CN110522769A - The extracting method and its application of aspongopus crude extract - Google Patents
The extracting method and its application of aspongopus crude extract Download PDFInfo
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- CN110522769A CN110522769A CN201910636772.7A CN201910636772A CN110522769A CN 110522769 A CN110522769 A CN 110522769A CN 201910636772 A CN201910636772 A CN 201910636772A CN 110522769 A CN110522769 A CN 110522769A
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- 241000660877 Coridius Species 0.000 title claims abstract description 51
- 239000000287 crude extract Substances 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 14
- 210000000087 hemolymph Anatomy 0.000 claims abstract description 17
- 239000000284 extract Substances 0.000 claims abstract description 11
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 claims abstract description 10
- 239000000137 peptide hydrolase inhibitor Substances 0.000 claims abstract description 10
- 206010006187 Breast cancer Diseases 0.000 claims description 9
- 208000026310 Breast neoplasm Diseases 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 210000001015 abdomen Anatomy 0.000 claims description 4
- 238000010257 thawing Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000007605 air drying Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 2
- 239000003560 cancer drug Substances 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 241000238631 Hexapoda Species 0.000 abstract description 7
- 239000004615 ingredient Substances 0.000 abstract description 6
- 238000009010 Bradford assay Methods 0.000 abstract description 2
- 239000003146 anticoagulant agent Substances 0.000 abstract description 2
- 229940127219 anticoagulant drug Drugs 0.000 abstract description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 206010028980 Neoplasm Diseases 0.000 description 9
- 239000003814 drug Substances 0.000 description 7
- 239000013641 positive control Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 210000004072 lung Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 206010027458 Metastases to lung Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000008901 benefit Effects 0.000 description 2
- 239000004519 grease Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 description 1
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 241000628925 Coridius chinensis Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000219112 Cucumis Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 241000663202 Dinidoridae Species 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 241000578422 Graphosoma lineatum Species 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229960004117 capecitabine Drugs 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229940126701 oral medication Drugs 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
- A61K35/64—Insects, e.g. bees, wasps or fleas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Insects & Arthropods (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Animal Husbandry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses the extracting methods and its application of a kind of aspongopus crude extract.The present invention does not use the anti-coagulants to play a role to haemocyte when extracting crude extract, main purpose is to keep to one of extract medicinal ingredient and (protease inhibitors is added), so being properly termed as the extract is aspongopus crude extract, rather than hemolymph.Another every 10g insect can at least extract crude extract 1ml, and amount is also more, measure protein sample concentration at least 70mg/ml additionally by Bradford method, protein content is higher.
Description
Technical field
The present invention relates to insect crude extract technical fields, and in particular to a kind of extracting method of aspongopus crude extract.
Background technique
Aspongopus Aspongopus chinensis (Dallas, 1851) is Semiptera Hemiptera Dinidoridae
Dinidoridae insect is commonly called as black pocket worm, the black stinkbug of melon, wind plate worm, insects, beats wind worm and wind bar worm etc., be distributed in Guizhou,
Anhui, Fujian, Guangdong, Jiangsu, Jiangxi, Sichuan, Shanghai, Yunnan, Tibet, Taiwan, Vietnam, Laos, Indonesia, Japan, Burma,
The Malay Peninsula and India.Version Chinese Pharmacopoeia in 2015, aspongopus are written with medicinal material and medicine materical crude slice classification, have regulating qi-flowing for relieving pain, and middle benefit gas helps
Positive effect.
Chinese medicine aspongopus is the dry adult of aspongopus, and the beginning is recorded in Compendium of Material Medica, " originates in Guizhou Yongning and defend Chishui River
In ... it flies out after to the Waking of Insects (3rd solar term), it is unavailable.Smell: salty, temperature, it is nontoxic, diaphragm gastral cavity stagnant gas is cured mainly, spleen-kidney deficiency strengthens member
Sun ".Aspongopus compatibility recipe is used clinically for the treatment of a variety of diseases such as epigastric pain, endometriosis, is also used for controlling
Pain caused by cancer is treated, there is good therapeutic effect.
