CN1451393A - Clam extract, and preparing process and use thereof - Google Patents
Clam extract, and preparing process and use thereof Download PDFInfo
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- CN1451393A CN1451393A CN02117433A CN02117433A CN1451393A CN 1451393 A CN1451393 A CN 1451393A CN 02117433 A CN02117433 A CN 02117433A CN 02117433 A CN02117433 A CN 02117433A CN 1451393 A CN1451393 A CN 1451393A
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Abstract
A clam extract for treating liver cancer, lung cancer and breast cancer is prepared from clam shell through homogenizing the mashed clam in distilled water at 0-4 deg.C, centrifugal separation of supernatant, extracting in solution of ammonium sulfate, centrifugal separation of supernatant, filtering, chromatography to collect polypeptide, and drying.
Description
Technical field
The present invention relates to a kind of extract that in animal body, obtains and preparation method thereof and application, particularly relate to a kind of clam extract and preparation method thereof and application.
Background technology
Conch Meretricis seu Cyclinae (Meretrix meretrix Linnaeus) belongs to the Veneridae animal, and main product is coastal in China north and south, is one of traditional Chinese crude drug.Shennong's Herbal record: " Conch Meretricis seu Cyclinae master malignant boil ".The coastal fisherman of China is just spread the proved recipe of useful sea clam treatment tumor a long time ago.Theory of Chinese medical science thinks that Conch Meretricis seu Cyclinae has the effect of hard masses softening and resolving, strengthening vital QI to eliminate pathogenic factors, can treat goiter, tumor, lump in the abdomen etc., is used for the treatment of pulmonary carcinoma, hepatocarcinoma, gastric cancer clinically, and curative effect is better.
The ocean Conch Meretricis seu Cyclinae contains multiple compositions such as protein, polypeptide, polysaccharide, nucleic acid, agglutinin, contains the active substance that suppresses tumor growth in the clam extract, thereby is usually used in tumor treatment.Shanghai Second Medicine University subsidiary Renji Hospital Ke Ling etc. observe the effect of ocean clam extract clinical treatment pulmonary carcinoma, find that it can improve symptom to the advanced lung cancer patient, prolong patient's life, and do not have bad side reaction (marine drug, 2,22-24,1987); Report clam extract animal experiment such as the old Hanyuan of Shanghai Inst. of Cytobiology, Chinese Academy of Sciences and the anticancer result of clinical trial, find (the treatment and prevention of tumour research of its inhibitory rate to 59% to zoografting S-180 tumor, 4,3-7,1980), can obviously prolong life span to advanced liver cancer, Patients with Gastric Cancer; The Luo Yun of Anhui Prov. Medical Science Inst. completely waits the part pharmacological action of report Conch Meretricis seu Cyclinae meat cold extraction thing, find that Conch Meretricis seu Cyclinae meat can obviously improve the mice spleen lymphocytes proliferation reaction, promote mouse anti SRBC antibody-producing capacity, the expository writing clam meat can strengthen mice body fluid and cellular immune function (Chinese marine drug, 2,14-17,1996).The many activity with the research clam extract of these reports are purpose, to active component in the extract and extracting method thereof, especially have anti-tumor activity for which kind of composition in the Conch Meretricis seu Cyclinae and study less.
Summary of the invention
The purpose of this invention is to provide a kind of clam extract with tumor-suppression activity.
A kind of clam extract, it is to be prepared by the method that comprises the steps:
1) under 0-4 ℃ of following condition with Conch Meretricis seu Cyclinae meat distilled water homogenate, centrifuging and taking supernatant;
2) under 0-4 ℃ of condition, centrifugal with the above-mentioned supernatant of 10-40% ammonium sulfate extracting, collect supernatant and filter after obtain filtrate;
3) filtrate is carried out column chromatography, be collected in 215 and/or 280nm under the polypeptide fractions of absworption peak is arranged, dry finished product.
