CN1371914A - Cockroach polypeptide tracted from cockroach and extraction method thereof - Google Patents
Cockroach polypeptide tracted from cockroach and extraction method thereof Download PDFInfo
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- 238000000605 extraction Methods 0.000 title claims description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 30
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- 238000000034 method Methods 0.000 claims description 13
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Abstract
The present invention provides a cockroach polypeptide extracted by acetic acid as solvent. Said cockroach polypeptide possesses the obvious action of inhiting and "killing" liver cancer cell.
Description
The present invention relates to Blatta seu periplaneta extract and extracting method thereof.Particularly, the present invention relates to the cockroach polypeptide and the extracting method thereof that extract from cockroach as solvent with acetate.
Cockroach survives in the shelling mode, but is infected especially easily after each shelling, so have to develop good antibacterial ability.With the standard of insect, cockroach is long-lived especially, can survive several years, considerably beyond fly, butterfly, the moth with mathematic(al) expectation in week.Scientists finds that cockroach has powerful immunity system to make it be able to longevity, and there is memory capability in this system simultaneously, can avoid superinfection.
Cockroach has multiple pharmaceutical use.Cockroach has the different protein of 50-60 kind to participate in complicated immune response, and external some are being learned the person and just utilized the immune response of this complexity and participating in the anti-liver cancer of protein research cockroach of immunity and the two aspect work that human body rejects to special albumen.
Can from cockroach, extract various active ingredients with solvent, the general at present ethanol that adopts extracts the active ingredient with pharmaceutical use as solvent more from cockroach, these active ingredients can be used for treating outer wound face, female genital diseases, cardiovascular disorder and hepatitis B etc.Chinese patent CN87100537 discloses " a kind of preparation method who treats outer wound face medicine ", and this method comprises the dry back coarse reduction of cockroach insect soma; Use alcohol solution dipping, extract effective medicinal components wherein, separate filter residue and filtrate then, and filter residue is extracted repeatedly; Concentrate gained filtrate, reclaim ethanol, obtain thick thing; The treated outer wound face medicine of a kind of treatment of making.Chinese patent 94118839 has disclosed and has extracted from cockroach the sticking propylhomoserin of the medicable active ingredient of cardiovascular diseases, behind the employing extraction using alcohol, removes degrease, makes water-soluble medicinal extract, and with compound nucleosides compatibility, the medicine of making is effective to the treatment cardiovascular disorder.Chinese patent 94108943 discloses with extraction using alcohols, degreasing, charcoal absorption, branch molten, obtains the contract mixture of amino acids of mucopolysaccharide in the cockroach, is mainly used in the treatment of hepatitis B.
Cockroach polypeptide is the proteinic degradation product of cockroach, and polypeptide further is degraded to amino acid.
The purpose of this invention is to provide the cockroach polypeptide that extracts from cockroach as solvent with acetate, be to adopt acetate to soak the cockroach powder 24-72 hour in the room temperature sealing as solvent, extracting solution after the immersion is heated into suspension in 80-100 ℃ of water-bath, the active ingredient that obtains behind this suspension is handled in freeze-drying.This cockroach polypeptide has the effect of significant inhibition and " killing " liver cancer cell.
Second purpose of the present invention provides the method for extracting cockroach polypeptide from cockroach, and this comprises the following steps:
(1) in an encloses container, the cockroach powder is immersed in the 0.05-0.2M acetic acid solution, sealing was soaked 24-48 hour;
(2) remove by filter filter residue, the filtrate that obtains is placed 80-100 ℃ of water-bath, heating makes the outstanding liquid that mixes;
(3) suspension is handled through freeze-drying, makes the cockroach polypeptide powder.
At first in 80-100 ℃ of baking oven behind the heating, drying cockroach, pulverize cockroach after the oven dry with 8000-10000 rev/min high speed stamp mill, grind into 80-100 purpose powder.