Summary of the invention
Technical problem to be solved by the invention is to provide the extracting method and its application of a kind of aspongopus crude extract, it
With fabulous recovery rate, and extract has breast cancer and significantly inhibits effect.
The present invention is implemented as follows: the extracting method of aspongopus crude extract, comprises the technical steps that:
1) acquisition and pretreatment of aspongopus: after aspongopus is cleaned up with clear water, -80 DEG C of refrigerators is placed in and are saved；
2) aspongopus crude extract extracts: taking out the aspongopus after frost be put into 4 DEG C of refrigerator defrostings first, thaw and complete
After remove cephalothorax, take abdomen carry out physical squeezing, the content squeezed out is placed in the centrifuge tube equipped with protease inhibitors
In, after content and protease inhibitors are mixed well, 10min is centrifuged at 12000 × g, 4 DEG C, after centrifugation, from
It is three layers after the heart, draws middle layer dark red solution and be placed in a new centrifuge tube, and protease inhibitors is added,
It takes dark red solution up to aspongopus crude extract in 10000 × g, 4 DEG C of centrifugation 5min, is stored in -80 DEG C of refrigerators.
It is after the aspongopus crude extract for obtaining step 2) is more than -80 DEG C of refrigerator pre-freeze 12h, nine after pre-freeze are fragrant
Worm hemolymph, which is put into vacuum freeze drier, to be freeze-dried, and aspongopus hemolymph freeze-dried powder is obtained.
The temperature of freeze-drying is -40~-30 DEG C, and drying time is 1-20h, after freeze-drying is at freeze-dried powder, is protected from light
Air drying saves.
Application of the aspongopus crude extract in preparation treatment breast cancer medicines.
Present inventor formerly has the application of one entitled " processing method and application of aspongopus ", with the Shen
It please compare, advantage of the invention essentially consists in the maximum activity ingredient for maintaining drug, and has found new internal antitumor work
Property, it was demonstrated that its intracorporal validity.Compared with the patent of earlier application, the present invention in terms of extracting insect extract just like
Lower difference does not use cotton to be absorbed and filter during physical squeezing, to guarantee mentioning to the maximum extent for medicinal ingredient
Take out, low-temperature centrifugation process, simplify centrifuging process, using the ultracentrifugation of single tube, and carry out different rotating speeds twice from
The heart is convenient for separated component.This step can also generate by-product, and aspongopus grease avoids destruction of the chemical mode to grease,
To carry out the research of next step.In the present invention, it is also contemplated that storage temperature and extract part, experiment show -80 DEG C
Medicinal ingredient can more obtain retaining to the greatest extent and abdomen is main medicinal ingredient place.
The present invention does not use the anti-coagulants to play a role to haemocyte when extracting crude extract, and main purpose is to mentioning
One of object medicinal ingredient is taken to keep and (protease inhibitors is added), it is possible to being known as the extract is aspongopus crude extract,
Rather than hemolymph.Another every 10g insect can at least extract crude extract 1ml, and amount is also more, measure additionally by Bradford method
Protein sample concentration at least 70mg/ml, protein content are higher.
Detailed description of the invention
Fig. 1 is flow chart of the invention；
Fig. 2 is mouse experiment tumor volume difference figure of the invention；
Fig. 3 is mouse experiment tumor weight disparity map of the invention；
Fig. 4 is the figure that mouse experiment lung of the invention shifts；
Fig. 5 is part knurl photo of the invention.
Specific embodiment
Embodiment 1: the extracting method of aspongopus crude extract comprises the technical steps that:
1) acquisition and pretreatment of aspongopus: aspongopus is cleaned up with clear water, is placed in -80 DEG C of refrigerators and is saved；
2) aspongopus hemolymph extracts: taking out the aspongopus after frost be put into 4 DEG C of refrigerator defrostings first, thaw and complete
After remove cephalothorax, take abdomen carry out physical squeezing, the content squeezed out is placed in the centrifuge tube equipped with protease inhibitors
In, after content and protease inhibitors are mixed well, 10min is centrifuged at 12000 × g, 4 DEG C, after centrifugation, from
It is divided into three layers after the heart, draws middle layer dark red solution and be placed in a new centrifuge tube, and protease inhibition is added
Agent takes dark red solution up to aspongopus hemolymph, i.e. aspongopus crude extract, saves in 10000 × g, 4 DEG C of centrifugation 5min
In -80 DEG C of refrigerators.
