WO2014161240A1

WO2014161240A1 - Preparation method and application of anti-tumor active polypeptide component in scapharca subcrenatae

Info

Publication number: WO2014161240A1
Application number: PCT/CN2013/078970
Authority: WO
Inventors: 于荣敏
Original assignee: Yu Rongmin
Priority date: 2013-04-01
Filing date: 2013-07-08
Publication date: 2014-10-09
Also published as: CN103254303A

Abstract

Disclosed are a preparation method and an application of an anti-tumor active polypeptide component in scapharca subcrenata. The preparation method comprises: shelling the scapharca subcrenata, washing the content, homogenate and extraction after adding a buffer solution, centrifugal dregs removal, taking the upper-layer clear liquid, fractional precipitation with ammonium sulfate, and dialysis and desalination, to obtain an active component; then, the active component is subjected to chromatographic isolation with an ion exchanger, dialysis and desalination, and freezing and drying, to obtain a group of polypeptide components having high anti-tumor activity. In the present invention, an extraction and separation process is used in combination with an active tracking method, to obtain a glycopeptide component with the molecular weight of 10.0-27.0kDa and acting strong inhibition on multiple kinds of tumor cells. The method can be applied in development and application of anti-tumor drugs.

Description

Preparation method and use of anti-tumor active polypeptide component in buttercup

The invention discloses a preparation and separation method for obtaining strong anti-tumor active components from a series of extraction, separation and chromatographic purification steps of marine organisms, and a test for the pharmacological activity of the components on some tumor cell lines. The invention belongs to the field of medical technology.

Background technique

The buttercup belongs to the mollusk door, the scorpion scorpion, the snail, the scorpion. It is distributed along the coasts of China, North Korea and Japan, with more offshore China and the East China Sea. The buttercup is rich in resources, high in nutritional value, and rich in vitamin B12. Its shell and meat have a long history of medicine, such as its shell as a traditional Chinese medicine.

According to literature search, people's research on edulis mainly focuses on the separation of polysaccharide components and their anti-oxidation or immunological activities, as well as the antibacterial activity of glycoproteins. Although there are sporadic reports on the anti-tumor aspect of the protein polypeptides contained therein, such as: Chinese Patent Application No. 98110478.9 discloses a "anti-cancer marine organism extract" technique, the method used is too simple, and the obtained material is complicated and cannot be Quantification, because of its own limitations, is not conducive to the development and utilization of drugs; and Chinese Patent Application No. 2005100043797.4 discloses a "a scorpion anti-cancer peptide extract and its preparation method" and 200610043222.7 discloses a "from "Methods for extracting small-molecule active polypeptides from edulis", these two patents mainly study the extraction and activity of small molecular peptides with a molecular weight of less than 3,000 in the edulis, and Chinese Patent Application No. 200510043846.4 discloses "a ranunculus peptide" An extract of the class and a preparation method thereof, which is a mixture of glycopeptides having a molecular weight of less than 100OOD in the edulis. However, the above-mentioned patents have narrowly studied the active substances of edulis, and no reports on their anti-tumor activity in vitro have been reported.

Summary of the invention

In view of the above-mentioned deficiencies and shortcomings of the prior art, the present invention aims to invent a simple and effective method for extracting and separating and simultaneously carrying out an activity test, and preparing a high purity and active ingredient of an active component from a marine organism. The peptide component of the relatively high-efficiency anti-tumor activity has the advantages of good solubility, good stability, and small toxic side effects. The present invention has many technological advances compared to the prior art:

1. The present invention extracts an active substance from a ranunculus by using a homogenization technique after mixing with a buffer solution and an ultrasonic method, thereby sufficiently dissolving the active ingredient in the extract and shortening the extraction time.

2. The present invention firstly separates the extract by ammonium sulfate precipitation method. The separation method has wide application range and low price, and can remove a large amount of inactive impurities in one step, and plays a good role in separation and impurity removal.

3. The invention adopts ion exchange chromatography to compare with the separation method of the gel column used in other patents, and the ion exchange method has the advantages of large sample volume, simple method and good repeatability. 4. The present invention uses the MTT method to track activity in each separation step of the polypeptide protein, and has the advantages of high efficiency and accurate determination of the optimal active portion.