Traditional biochemical extracting method adopts organic solvent to extract.Different solvents can extract different active ingredients.At present, generally adopt ethanol from cockroach, to extract active ingredient as solvent.Active ingredient is generally the sticking propylhomoserin of cockroach in the cockroach of extraction using alcohol.The inventor examines or check the effect of PBS, acetate, acetone, ethanol and five kinds of solvent extraction cockroach polypeptides of DMSO, method is to use 40 milliliters of above-mentioned five kinds of solvents in 4 ℃ of soaked overnight respectively in 1 gram cockroach powder, used whizzer in second day, centrifugation is 15 minutes under 4000rpm, abandon sediment, measure the optical density(OD) of supernatant liquor then at 260nm and 280nm with spectrophotometer, calculate the protein concentration of cockroach powder solution, found that acetate solubleness the best to cockroach polypeptide, cockroach polypeptide concentration can reach 3mg/ml.
In an encloses container, be 1 according to cockroach powder and solvent ratios: 5-10, the cockroach powder is immersed in the 0.05-0.2M acetic acid solution, seal this container, soaked 24-72 hour, soaked preferably 24-48 hour; Remove by filter filter residue, the filtrate that obtains was placed 80-100 ℃ of water-bath 0.5-1.0 hour, make the outstanding liquid that mixes; Suspension is handled through freeze-drying, makes the cockroach polypeptide powder.
By Shanghai Inst. of Cytobiology, Chinese Academy of Sciences cockroach polypeptide is carried out cancer cell in vitro propagation inhibition test, the cockroach polypeptide that extracts with the inventive method is assigned in the nutritive medium, cultivate liver cancer cell 7404 and 7721, cockroach polypeptide all has obvious suppression and " killing " effect to these two kinds of cell growths under 100 μ g/ml or more heavy dose of condition.
According to LD
50Test method is given gastric infusion of mouse with the cockroach polypeptide that the present invention extracts, and carries out acute toxicity test, records LD after 7 days
50＞9.6g/kg.Therefore use almost non-toxic side effect clinically.
Carry out chronic toxicity test with dog, take 1 year (24g/kg, 3 times/day, the ante cibum) of cockroach polypeptide that the present invention extracts continuously, none death.To detections such as its blood picture, liver, renal function, electrocardiogram(ECGs, do not see influence.Dissect after 1 year, do not cause parenchymatous organ's pathology damage.
The animal tumor model test: test-results shows that cockroach polypeptide is effective in cure to S-180 solid-type tumour, and tumor control rate is greater than 40%.
The scavenger cell test: this evidence, oncopeptide can improve Turnover of Mouse Peritoneal Macrophages phagocytic rate and phagocytic index.
The cockroach polypeptide that the present invention extracts has unusual effect to suppressing with " killing " liver cancer cell.The 300mg cockroach polypeptide is incapsulated, make capsule medicine.Give and suffer from hepatocarcinoma patient examination clothes (300mg/ grain, 5/time, 3 times/day), minimum taking continuously 1 year, the longest taking continuously 3 years, patient's liver cancer symptom, index are all got better commentaries on classics, especially alpha-fetoprotein index by more than the 400 μ g/L, are reduced to below the 40 μ g/L.
It is shown in Figure 1 that to be 7721 cells be equivalent to dissolve cockroach polypeptide and cultivate 3 days growing state in the conventional nutrient solution of the HAC of isoconcentration only containing.
Shown in Figure 2 is the 7721 cell growing states of handling through 100 μ g/ml 3 days.
Shown in Figure 3 is that 7404 cells form colony at the soft agar layer.
Shown in Figure 4 is the situation that no longer forms colony through 7404 cells that 100 μ g/ml handle at the soft agar layer.
Can further understand the present invention by following embodiment.
Embodiment
Cockroach is placed 90 ℃ of baking ovens, heating, drying 2 hours.Use the high speed stamp mill, under 10000 rev/mins of speed, pulverize the cockroach of oven dry, each 5 minutes, carry out 3 times.Making granularity is 80-100 purpose powder.
