CN101037469A - Method for extracting small molecule active peptides from scapharca subcrenata - Google Patents

Abstract

The invention relates to a method for extracting small molecule active polypeptides from cockles. Shell the cockles, wash, homogenize, add distilled water to extract at low temperature, flocculate, centrifuge to remove slag, take the supernatant, freeze-dry the supernatant, perform gel column chromatography, collect each peak, and then conduct ion exchange column chromatography Chromatography achieves separation and purification, collects active peaks, and after freeze-drying, desalts with a gel column to obtain natural active peptides with high purity. The obtained small molecular peptides are further purified with a C18 reverse-phase column to obtain a purity of 95%. The above active peptides. The molecular weight of the clam series small molecule peptides obtained by the method of the invention is 500-3000 Da, and the biological activity is effectively protected during the extraction process. The active peptide has significant tumor-suppressing activity, and the anti-tumor experiment in vitro shows that the tumor-suppressing rate reaches 52%, and can be used for the development and application of new anti-tumor drugs.

Description

Method for extracting small molecule active polypeptide from cockles

Technical field:

The invention relates to a method for extracting small-molecule active peptides from cockles, in particular to separating and purifying the active peptides from cockles by column chromatography, and belongs to the technical field of marine natural product extraction and medicine.

Background technique:

Hairy clams (Arca subcrenata Lischke; Scapharca subcrenata) belong to the mollusk phylum, clambranchia, order Dendonta, clam family, is one of the abundant marine organisms in my country, and has a long history of medicinal use. Its shell is named corrugated fruit, which has been recorded in past materia medica. It has the functions of reducing phlegm, softening hardness, reducing acid and relieving pain. The cockle meat is a soft part, which is mostly contained in dietotherapy herbal medicines. The clam as a whole is used as medicine, which has the function of shell and meat. It can nourish the blood and warm the middle, strengthen the spleen and stomach, and is suitable for the syndrome of blood deficiency and spleen weakness. Pharmacological experiments have proved that the extract of cockles has a good therapeutic effect on the models of hemorrhagic anemia, hemolytic anemia and bone marrow dysfunction anemia in mice. Clam meat is rich in protein, fat, carbohydrates, and contains riboflavin, niacin, vitamin E, and various trace elements. It also contains glycogen, rich in various amino acids, and has good medicinal value. Therefore, it is of great significance to rationally utilize marine resources and develop cockles as medical and health products.

According to the retrieved literature, there are few studies on extracting active substances from cockles in foreign countries, and there are almost no reports. There are relevant domestic literatures reporting the extraction and activity research of active substances in cockles. For example, the patent document "An Anticancer Marine Biological Extract" (CN1248442A) reports the research on extracting anticancer drugs from cockles. The patent document "Polypeptide protein active substances in cockles and its preparation method and application" (CN1600792A) also reports the preparation method and application of polypeptide protein active substances in cockles. The above-mentioned patent documents describe in detail the extraction process and pharmacological activity research of active substances in cockles, but more in-depth research on active substances, such as structural analysis and research on physical and chemical properties, has not been reported.

Invention content:

The purpose of the present invention is to extract active ingredients from natural marine products by means of modern separation and extraction technology, especially to provide a small molecular peptide substance derived from cockles, including preparation methods and biological activity research and its active substances. Further separation and purification and structural analysis.

In order to realize the above-mentioned purpose of the invention, the extraction steps of the present invention are as follows:

Shell the cockles, wash, homogenize, add distilled water to extract at low temperature, flocculate, centrifuge to remove slag, take the supernatant, freeze-dry the supernatant, and perform gel column chromatography with Sephadex G25, respectively. Collect each peak, and then use anionic dextran gel DEAE-SephadexA25 for ion exchange column chromatography to achieve separation and purification, collect active peaks, freeze-dry, and desalt with a gel column to obtain a series of natural small molecules with high purity For active peptides, the obtained small molecular polypeptides were further purified with a C18 reverse-phase column to obtain small molecular active peptides with a purity of more than 95%, and their structures were analyzed.

The small-molecule active peptide is a naturally extracted substance and belongs to an oligopeptide with a small molecular weight. The molecular weight of the small-molecule peptides obtained by the preparation method of the present invention is 500-3000 Da, and the biological activity of the clam is effectively protected during the extraction process. . The active peptide has significant tumor-suppressing activity, and anti-tumor experiments in vitro show that the tumor-suppressing rate reaches 52%.