5. The present invention further identifies its final component by using some biotechnological methods such as electrophoresis, and clarifies the main components and some related properties.

The preparation steps of the present invention are as follows:

The fresh buttercup is husked, the contents are removed, washed, buffered, homogenized, extracted, centrifuged, supernatant taken, ammonium sulfate fractionated, dialyzed, dried, separated by column chromatography, separately collected for activity peaks, freeze-dried Concentration, dialysis desalting, and lyophilization yield a set of polypeptide components having a high antitumor activity with a molecular weight ranging from 10.0 to 27.0 kDa.

The active component prepared by the invention is a material obtained by a natural extraction method, which is always carried out in an aqueous solution during the preparation process, is not doped with organic poisonous substances and other pollutants, and the whole preparation process is environmentally friendly and hygienic, and adopts the MTT method. Screening of tumor cell lines showed that it has a strong inhibitory effect on the proliferation and growth of various tumor cell lines, and the highest inhibition rate in vitro is over 90%, which can be applied to medical purposes.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 is an electropherogram of the intermediate active component I after salting out in Example 1, and M represents the protein standard used, and its molecular weight distribution is 14.4 kDa, 20 kDa, 26 kDa, 35 kDa, 45 kDa, 66.2 kDa and 94 kDa.

2 is an electrophoresis pattern of the target component II after chromatographic column chromatography in Example 1, and M represents the protein standard used, and its molecular weight distribution is 14.4 kDa, 20 kDa, 26 kDa, 35 kDa, 45 kDa. , 66.2 kDa and 94 kDa. Specific implementation examples:

The invention is further described below in conjunction with the embodiments.

Example 1 :

The shells were peeled and the contents were taken, washed three times with low-temperature distilled water, and 150 g was weighed after being dried, and the extraction buffer (pH=8.0, 0.03 M, sodium phosphate buffer) was added at a solid-liquid ratio of 1:3. The high-speed tissue masher was homogenized at 8000 rpm for 3 min to a fine and non-blocky shape. The homogenate was ultrasonically extracted in a low-frequency ultrasound machine for 40 min, using a low-temperature high-speed centrifuge, 10000 rpm, 4 ° C, and centrifuged for 30 min. To remove the slag, take the supernatant and separate the salt by precipitation. After adding the volume of the supernatant to the ice bath, add 70% saturated ammonium sulfate, continue stirring for a period of time, then remove the precipitate by high speed centrifugation. The supernatant was further salted out according to the volume of the supernatant and added to 100% saturated ammonium sulfate. After stirring for a while, the mixture was centrifuged at 10,000 rpm, 4 ° C for 30 min, and the precipitate was collected. The precipitate was dissolved in a small amount of extract and placed in a 3000 Da dialysis bag for 24 h, and the peripheral distilled water was replaced every 6 h. The intermediate active component I after dialysis and desalting was further separated and purified by ion exchange chromatography. The anion exchanger was DEAE-Sepharose Fast Flow, and the 0.030 M Tris-HCl buffer having a pH of 8.0 was used as the initial buffer. The concentration of sodium chloride is eluted in stages, and the active peak is collected and collected in A _28Q , desalted by dialysis, and freeze-dried to obtain polypeptide substance II, which is a polypeptide component with high antitumor activity. Taking component I and component II, the protein distribution of the components obtained in each separation step was observed by SDS-PAGE gel electrophoresis. It was observed from the electropherogram that there were many distinct bands in the component I, and the molecular weight distribution. Wide, and the target component II after ion exchange column chromatography contains only one distinct band, indicating that it contains a main component and several trace components, and its molecular weight ranges from 10.0 to 27.0 kDa.

Example 2:

In the whole experiment, the activity of the components in the extraction process was detected by MTT method.