In 1 liter of port grinding bottle, according to 1: 8 solid-to-liquid ratio, put into cockroach powder and 800 gram 0.1M acetic acid solutions that 100 gram oven dry are pulverized, the sealing back was in soaking at room temperature 30 hours.Filtering separation is abandoned filter residue.Filtrate is placed 90 ℃ of water-baths, heated 1.0 hours, obtain a suspension.This suspension is handled to such an extent that pressed powder 30 restrains through freeze-drying.
Cockroach is placed 80 ℃ of baking ovens, heating, drying 10 hours.Use the high speed stamp mill, under 10000 rev/mins of speed, pulverize the cockroach of oven dry, each 5 minutes, carry out 3 times.Making granularity is 80-100 purpose powder.
In 1 liter of port grinding bottle, according to 1: 10 solid-to-liquid ratio, put into cockroach powder and 1000 gram 0.05M acetic acid solutions that 100 gram oven dry are pulverized, the sealing back was in soaking at room temperature 48 hours.Filtering separation is abandoned filter residue.Filtrate is placed 100 ℃ of water-baths, heated 0.5 hour, obtain a suspension.This suspension is handled to such an extent that pressed powder 25 restrains through freeze-drying.
Cockroach is placed 100 ℃ of baking ovens, heating, drying 2 hours.Use the high speed stamp mill, under 8000 rev/mins of speed, pulverize the cockroach of oven dry, each 5 minutes, carry out 3 times.Making granularity is 80-100 purpose powder.
In 1 liter of port grinding bottle, according to 1: 5 solid-to-liquid ratio, put into cockroach powder and 500 gram 0.15M acetic acid solutions that 100 gram oven dry are pulverized, the sealing back was in soaking at room temperature 24 hours.Filtering separation is abandoned filter residue.Filtrate is placed 80 ℃ of water-baths, stir heating down 1.0 hours, obtain a suspension.This suspension is handled to such an extent that pressed powder 30 restrains through freeze-drying.
Preparation embodiment 4
In 1 liter of port grinding bottle, according to 1: 5 solid-to-liquid ratio, put into 100 gram particle degree and be 80-100 purpose cockroach powder and 500 gram 0.10M acetic acid solutions, the sealing back was in soaking at room temperature 72 hours.Filtering separation is abandoned filter residue.Filtrate is placed 80 ℃ of water-baths, stir heating down 1.0 hours, obtain a suspension.This suspension is handled to such an extent that pressed powder 30 restrains through freeze-drying.
The cockroach polypeptide of producing with above-mentioned preparation embodiment carries out following experiment.
1. cancer cell in vitro is bred inhibition test
Undertaken by Shanghai Inst. of Cytobiology, Chinese Academy of Sciences.
The cockroach polypeptide that extracts among the above-mentioned preparation embodiment is assigned in the nutritive medium with the dosage of 25 μ g/ml, 50 μ g/ml, 100 μ g/ml and 200 μ g/ml respectively, cultivate liver cancer cell 7404 and 7721, under phase microscope, observe the cell growing state of two kinds of hepatic cell lines day by day.The cell inoculation number is 1 * 10
4/ ml.Cultivate a week, this tests triplicate, finds that cockroach polypeptide all has different inhibition and " killing " effect to two kinds of cell growths under heavy dose of condition.
The growing state of 7721 cells sees Table 1.
Table 1
The cockroach polypeptide of various dose is to the growth effect of 7712 cells
| Cockroach polypeptide dosage (μ g/ml) | ??0 | ????25 | ????50 | ????100 | ????200 |
| Cell count (ten thousand/ml) | ??35.3 | ????35 | ????34 | ????1.7 | ????0.2 |
As shown in Table 1,25 μ g/ml and 50 μ g/ml dosage groups after 7 days counting cells compare no significant difference with control group.The cellular control unit number is 33.5 ten thousand/ml, and the cell count of 100 μ g/ml and 200 μ g/ml dosage groups is respectively 1.7 ten thousand/ml and 0.2 ten thousand/ml, has compared notable difference with control group.