In said process, described under 0-4 ℃ of following condition that Conch Meretricis seu Cyclinae meat is used during with the distilled water homogenate be to be Conch Meretricis seu Cyclinae meat 1-3 0 ℃ distilled water doubly, the homogenate time is 25-35 minute.
Describedly carry out at twice with 10-40% ammonium sulfate extracting supernatant, the weight ratio of supernatant and ammonium sulfate is 1 for the first time: 0.5-1: 3; The weight ratio of supernatant and ammonium sulfate is 1 for the second time: 0.2-1: 2, and mixing time was respectively 0.5-1 hour; The supernatant that obtains after centrifugal obtains filtrate after ultrafiltration.
Filler in the described column chromatography is a Sephadex G-25 polydextran gel, and mobile phase is distilled water; The cryodesiccated mode of dry employing.
Second purpose of the present invention provides the method that a kind of preparation has the clam extract of tumor-suppression activity.
A kind of method for preparing clam extract may further comprise the steps:
1) under 0-4 ℃ of following condition with Conch Meretricis seu Cyclinae meat distilled water homogenate, centrifuging and taking supernatant;
2) under 0-4 ℃ of condition, centrifugal with the above-mentioned supernatant of 10-40% ammonium sulfate extracting, collect supernatant and filter after obtain filtrate;
3) filtrate is carried out column chromatography, be collected in 215 and/or 280nm under the polypeptide fractions of absworption peak is arranged, dry finished product.
In said process, described under 0-4 ℃ of following condition that Conch Meretricis seu Cyclinae meat is used during with the distilled water homogenate be to be Conch Meretricis seu Cyclinae meat 1-3 0 ℃ distilled water doubly, the homogenate time is 25-35 minute.
Describedly carry out at twice with 10-40% ammonium sulfate extracting supernatant, the weight ratio of supernatant and ammonium sulfate is 1 for the first time: 0.5-1: 3; The weight ratio of supernatant and ammonium sulfate is 1 for the second time: 0.2-1: 2, and mixing time was respectively 0.5-1 hour; The supernatant that obtains after centrifugal obtains filtrate after ultrafiltration.
Filler in the described column chromatography is a Sephadex G-25 polydextran gel, and mobile phase is distilled water; The cryodesiccated mode of dry employing.
The 3rd purpose of the present invention provides the application of the clam extract with tumor-suppression activity.
Above-mentioned clam extract with the treatment effective dose is the inhibition tumour medicine of active ingredient, particularly treats the medicine of hepatocarcinoma, pulmonary carcinoma and breast carcinoma, and tumor is had good inhibition and therapeutical effect.
During concrete the application, in medicine, can also add one or more pharmaceutically acceptable carriers.Described carrier comprises diluent, excipient, filler, binding agent, wetting agent, disintegrating agent, absorption enhancer, surfactant, absorption carrier, lubricant of pharmaceutical field routine etc., can also add flavouring agent, sweeting agent etc. in case of necessity.
Medicine of the present invention can be made various ways such as tablet, powder, granule, capsule, oral liquid and injection.The medicine of above-mentioned various dosage forms all can be according to the conventional method preparation of pharmaceutical field
Amount of drug of the present invention is generally 0.1-1.0mg/kg body weight every day.
The present invention passes through modern bioseparation technology, extraction, separation and purification bioactive peptide from Conch Meretricis seu Cyclinae, and its extracting method and product have following characteristic:
1, is raw material with fresh Conch Meretricis seu Cyclinae meat, carries out low-temperature homogenate and centrifugal treating, guarantee that the Conch Meretricis seu Cyclinae polypeptide active do not suffer a loss.
2, the method that adopts ammonium sulphate precipitation, ultrafiltration and column chromatography technology to combine can access purer bioactive peptide component.In the extract that obtains through homogenate, after centrifugal except that polypeptide, still have macromolecular protein, other impurity such as polysaccharide, by sulphuric acid amine fractional precipitation, can remove foreign protein, by ultrafiltration, other macromole impurity such as polysaccharide both can have been removed, can concentrate again, pass through the polydextran gel column chromatography again, can reach desalination and remove the purpose of other small molecular weight impurities.