Detailed ways:

The present invention will be further described below in conjunction with the examples.

Shell the cockles, take the cockle meat, wash with water at 4°C, homogenate, add distilled water at a low temperature (4°C) for extraction for 2 hours according to the weight ratio of 1:1, flocculate, centrifuge to remove slag, take the supernatant, and After the supernatant was freeze-dried, use Sephadex G25 for gel column chromatography (0.03mol/l NH ₄ HCO ₃ buffer elution), collect each peak separately, take the peak with biological activity and then use anion Sephadex DEAE-Sephadex A25 ion-exchange column chromatography (PH8.5 Tris-HCl buffer, 0 ~ 0.03mol/NaCl gradient elution), collect active peaks, after freeze-drying, use Sephadex Sephadex G15 gel column desalted to obtain natural small molecule active peptides with high purity. The active peptide is a naturally extracted substance without any chemical modification, and the extraction process effectively protects its biological activity.

The activity of the finally obtained extract was studied by animal experiment (tumor inhibitory effect of marine biological extract on S180 mice). The experimental method is as follows: take male Kunming mice, weighing 19-21 g, and divide them into random groups, 10 mice in each group. Each mouse was inoculated with 0.2ml of tumor fluid of S180 Ehrlich ascites carcinoma in the left armpit. The next day, normal saline was used as a negative control and 5-fluorouracil was used as a positive control for intraperitoneal injection. Eight days after the injection, the animals were weighed, and the subcutaneous tumor mass was dissected and weighed. The curative effect of the drug on solid tumors is expressed by the tumor weight inhibition percentage, and the calculation formula is as follows: tumor weight inhibition percentage (%)=(1-T/C)×100%, T: average tumor weight of the treatment group, C: average weight of the negative control group Tumor weight.

Table 1 The effect of drugs on the growth of S180 solid tumor in mice group dose number of surviving animals Tumor weight (x±s, g) Tumor inhibition rate Blank control group 10 1.04±0.41 cyclophosphamide 10 0.68±0.36 34.62 sample 1 high 20 7 0.41±0.16 51.85 middle 10 8 0.44±0.20 46.48 Low 50 5 0.67±0.38 35.58 sample 2 high 20 10 1.02±0.51 45.96 middle 10 9 0.83±0.41 42.12 Low 50 10 0.74±0.37 39.52

The results of in vitro anti-tumor experiments show that the natural active peptide extracted by this method has a high tumor inhibition rate. Therefore, the natural active peptide extracted by the present invention has anti-tumor activity and may become a new type of anti-tumor drug.

Sample 1 and sample 2 are further refined with the method of high-performance liquid phase RP-HPLC (reversed-phase high-performance liquid phase), obtain the pure product that purity is greater than 95%, use MALDI-TOF-MS (matrix-assisted laser desorption/ionization-time of flight) The mass spectrometry) technology determined the molecular weight range of 500-3000Da, and the amino acid composition, structure and sequence of some active peptides were determined by techniques and means such as ESI-MS (electrospray ionization mass spectrometry) and MS/MS (tandem mass spectrometry).

Claims (4)

1. A method for extracting small molecule active polypeptides from cockles, characterized in that the extraction steps are as follows: Shell the cockles, wash, homogenate, add distilled water to extract at low temperature, flocculate, centrifuge to remove slag, take the supernatant, freeze-dry the supernatant, perform gel column chromatography, collect each peak separately, and then perform ion exchange Column chromatography realizes separation and purification, collects active peaks, and after freeze-drying, desalts with a gel column to obtain a series of natural active peptides with high purity. The obtained small molecule peptides are further purified with a reversed-phase column to obtain a purity of 95 % more active peptides. 2. The method for extracting small-molecule active polypeptides from cockles according to claim 1, characterized in that the obtained cockles small-molecule peptides have a molecular weight of 500-3000Da. 3. The method for extracting small molecule active polypeptides from cockles according to claim 1, characterized in that after washing the cockle meat with water at 4°C, homogenate, add distilled water according to the weight ratio of 1:1, and extract at 4°C 2 hours. 4. The method for extracting small molecule active polypeptides from cockles according to claim 1, characterized in that after the supernatant is lyophilized, gel column chromatography and buffer elution are performed with Sephadex G25 , after collecting each peak respectively, take the peak with biological activity and then use ion exchange column chromatography, pH8.5 Tris-HCl buffer solution, 0～0.03mol/NaCl gradient elution, collect each peak with activity, after lyophilization, Gel column desalting.