, **P<0.01; compared with the model control group, ^A P<0.05.

[0024] 2. 2 The effect of oral turtle liquid on the reduction of bone marrow nucleated cells and the decrease of bone marrow DNA content in radiation

The test results are shown in Table 2. Compared with the blank control group, the number of nucleated cells in the bone marrow of the model control group was significantly reduced, and the DNA content of the bone marrow cells was significantly decreased. Compared with the model control group, the number of nucleated cells in the bone marrow of the medium and high dose group of the turtle liquid oral solution was obvious. Increased, the DNA content of bone marrow cells increased significantly, suggesting that the turtle liquid oral solution has better anti-irradiation-induced bone marrow damage.

Table 2 Effect of thyroid peptide oral solution on radiation-induced reduction of bone marrow nucleated cells and decrease of bone marrow cell DNA content (t _s )

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Compared with the blank control group, *P<0.05, **P<0.01; compared with the model control group, ^A P<0.05, ^AA P<0.

[0026] 2. The effect of 3 turtle liquid oral solution on the decrease of superoxide dismutase (SOD) activity induced by radiation

The test results are shown in Table 3. Compared with the blank control group, the SOD activity in the serum and liver tissues of the model control group was significantly decreased. Compared with the model control group, the SOD activity in the serum and liver tissues of the medium and high dose groups of the turtle liquid oral liquid was significantly increased, suggesting that the turtle The peptide oral solution can better resist the damage of the body's redox reaction caused by the blessing.

Table 3 Effect of the squid peptide oral solution on the decrease of S0D activity in serum and liver tissue caused by radiation ( )

[0028] 2. 4 The effect of the turtle liquid oral solution on the blood hemolysin level in irradiated mice

The test results are shown in Table 4. Compared with the blank control group, the serum hemolysin level of the model control group was significantly lower. Compared with the model control group, the serum hemolysin level in the medium and high dose group of the turtle liquid oral liquid was significantly increased, suggesting that the turtle liquid oral liquid can Better against the damage of the body's immune system caused by irradiation.

Table 4 Effect of the squid peptide oral solution on the decrease of SOD activity in serum and liver tissue caused by radiation ( _3⁄4$ )

[0030] 3. Conclusion:

According to the recommended daily intake of the turtle powder oral powder complex powder 4. 0g crude drug / 60kg body weight expansion 5, 10 and 20 times set low, medium and high dose groups, namely 0. 333g crude drug, 0. 667g crude drug, 1. 334 g of crude drug/kg body weight, another ⁶ °Co irradiation model control group and blank control group. The corresponding drug was given by gavage for 30 days. The experimental results were judged to be significant by P<0.05. The animal experiment showed that the medium and high dose of the turtle liquid oral solution can significantly increase the WBC value of the irradiated mice; the middle and high dose groups of the turtle liquid oral liquid can increase the number of nucleated cells and the DNA content of the bone marrow cells of the mice after irradiation; The medium and high dose group of the turtle liquid oral liquid can increase the activity of SOD in serum and liver tissue of mice. The dose of sputum in the squid peptide oral liquid can significantly increase the serum hemolysin level of the irradiated mice, and it is believed that the squid peptide oral liquid has radiation damage. Auxiliary protection. detailed description

[0031] Embodiment 1

First, the turtle oligopeptide production method:

1. Using Chinese sturgeon as raw material, use 100kg fresh turtle, remove internal organs and oil, stir it, add 1000L pure water, and decoct at 100 ° C for 2h;

The 5% of the flavored protease 0. 5kg, 5% of the flavor of the broth of the broth of the broth of the broth of the broth of the broth of the broth. 5', the obtained enzymatic hydrolysate was heated to 100 ° C, and the enzyme was 30 tnin;

I. The temperature of the enzymatic solution is 1. 8 bar, the temperature of the enzymatic solution is 1. 8 bar, the temperature of the enzymatic solution is 1. 8 bar, the temperature of the enzymatic solution is 1. 8 bar, the temperature of the enzymatic hydrolyzate is 30. C was subjected to ultrafiltration separation, and the film was concentrated, and spray dried to obtain 11.6 kg of powdered torpedopeptide dry powder, and 290 liters of pure water was added to prepare a squid peptide oral solution.

[0032] Second, the determination of the oligopeptide content of soft-shelled turtles:

Method summary

Low molecular weight protein hydrolysates (including peptides and free amino acids) are soluble in trichloroacetic acid solutions: high molecular weight proteins are readily precipitated in trichloroacetic acid solutions. After the sample is dissolved in the trichloroacetic acid solution, the precipitated protein substance is separated by centrifugation, and the acid-soluble protein content in the centrifuged supernatant is determined. The acid-soluble protein content in the supernatant minus the free amino acid content is the content of the oligopeptide.