The present invention will be further described below in conjunction with specific embodiment.
The specific embodiment
Embodiment 1,
One, the extraction of active substance
Get the fresh Conch Meretricis seu Cyclinae meat of 500 grams, adds 1000 milliliters of distilled water that are cooled to 0 ℃, with homogenizer homogenate 30 minutes, under 4 ℃ of conditions centrifugal 10 minutes, supernatant added 1000 milliliter of 10% ammonium sulfate, stirs extraction 0.5 hour under 2 ℃ of conditions; Centrifugal 0.5 hour; Supernatant adds 1000 milliliter of 40% ammonium sulfate again, stirs under 2 ℃ of conditions and extracts 0.5 hour; Centrifugal 0.5 hour; Merge supernatant, with ultrafilter with this supernatant concentration, molecular retention amount 10000 dalton; Multiple concentrated solution is carried out Sephadex G-25 polydextran gel column chromatography, use the distilled water eluting, be collected in 215 and 280nm under the polypeptide fractions of absworption peak is arranged, merge, vacuum lyophilization, clam active peptide extract 6.2 grams, yield 1.24%.
Two, active substance is to the inhibitory action of tumor cell
Above-mentioned clam active peptide extract dissolved in distilled water, be made into the 20mg/ml mother solution ,-20 ℃ of preservations are standby.Diluting with culture medium during experiment, is 100 μ g/ml with concentration finally.The different tumor cells that grow into logarithmic (log) phase are digested to cell suspension with Digestive system, and with every hole 10
3-10
4Individual cell, the amount of 200 μ l is inoculated into 96 well culture plates.After 24 hours, remove the culture medium of cultivating in the plate hole, be changed by fresh bioactive peptide medicinal liquid, every hole 200 μ l.Dosing was cultivated respectively 3 days after handling, and every then hole adds the MTT solution of 20 μ l, and 37 ℃ are continued to cultivate 4 hours.The careful supernatant of removing culture in the hole, every then hole adds the DMSO of 200 μ l, and concussion is 20 minutes on the horizontal shaking table, and the assurance crystallization is fully dissolved.Detect the absorbance value in each hole at the 490nm place, calculate suppression ratio.The result shows, is 63.1% to the suppression ratio of human liver cancer cell Hep G2, is 54.7% to the suppression ratio of human lung carcinoma cell, is 67.5% to the suppression ratio of human breast cancer cell MCF-7.
Three, active substance is to the inhibitory action of mouse entity tumor
Adopt mice right hind subcutaneous vaccination S
180The solid tumor cell suspension is pressed 6mg/kg body weight intravenously administrable with above-mentioned extract, and the result shows S
180The suppression ratio of solid tumor is 59.2%.
Embodiment 2:
One, the extraction of active substance
Get the fresh Conch Meretricis seu Cyclinae meat of 1000 grams, adds 3000 milliliters of distilled water that are cooled to 0 ℃, with homogenizer homogenate 25 minutes, under 4 ℃ of conditions centrifugal 10 minutes, supernatant added 3000 milliliter of 10% ammonium sulfate, stirs extraction 1 hour under 4 ℃ of conditions; Descended centrifugal 20 minutes at 4 ℃; Supernatant adds 1000 milliliter of 25% ammonium sulfate again, stirs under 4 ℃ of conditions and extracts 1 hour; Centrifugal 10 minutes; Merge supernatant, should concentrate molecular retention amount 10000 dalton for the soil clear liquid with ultrafilter; Again concentrated solution is carried out Sephadex G-25 polydextran gel column chromatography, use the distilled water eluting, be collected in the polypeptide fractions that absworption peak is arranged under the 280nm, merge, vacuum lyophilization gets clam active peptide extract 14.7 grams, yield 1.47%.