Tumor cells in the logarithmic growth phase (adherent cells were trypsinized), and the cell suspension was blown with complete medium to adjust the cell concentration to 4×10 ³ /ml. The cell suspension was inoculated on a 96-well cell culture plate at 100 μl per well, and cultured in a 37 ° C, 5 % CO ₂ , 100% relative humidity incubator for 24 ho, and the culture medium in the plate was aspirated. The culture medium was diluted into different concentrations of the sample and added to the 96-well plate. Four sample concentration groups were set up, and each concentration was set to 3 parallel wells. The culture medium well without the sample was used as the negative control group and placed in the C0 ₂ incubator. Cultivate for 48 h. After aspirating the liquid, 20 μM MTT was added to each well, and after 5 h, it was poured out and the stop solution DMSO 100 μl was added to each well, and left at room temperature for 30 min. The OD value was measured at 570 nm on a microplate reader. Cisplatin was used as a positive control. The IC ₅ o value was calculated using pharmacological methods.

The activity results of the intermediate component I by salting out are shown in Table 1:

Table 1. Component I activity results

Cell line GLC-82 H460 A549 Hela HepG2

IC ₅₀ (g/ml) 153.0 432.8 199.0 384.0 408.9 The component I of the intermediate component I was separated by ion exchange column chromatography. The antitumor activity results are shown in Table 2:

Table 2. Component II activity results

Tumor strain Hela HepG2

IC50 (g/ml) 214.0 624

The intermediate component I produced by the present invention is sensitive to lung cancer cells (GLC-82, SPC-A-K NCI-H460 and A549), cervical cancer Hela, and liver cancer HepG2. Moreover, the target active peptide component II which was further separated by chromatography on the component I was more effective in inhibiting cervical cancer Hela and liver cancer HepG2, and the inhibitory activity against hepatoma HepG2 was increased by 6 times. The active polypeptide component has a protein content of more than 80%, high water solubility, and a clear color of the sample, and is suitable for preparing a water-soluble lyophilized powder, and is suitable for intravenous use in clinical practice.

Claims

WO 2014/161240 Claims PCT/CN2013/078970

1. A preparation method and use of the anti-tumor active polypeptide component of the clam, which is characterized by: peeling the fresh clam, taking out the contents, washing, adding buffer, homogenizing, extracting, centrifuging, taking the supernatant, Ammonium sulfate fractionated precipitation, dialysis, drying, using ion exchange chromatography, collecting active peaks respectively, freeze-drying and concentration, dialysis desalting, and freeze-drying to obtain anti-tumor active polypeptide components with a molecular weight range of 10.0 to 27.0 kDa.

2. A method for preparing and separating anti-tumor active polypeptide components in clams according to claim 1, characterized in that the added buffer is 0.01~0.05 M, sodium phosphate, potassium phosphate or Tris with a pH of 6.0~9.0 Alkaline buffer solution has a solid-liquid ratio of 1:1 to 1:4.

3. A method for preparing and separating anti-tumor active polypeptide components in clams according to claim 1, characterized in that the extraction method is ultrasonic and stirred with a stirrer.

4. A method for preparing and separating anti-tumor active polypeptide components in clams according to claim 1, characterized in that ammonium sulfate is used for graded precipitation, and the precipitation method is three-stage, medium-low, or two-stage precipitation, and the high-grade precipitation is sulfuric acid. Ammonium concentration range is 60~100%.

5. A method for preparing an anti-tumor active polypeptide component in clams according to claim 1, characterized in that the column chromatography separation is an anionic chromatography column, and its filler is DEAE-Sephorose, DEAE- Cellulose, etc., the initial elution buffer is 0.020~0.050 M Tris-HCl and sodium phosphate buffer with a pH of 6.0~9.0, and elution is performed by increasing the concentration of sodium chloride.

6. A method for preparing and separating anti-tumor active polypeptide components in clams according to claim 1, characterized in that the desalting method used is desalting using a dialysis bag in the range of 1000 Da to 3000 Da, by using freeze-dried Concentrate by dialysis or freeze-drying method to dry the sample.

7. A method for preparing and separating anti-tumor active polypeptide components in clams according to claim 1, characterized in that the molecular weight range of the active polypeptide component is 10.0~27.0 kDa, which mainly includes one main component and several Trace ingredients.

8. Use of the anti-tumor polypeptide component in the clam according to claim 1 in the preparation of anti-tumor drugs.