3) after the aspongopus crude extract for obtaining step 2) is more than -80 DEG C of refrigerator pre-freeze 12h, by nine after pre-freeze
Fragrant worm hemolymph, which is put into vacuum freeze drier, to be freeze-dried, and aspongopus hemolymph freeze-dried powder is obtained.
The temperature of freeze-drying is -35 DEG C, and using culture dish, with a thickness of 2mm, drying time is 4-5h, freeze-drying
After freeze-dried powder, it is protected from light air drying preservation.
Application of the aspongopus crude extract in preparation treatment breast cancer medicines.
In order to further verify effect of the invention.Applicant carried out following experiments:
Test one: the increment of breast cancer cell is influenced in vitro.Mouse mammary carcinoma cell line 4T1 and human milk are chosen first
Adenocarcinoma cell HCC1937 selects the gradient degerming drug of various concentration, handles two kinds of cell lines, uses mtt assay, CCK-8 method
With the absorbance value of all-wave length microplate reader Multiskan GO measurement 490nm, 450nm, cell viability is calculated.The results show that nine
Fragrant worm hemolymph can inhibit the cell Proliferation vigor of two kinds of breast cancer cells, and be in dosage, time dependence.
Test two: establishing 4T1 mammary gland of mouse carcinoma animal model, and knurl is long to start that medication is subcutaneously injected to a certain extent, if
Negative control injection PBS and positive control injection chemotherapeutic drugs Cisplatin are set, the variation of vernier caliper record knurl during treatment,
After treatment end, mouse is sacrificed, dissects knurl, weighing and measurement volume, the transfer case of the organs such as observation lung liver kidney.As a result
It has been shown that, compared with negative control group, aspongopus hemolymph group and positive controls slow down the speed of growth of knurl, and
Tumor weight and volume after treatment end have statistical difference；Compared with positive controls, aspongopus hemolymph group is in tumor
Body weight and volume no difference of science of statistics.Each group is without obvious organ metastasis.So this tests provable aspongopus hemolymph
It can inhibit the growth of internal breast cancer.Test three: establishing 4T1 mouse breast cancer model, and knurl is long to be started subcutaneously to a certain extent
Oral medication has deliberately delayed administration time compared to test two, it is expected to observe internal transfer case.Negative control is set
The oral distilled water of group and positive controls take orally chemotherapeutics capecitabine, the change of vernier caliper record knurl during treatment
Change, after treatment end, sacrifice mouse, dissects knurl, weighing and measurement volume, the transfer case of the organs such as observation lung liver kidney.Knot
Fruit shows, compared with negative control group, aspongopus hemolymph group and positive controls slow down the speed of growth of knurl, and
And tumor weight and the equal no difference of science of statistics of volume after treatment end.Compared with positive controls, aspongopus hemolymph group exists
Tumor weight and volume no difference of science of statistics.Observe that negative control group there are three mouse that Lung metastases occur, hence it is evident that tumor nodule
Be covered with entire lung, low dosage aspongopus hemolymph group has a mouse that Lung metastases occur, hence it is evident that tumor nodule compared to it is negative compared with
It is few.High dose aspongopus hemolymph group and positive controls do not observe Lung metastases.So this tests provable aspongopus blood
Lymph can inhibit the transfer of internal breast cancer middle and later periods.