Two, active substance is to the inhibitory action of tumor cell
Detect according to the described MTT method of embodiment 1, the result is an extract of the present invention to the suppression ratio of human liver cancer cell Hep G2 is 47.6%, is 56.3% to the suppression ratio of human lung carcinoma cell, is 53.1% to the suppression ratio of human breast cancer cell MCF-7.
Three, active substance is to the inhibitory action of mouse entity tumor
Adopt mice right hind subcutaneous vaccination S
180The solid tumor cell suspension is pressed the 6mg/kg intravenously administrable with above-mentioned extract, and the result shows S
180The suppression ratio of solid tumor is 51.8%.
Embodiment 3:
One, the extraction of active substance
Get the fresh Conch Meretricis seu Cyclinae meat of 500 grams, adds 1000 milliliters of distilled water that are cooled to 0 ℃, with homogenizer homogenate 35 minutes, under 4 ℃ of conditions centrifugal 30 minutes, supernatant added 800 milliliter of 10% ammonium sulfate, stirs extraction 1 hour under 4 ℃ of conditions; Descended centrifugal 50 minutes at 4 ℃; Supernatant adds 800 milliliter of 40% ammonium sulfate again, stirs under 4 ℃ of conditions and extracts 1 hour; Centrifugal 50 minutes; Merge supernatant, with ultrafilter with this supernatant concentration, molecular retention amount 10000 dalton; Again concentrated solution is carried out Sephadex G-25 polydextran gel column chromatography, use the distilled water eluting, be collected in the polypeptide fractions that absworption peak is arranged for 215 times, merge, vacuum lyophilization gets clam active peptide extract 5.2 grams, yield 1.04%.
Two, active substance is to the inhibitory action of tumor cell
Detect according to the described MTT method of embodiment one, the result is an extract of the present invention to the suppression ratio of human liver cancer cell Hep G2 is 45%, is 33.7% to the suppression ratio of human lung carcinoma cell, is 47.2% to the suppression ratio of human breast cancer cell MCF-7.
Three, active substance is to the inhibitory action of mouse entity tumor
Adopt mice right hind subcutaneous vaccination S
180The solid tumor cell suspension is pressed the 6mg/kg intravenously administrable with above-mentioned extract, and the result shows S
180The suppression ratio of solid tumor is 36.7%.
Claims (12)
1, a kind of clam extract, it is to be prepared by the method that comprises the steps:
1) under 0-4 ℃ of following condition with Conch Meretricis seu Cyclinae meat distilled water homogenate, centrifuging and taking supernatant;
2) under 0-4 ℃ of condition, centrifugal with the above-mentioned supernatant of 10-40% ammonium sulfate extracting, collect supernatant and filter after obtain filtrate;
3) filtrate is carried out column chromatography, be collected in 215 and/or 280nm under the polypeptide fractions of absworption peak is arranged, dry finished product.
2, clam extract according to claim 1 is characterized in that: described under 0-4 ℃ of following condition that Conch Meretricis seu Cyclinae meat is used during with the distilled water homogenate be to be Conch Meretricis seu Cyclinae meat 1-3 0 ℃ distilled water doubly, the homogenate time is 25-35 minute.
3, clam extract according to claim 1 is characterized in that: describedly carry out at twice with 10-40% ammonium sulfate extracting supernatant, the weight ratio of supernatant and ammonium sulfate is 1 for the first time: 0.5-1: 3; The weight ratio of supernatant and ammonium sulfate is 1 for the second time: 0.2-1: 2, and mixing time was respectively 0.5-1 hour; The supernatant that obtains after centrifugal obtains filtrate after ultrafiltration.
4, clam extract according to claim 1 is characterized in that: the filler in the described column chromatography is a Sephadex G-25 polydextran gel, and mobile phase is distilled water; The cryodesiccated mode of dry employing.