The result that 7404 cells are handled with the cockroach polypeptide of above-mentioned various dose slightly is different from 7721 cells, the results are shown in table 2.
Table 2
The cockroach polypeptide of various dose is to the growth effect of 7404 cells
| Cockroach polypeptide dosage (μ g/ml) | ??0 | ??25 | ??50 | ????100 | ????200 |
| Cell count (ten thousand/ml) | ??21.3 | ??18.0 | ??17.0 | ????0 | ????0 |
As shown in Table 2, (25 μ g/ml, 50 μ g/ml) cultivated 3 days under the cockroach polypeptide low dosage, and cell just has the minority phenomena of mortality.And cultivated 3 days at high dose group 100 μ g/ml, just find all dead and cracking (seeing Fig. 1 and Fig. 2) of cell.Show that cockroach polypeptide is more responsive to the growth-inhibiting or the comparison 7721 of " killing " effect of 7404 cells.
7721 and 7404 cells to the cultivation of 100 μ g/ml cockroach polypeptides, these two kinds of cells all can not form colony on soft agar, and control group still can form colony growth (seeing Fig. 3 and Fig. 4), shows that cockroach polypeptide can effectively kill liver cancer cell with the dosage of 100 μ g/ml.
2. acute toxicity (LD
50) test
Undertaken by Shanghai Drug Control Inst..
Give gastric infusion of mouse with the cockroach polypeptide that extracts among the above-mentioned preparation embodiment, observed 7 days, record LD
50Therefore＞9.6g/kg uses almost non-toxic side effect clinically.
3. chronic toxicity test
Dog is taken 1 year (24g/kg, 3 times/day, the ante cibum) of cockroach polypeptide of extracting among the above-mentioned preparation embodiment, none death continuously.To detections such as its blood picture, liver, renal function, electrocardiogram(ECGs, do not see influence.Dissect after 1 year, do not cause parenchymatous organ's pathology damage.
4. animal tumor model test
According to following method small white mouse is carried out sarcoma S 180 experiments.Get 40 of the small white mouses of body weight 18-22 gram, 10 is a test group, totally three test group, and all the other 10 is control group.The cockroach polypeptide of test group to extract among the above-mentioned preparation embodiment of 1g/kg dosage abdominal injection polypeptide, the control group injecting normal saline, once a day, and continuous 14 days, dissected in the 15th day, calculate tumor control rate according to following formula.
Inhibiting rate=(the average knurl of the average knurl weight-test group of control group is heavy)/average knurl of control group heavy * 100%
This test serves as effective with tumor control rate greater than 30%.The inhibiting rate of three test group is respectively 45%, 48% and 42%.Greater than 30%, for effectively.
Experimental result shows that cockroach polypeptide is effective in cure to S-180 solid-type tumour.
5. scavenger cell test
According to following method small white mouse is carried out animal peritoneal macrophage phagocytic rate and phagocytic index experiment.
Get body weight and be 20 of the small white mouses of 20-25 gram, 10 is test group, and 10 is control group.Test group is injected the cockroach polypeptide that extracts among the above-mentioned preparation embodiment with 40g/kg dosage, and the control group injecting normal saline once a day, continuous 5 days, drips the sheet method according to the observation macrophage function and tests.The test group phagocytic rate is 78.02 ± 2.01, and phagocytic index is 2.02 ± 0.10, and the control group phagocytic rate is 47.13 ± 3.20, and phagocytic index is 1.15 ± 0.05.Experimental result shows that cockroach polypeptide has the increase effect to the phagocytic rate and the phagocytic index of intraperitoneal mouse scavenger cell.