[0033] 2. Analysis step

2. 1 Determination of acid soluble protein content

Weigh 2g (accurate to 1mg) sample, add 10mL of 15% trichloroacetic acid solution, mix well, and let stand for 10min. After centrifuging the sample solution at 4000 rpm for 10 min, all the supernatants were taken and determined in the clear solution according to the method specified in GB/T 5009. 5。 The acid-soluble protein, protein conversion factor of 6.5. The test results are converted to dry basis based on the dry weight loss of the sample.

[0034] 2. 2 Determination of free amino acid content

Sample preparation: Weigh 20~30mg sample, accurate to 0. OOOlg, dissolved evenly with 3% sulfosalicylic acid solution. Transfer the sample solution to a 50 ml volumetric flask and bring up to volume. The sample solution was centrifuged at a speed of over 5min to obtain the supernatant 4000r / mi _n centrifuge, then 0. 45 μ m filter membrane serum, and the filtrate was transferred to a 50ml volumetric flask, after the volume as a sample detection instrument . The rest of the operation is the same as the method for determining the free amino acid content of GB 12292 fruit and vegetable juice.

[0035] 2. 3 Expression of results

The content of oligopeptides is calculated according to formula (1):

:j3⁄4 = ( 1 )

Where -

1-——the content of oligopeptide in the sample (on a dry basis), %;

4——The content of acid-soluble protein in the sample (on a dry basis), %;

A - free amino acid content in the sample (on a dry basis), %.

[0036] 2. 4 measurement results

The oligopeptide content of the soft-shelled turtle was determined, and the results are shown in Table 4.

Table 4 Determination of the content of oligopeptides in turtle peptide powder

[0038] 3. The proportion of peptides with a relative molecular mass of less than 1000 (effect gel filtration chromatography)

3. 1 method summary

It was determined by gel filtration chromatography. That is, the porous filler is used as the stationary phase, and the separation is performed according to the difference in molecular volume of the sample components, and the specific data processing software (ie, GPC) for measuring the relative molecular mass distribution by gel chromatography is detected under the ultraviolet absorption wavelength of the peptide bond at 220 nm. Software), the chromatogram and its data are processed to calculate the relative molecular mass and distribution range of the oligopeptide.

[0039] 3. 2 reagent

Acetonitrile: chromatographically pure; trifluoroacetic acid: analytically pure; water: ultrapure water or double distilled water.

[0040] Standards for relative molecular mass calibration curves: cytochrome C (cyyochrome, layer 12500); aprotinin (W6500); bacitracin (MW1450); ethionine-alanine-tyrosine - arginine (Li 451); e-amino acid mono-alanine (purchased 189).

[0041] 3. 3 instruments and equipment

High performance liquid chromatograph: equipped with a UV detector and a chromatographic workstation or integrator with GPC data processing software; mobile phase vacuum filtration degassing device; ultrasonic oscillator; analytical balance: sensitivity 0. 0001g.

[0042] 3. 4 chromatographic conditions and system adaptability experiments

Column: TSKgel G2000 SWXL 300 draw X 7. 8 or other similar types of gel column suitable for the determination of proteins and peptides; mobile phase: acetonitrile: water: trifluoroacetic acid, 45: 55: 0. 1 (volume ratio) detection wavelength: UV220nm; flow rate: 0. 5ml / min; column temperature: 30 ° C; injection volume: 10 μ 1 .

[0043] In order to meet the detection requirements of the chromatographic system, it is stipulated that under the above chromatographic conditions, the column efficiency of the gel column, that is, the number of theoretical plates (Ν) according to the tripeptide standard (ethine-alanine-alanine) Peak calculation of not less than 5000, the distribution of oligopeptides The number (Kd) should be between 0 and 1.

[0044] 3. 5 relative molecular mass calibration curve production

The solution of the above-mentioned different relative molecular mass of the peptide standard solution having a pore size of 0. 2-0. 5 μ m polytetrafluoroethylene or nylon filter membrane after filtration Inject, and obtain a chromatogram of the series of standards. The relative molecular mass calibration curve and its equation are obtained by plotting the relative molecular mass logarithm (IgMW) versus retention time or by linear regression.

[0045] 3. 6 sample preparation

The sample is taken up in a volume of 0. 2-0. 5 μ πι polytetrafluoroethylene or nylon filtered. The sample is weighed in a volume of 0. 2-0. 5 μ πι polytetrafluoroethylene or nylon. After the membrane is filtered, the machine is injected.