5, a kind of method for preparing clam extract may further comprise the steps:
1) under 0-4 ℃ of following condition with Conch Meretricis seu Cyclinae meat distilled water homogenate, centrifuging and taking supernatant;
2) under 0-4 ℃ of condition, centrifugal with the above-mentioned supernatant of 10-40% ammonium sulfate extracting, collect supernatant and filter after obtain filtrate;
3) filtrate is carried out column chromatography, be collected in 215 and/or 280nm under the polypeptide fractions of absworption peak is arranged, dry finished product.
6, method according to claim 5 is characterized in that: described under 0-4 ℃ of following condition that Conch Meretricis seu Cyclinae meat is used during with the distilled water homogenate be to be Conch Meretricis seu Cyclinae meat 1-3 0 ℃ distilled water doubly, the homogenate time is 25-35 minute.
7, method according to claim 5 is characterized in that: describedly carry out at twice with 10-40% ammonium sulfate extracting supernatant, the weight ratio of supernatant and ammonium sulfate is 1 for the first time: 0.5-1: 3; The weight ratio of supernatant and ammonium sulfate is 1 for the second time: 0.2-1: 2, and mixing time was respectively 0.5-1 hour; The supernatant that obtains after centrifugal obtains filtrate after ultrafiltration.
8, method according to claim 5 is characterized in that: the filler in the described column chromatography is the SephadexG-25 polydextran gel, and mobile phase is distilled water; The cryodesiccated mode of dry employing.
9, a kind of antitumor drug, its active component are the described clam extracts of claim 1.
10, medicine according to claim 9 is characterized in that: described medicine is the medicine of treatment hepatocarcinoma.
11, medicine according to claim 9 is characterized in that: described medicine is the treatment lung cancer drugs.
12, medicine according to claim 9 is characterized in that: described medicine is the medicine of treatment breast carcinoma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021174334A CN1257183C (en) | 2002-04-19 | 2002-04-19 | Clam extract, and preparing process and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021174334A CN1257183C (en) | 2002-04-19 | 2002-04-19 | Clam extract, and preparing process and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1451393A true CN1451393A (en) | 2003-10-29 |
| CN1257183C CN1257183C (en) | 2006-05-24 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB021174334A Expired - Fee Related CN1257183C (en) | 2002-04-19 | 2002-04-19 | Clam extract, and preparing process and use thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102125686A (en) * | 2011-01-31 | 2011-07-20 | 中国科学院海洋研究所 | Application of hard clam polypeptide |
| CN105769926A (en) * | 2016-02-28 | 2016-07-20 | 汉中领维生物科技发展有限公司 | Skin mucus extracts of andrias davidianus Blanchard for preparing anti-breast cancer drugs and application thereof |
| CN106676153A (en) * | 2016-11-21 | 2017-05-17 | 浙江海洋大学 | Preparation method and application of meretrix lusoria blood-pressure-reducing peptide |
| CN106916868A (en) * | 2015-12-25 | 2017-07-04 | 无限极（中国）有限公司 | A kind of white clam polypeptide with anti-oxidant in vivo and neuroprotective function and preparation method and application |
-
2002
- 2002-04-19 CN CNB021174334A patent/CN1257183C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102125686A (en) * | 2011-01-31 | 2011-07-20 | 中国科学院海洋研究所 | Application of hard clam polypeptide |
| CN102125686B (en) * | 2011-01-31 | 2013-06-19 | 中国科学院海洋研究所 | Application of hard clam polypeptide |
| CN106916868A (en) * | 2015-12-25 | 2017-07-04 | 无限极（中国）有限公司 | A kind of white clam polypeptide with anti-oxidant in vivo and neuroprotective function and preparation method and application |
| CN105769926A (en) * | 2016-02-28 | 2016-07-20 | 汉中领维生物科技发展有限公司 | Skin mucus extracts of andrias davidianus Blanchard for preparing anti-breast cancer drugs and application thereof |
| CN106676153A (en) * | 2016-11-21 | 2017-05-17 | 浙江海洋大学 | Preparation method and application of meretrix lusoria blood-pressure-reducing peptide |
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| Publication number | Publication date |
|---|---|
| CN1257183C (en) | 2006-05-24 |
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