Claims (7)
1. cockroach polypeptide that from cockroach, extracts, it is characterized in that described cockroach polypeptide is to adopt acetate to soak the cockroach powder 24-72 hour as solvent in the room temperature sealing, extracting solution after the immersion is heated into suspension in 80-100 ℃ of water-bath, the active ingredient that obtains behind this suspension is handled in freeze-drying.
2. method of extracting cockroach polypeptide from cockroach, described method comprises the following steps:
(1) in an encloses container, with the cockroach powder in soaking at room temperature in acetic acid solution, sealing was soaked 24-72 hour;
(2) remove by filter filter residue, the filtrate that obtains is placed 80-100 ℃ of water-bath, heating makes the outstanding liquid that mixes;
(3) suspension is handled through freeze-drying, makes the cockroach polypeptide powder.
3. the method for extraction cockroach polypeptide as claimed in claim 2 is characterized in that described acetic acid concentration is in the 0.05-0.2M scope.
4. the method for extraction cockroach polypeptide as claimed in claim 2 is characterized in that described cockroach powder size is the 80-100 order.
5. the method for extraction cockroach polypeptide as claimed in claim 2, the weight ratio that it is characterized in that described cockroach powder and acetate is 1: the 5-10 scope.
6. the method for extraction cockroach polypeptide as claimed in claim 2 is characterized in that described cockroach powder seals immersion 24-48 hour in room temperature in the 0.05-0.2M acetic acid solution.
7. the method for extraction cockroach polypeptide as claimed in claim 2 is characterized in that described extracting solution heated 0.5-1.0 hour in 80-100 ℃ of water-bath.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006056097A1 (en) * | 2004-11-23 | 2006-06-01 | Weichang You | A pharmaceutical composition for treating senile dementia |
| CN1331481C (en) * | 2004-07-08 | 2007-08-15 | 严少宏 | Method for extracting medicinal and edible substance from dictyoptera |
| CN100525781C (en) * | 2006-09-30 | 2009-08-12 | 耿福能 | American falx extract, medicinal composition containing it and its making method |
| CN107266532A (en) * | 2016-04-06 | 2017-10-20 | 暨南大学 | A kind of novel polypeptide PAP1 and its application |
| CN107693546A (en) * | 2016-12-16 | 2018-02-16 | 四川好医生攀西药业有限责任公司 | A kind of medicine containing American cockroach and Sorafenib and its application |
| CN109394804A (en) * | 2018-12-10 | 2019-03-01 | 曾付英 | A kind of Chinese medicine composition for the treatment of cancer disease |
| CN114249619A (en) * | 2022-01-20 | 2022-03-29 | 黑龙江省昊千生物科技有限公司 | Foliar application fertilizer for rapidly increasing selenium content of crops |
-
2001
- 2001-02-28 CN CN 01105504 patent/CN1199991C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1331481C (en) * | 2004-07-08 | 2007-08-15 | 严少宏 | Method for extracting medicinal and edible substance from dictyoptera |
| WO2006056097A1 (en) * | 2004-11-23 | 2006-06-01 | Weichang You | A pharmaceutical composition for treating senile dementia |
| CN100525781C (en) * | 2006-09-30 | 2009-08-12 | 耿福能 | American falx extract, medicinal composition containing it and its making method |
| CN107266532A (en) * | 2016-04-06 | 2017-10-20 | 暨南大学 | A kind of novel polypeptide PAP1 and its application |
| CN107693546A (en) * | 2016-12-16 | 2018-02-16 | 四川好医生攀西药业有限责任公司 | A kind of medicine containing American cockroach and Sorafenib and its application |
| CN109394804A (en) * | 2018-12-10 | 2019-03-01 | 曾付英 | A kind of Chinese medicine composition for the treatment of cancer disease |
| CN114249619A (en) * | 2022-01-20 | 2022-03-29 | 黑龙江省昊千生物科技有限公司 | Foliar application fertilizer for rapidly increasing selenium content of crops |
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| CN1199991C (en) | 2005-05-04 |
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