Claims (4)
1. a kind of extracting method of aspongopus crude extract, which is characterized in that comprise the technical steps that:
1) acquisition and pretreatment of aspongopus: after aspongopus is cleaned up with clear water, -80 DEG C of refrigerators is placed in and are saved；
2) aspongopus crude extract extracts: taking out the aspongopus after frost be put into 4 DEG C of refrigerator defrostings first, remove after the completion of thawing
Cephalothorax takes abdomen to carry out physical squeezing, the content squeezed out is placed in the centrifuge tube equipped with protease inhibitors, by content
After object and protease inhibitors mix well, it is centrifuged 10min at 12000 × g, 4 DEG C, after centrifugation, is after centrifugation
It three layers, draws middle layer dark red solution and is placed in a new centrifuge tube, and protease inhibitors is added, in 10000 × g, 4
DEG C centrifugation 5min, take dark red solution up to aspongopus crude extract, be stored in -80 DEG C of refrigerators.
2. the extracting method of aspongopus crude extract according to claim 1, it is characterised in that: in obtain step 2) nine
After fragrant worm crude extract is more than -80 DEG C of refrigerator pre-freeze 12h, the aspongopus crude extract after pre-freeze is put into vacuum freeze drier
It is freeze-dried, obtains aspongopus hemolymph freeze-dried powder.
3. the extracting method of aspongopus crude extract according to claim 2, it is characterised in that: the temperature of freeze-drying be-
40~-30 DEG C, drying time is 1-20h, after freeze-drying is at freeze-dried powder, is protected from light air drying preservation.
4. a kind of aspongopus crude extract that extracting method according to claim 1 prepares treats breast cancer drug in preparation
Application in object.
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| CN201910636772.7A CN110522769A (en) | 2019-07-15 | 2019-07-15 | The extracting method and its application of aspongopus crude extract |
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| CN201910636772.7A Pending CN110522769A (en) | 2019-07-15 | 2019-07-15 | The extracting method and its application of aspongopus crude extract |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111285938A (en) * | 2020-03-02 | 2020-06-16 | 贵州大学 | Extraction method and application of aspongopus crude polysaccharide |
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|---|---|---|---|---|
| CN1559447A (en) * | 2004-02-18 | 2005-01-05 | 中国人民解放军军需大学 | Extracte of Aspongopus chinensis Dallas and Extraction method therefor medicinal use and preparn. thereof |
| CN102670660A (en) * | 2012-05-30 | 2012-09-19 | 贵州大学 | Processing method and application of aspongopus |
| CN108148110A (en) * | 2018-03-07 | 2018-06-12 | 贵州大学 | A kind of method of fast separating and purifying aspongopus antitumor protein components |
| CN108185107A (en) * | 2018-01-05 | 2018-06-22 | 佛山市聚成生化技术研发有限公司 | A kind of preparation method of aspongopus albumen powder and application |
-
2019
- 2019-07-15 CN CN201910636772.7A patent/CN110522769A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1559447A (en) * | 2004-02-18 | 2005-01-05 | 中国人民解放军军需大学 | Extracte of Aspongopus chinensis Dallas and Extraction method therefor medicinal use and preparn. thereof |
| CN102670660A (en) * | 2012-05-30 | 2012-09-19 | 贵州大学 | Processing method and application of aspongopus |
| CN108185107A (en) * | 2018-01-05 | 2018-06-22 | 佛山市聚成生化技术研发有限公司 | A kind of preparation method of aspongopus albumen powder and application |
| CN108148110A (en) * | 2018-03-07 | 2018-06-12 | 贵州大学 | A kind of method of fast separating and purifying aspongopus antitumor protein components |
Non-Patent Citations (1)
| Title |
|---|
| 杨佳琪: "CCK-8法检测九香虫血淋巴对人乳腺癌MCF-7细胞增殖的抑制作用", 《环境昆虫学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111285938A (en) * | 2020-03-02 | 2020-06-16 | 贵州大学 | Extraction method and application of aspongopus crude polysaccharide |
| CN111285938B (en) * | 2020-03-02 | 2021-07-06 | 贵州大学 | Extraction method and application of aspongopus crude polysaccharide |
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Application publication date: 20191203 |