[0046] 3. 7 calculation of relative molecular mass

The sample solution prepared in 3. 6 was analyzed under the above chromatographic conditions. Then, using GPC data processing software, the chromatographic data of the sample is substituted into the calibration curve equation to calculate the relative molecular mass of the peptide in the sample and its distribution range. The peak area normalization method was used to calculate the sum of the relative percentages of the peak areas of the peptides having a molecular weight range of less than 1000.

[0047] 3. 8 measurement results

The molecular weight distribution range of the soft-shelled peptides of turtles is shown in Table 8.

Table 8 turtle oligopeptide results

The above results indicate that the molecular weight of soft-shelled peptides is mainly concentrated in 140-1000, accounting for more than 70%.

Embodiment 2

Turtle peptide preparation method:

First, the preparation method of the turtle peptide, the steps are as follows:

1. Using Chinese sturgeon as raw material, use 100kg of fresh turtle, stir and fry lh;

05%的味 protease,60°。 The cooling to 40 ° C, according to the weight of the raw turtle, 0.2% of the ratio of alkaline protease, 60 ° C constant temperature digestion lh, and then according to the weight of the raw turtle, 0. 05% flavor protease, 60 ° C, the enzymatic hydrolysate was heated to 85 ° C, and the enzyme was killed for 10 min;

3. The enzymatic hydrolysate is centrifuged and filtered, and the supernatant liquid is purified by a membrane having a molecular weight cut off of 0.50 Daltons, and the inlet pressure is 1. 2 bar, the outlet pressure is 1 bar, and the temperature of the solution is 20 °. 5公斤甲鱼peptide成品。 The C was subjected to ultrafiltration separation, the film was concentrated, and spray dried to obtain 15. 6 kg of turtle peptide finished product.

[0049] Second, the determination of turtle peptide content:

Determination of turtle peptide content:

According to the method of Example 1, the molecular weight of the soft-shell peptide of the soft-shelled turtle is mainly concentrated in 140-2000, accounting for more than 50%.

Embodiment 3

First, the preparation method of the turtle peptide, the steps are as follows:

1. Using Chinese sturgeon as raw material, use 100kg of fresh turtle, stir and boil for 4h;

2. Cool to 40 ° C, add alkaline protease at a ratio of 5% by weight of the raw turtle, heat-fermented at 40 ° C for 10 h, or add 2% neutral protease of the weight of the raw turtle, 40 ° C thermolyzed 4h, the obtained hydrolysate was heated to 120 ° C, enzyme inactivation 60mi _n; filtered, the filtrate was concentrated to obtain spray-dried.

[0051] Second, the determination of turtle peptide content:

According to the method of Example 1, the molecular weight of the turtle peptide was mainly concentrated below 10,000, accounting for more than 95%.

Example 4

First, the preparation method of soft-shelled oligopeptide, the steps are as follows:

1. Using Chinese sturgeon as raw material, use 100kg of fresh turtle, stir and simmer for 3h.

2. Cool to 62 ° C, add alkaline protease according to the ratio of 3% of the weight of the raw turtle, constant temperature solution at 62 ° C for 4 h, and then dilute the enzyme by 1% of the weight of the raw turtle, and incubate for 2 h at 55 °C. The obtained enzymatic hydrolysate was heated to 110 ° C, and the enzyme was killed for 40 min;

3. The enzymatic hydrolysate was centrifuged and filtered, and the supernatant was purified by a hollow ultrafiltration membrane with a molecular weight cutoff of 100,000 Daltons. The inlet pressure was 2.8 bar, the outlet pressure was 2 bar, and the temperature of the hydrolysate was 30 ° C. 5公斤甲甲的 oligomeric peptide finished product, obtained by ultrafiltration separation, film concentration, spray drying.

[0053] Second, the determination of oligopeptide content of soft-shelled turtles:

According to the method of Example 1, the molecular weight of the soft-shell peptide of the soft-shelled turtle is mainly concentrated at 300-700, accounting for more than 30%.

Claims

Claim

1. The application of the fi peptide in the preparation of a drug having a protective function against cadmium.

The turtle peptide according to claim 1, wherein the molecular weight distribution of the turtle peptide is within a range of 1000OODalton.

The turtle peptide according to claim 2, wherein the turtle peptide has a molecular weight distribution within 2000 Dalt _O n .

4. The turtle peptide according to claim 3, wherein the molecular weight distribution of the turtle peptide is in the range of 140 to 100 Odalton.

The soft-shelled peptide according to claim 4, wherein the molecular weight distribution of the turtle peptide is in the range of 300 to 700 Dalt _on .