CN101108879A - Method for extracting active ingredient of natural leech essence - Google Patents
Method for extracting active ingredient of natural leech essence Download PDFInfo
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- CN101108879A CN101108879A CNA2007100660961A CN200710066096A CN101108879A CN 101108879 A CN101108879 A CN 101108879A CN A2007100660961 A CNA2007100660961 A CN A2007100660961A CN 200710066096 A CN200710066096 A CN 200710066096A CN 101108879 A CN101108879 A CN 101108879A
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Abstract
The invention discloses an extract method of the effective constituent of the Natural hirudin. The method is that the blood-sucking leech is washed cleanly by water and is added with the ethanol water with content of 15 per cent to 25 per cent to soak for 30h to 50h, and then is filtered to gain the filter liquor and dregs of a decoction; the Poecilobdella manillensis waste residue is broken into pieces and is added with aqueous acetone solution with content of 15 per cent to 25 per cent to soak, and then is filtered to gain the filter liquor; after recovering ethanol and acetone from the two-time filter liquor in the vacuum, the filter liquor is combined and frozen and dried in the vacuum conventionally to gain the product. The invention is simple in process, low in cost, higher in yield, good in product quality and long in retention period. The ''three wastes'' are not discharged in the whole production process, and the product can work as the raw material in such three fields as medicine, hairdressing and health food.
Description
Technical field
The present invention relates to a kind of extracting method of r-hirudin, particularly from the hirudinaria manillensis Hirudinaria manillensis, extract the method for active ingredient of natural leech essence.
Background technology
Leech is the general designation of Annelita on the animal classification (Annelida) Hirudinea (Hirrdinea), is commonly called as ant sulphur, and English original text is [Leech].Known in the world have 500 approximately surplus kind, divide 4 orders.Have 2 orders, 8 sections, 33 genus in China, have nearly 100 kinds more than.The kind class of wherein sucking mammalian has the doctor leech of doctor leech section (Hirudinidae) to belong to the Haemadipsa (Haemadipsa) of (Hirudo) and Poecilobdella (Poecilobdeaal) and mountain leech section (Haemadipsidae).
And modern the hirudinaria manillensis The Chemical Constituents is started from the fifties latter stage in last century.Arrived the mid-80, along with Biomedical Development, the foreign scholar has carried out amino acid sequencing and molecular cloning to hirudinaria manillensis, makes the research of hirudinaria manillensis start climax in the world.The modern study proof contains polypeptide active components such as the most effective anticoagulative substance natural hirudin in hirudinaria manillensis Hirudinaria manillensis salivary gland secretion, and the content height.Can be divided into two classes: a class is the composition that directly acts on blood coagulation system, comprises thrombin inhibitors, and other suppresses the material of blood coagulation.Another kind of is other proteinase inhibitor and other activeconstituents.
On the south China the Changjiang river particularly area such as Guangdong and Guangxi Provinces, Hainan abundant Hirudinaria manillensis resource is arranged.But owing to domestic extraction, Separation Research to polypeptide class active substances such as natural hirudins exists very big difficulty for a long time, mostly be to get head after leech is put to death to add the chemical industry solvent extraction or wholely smash into behind the muddy that solubilizing agent extracts to pieces or live body normal saline extraction or living body freezing melt and dissolved back normal saline extraction or extract with extruding blood plasma the leech of mechanical means after inhaling full blood, the separation through complexity obtains the r-hirudin crude product then.These methods exist that extract yield is low, at the bottom of the purity, complex process, long, the high excessively drawback of cost of cycle.Therefore, the natural hirudin price of selling on the present domestic and international market is very expensive.Even have the personation of eurysome golden thread leech crude extract to sell for r-hirudin, cause great confusion for the utilization of leech medicine.
We retrieve some technical schemes the extraction open source literature of relevant r-hirudin, for example:
1, China produces the separation and the purifying of Hirudo japonica r-hirudin, Lee Yang Tong builds the autumn, the hydrobiont journal .1997 of Inst. of Hydrobiology, Chinese Academy of Sciences's [periodical name], 21 (2) .[digests] adopt ion-exchange chromatography the salivary gland secretion that extracts from my home-made Hirudo japonica live body to be separated and purifying with the gel filtration chromatography method, obtained more purified r-hirudin.
2, Chinese patent, method＜application number with affinity chromatography purification r-hirudin〉95117701＜applicant consortium as a juridical person Korean Institute of Science and Technology of Dongkook Pharmaceutical Co., Ltd＜＜digest the invention discloses a kind of from the solution that contains r-hirudin and other material the method for purification r-hirudin, it is characterized in that this method comprises the step that described solution is handled through the metal ion affinity chromatography, wherein cupric ion (Cu
2+) as metal ion, phosphate buffer is as elutriant.＜claim〉a kind of from the solution that contains r-hirudin and other material the method for purification r-hirudin, it is characterized in that described method comprises the step that described solution is handled through the metal ion affinity chromatography, wherein said metal ion is cupric ion (Cu
2+) and with phosphate buffer as elutriant.
3, Chinese patent, the extracting method of medical natural hirudiu,＜application number〉92107076＜applicant〉Hu Weiming＜address〉Changsha, Hunan chromium salt factory catalyzer subsidiary factory＜digest〉extracting method of medical natural hirudiu, relate to the extraction of animal effective constituent.The present invention adopts and extract r-hirudin from whole leech, earlier the leech slurry is heated back adjusting pH value to 4.4-4.6, re-adjustment pH value lower the temperature again to 6-7, centrifuge dehydration then, residue physiological saline repetitive scrubbing, mother liquor dialysis back is used water dissolution with 95% above ethanol precipitation after the precipitation vacuum-drying, dialysis once more, last vacuum-drying obtains elaboration.The r-hirudin processing method of extracting with the present invention is easy, and final product quality is qualified, and yield brings up to 4/1000ths, thereby greatly reduces product cost.＜claim〉a kind of extracting method of medical natural hirudiu, use the physiological saline lixiviate, the invention is characterized in: after (1) cleans impurity such as removing earth with leech, integral body is smashed into muddy to pieces, (2) the leech slurry is heated to 60-80 ℃, and keeps 2-3 minute, and the Glacial acetic acid of adding 10% is adjusted the pH value at 4.4-4.6 in (3) leech slurry after heating, be cooled to 25-30 ℃ immediately, add 10%Na again
2CO
3Adjust the pH value at 6-7, (4) the leech slurry is through centrifugal collection mother liquor, the physiological saline lixiviate successively of residue adding 0.9% 2-3 time, collect whole vat liquors, (5) the mother liquor dialysis is 36 hours, changed water once every 12 hours, (6) dialyzate is with 95% above ethanol sedimentation, and adding 95% ethanol earlier, to make its content be 61%, placed 12 hours, supernatant liquor inclines, it is 85% that the ethanol that adds same concentration again makes its content, places 24 hours, obtains the r-hirudin precipitation, (7) will precipitate cryodrying and get raw product, (8) raw product is with an amount of second distillation water dissolution.
4, Chinese patent, [application (patent) number] 03113566.8[title] cultural method of Hirudinaria manillensis and the extractive technique of r-hirudin [application (patent right) people] Zhou Weihai [address] Dongge Road,, Nanning City, Guangxi Zhuang Autonomous Region extended line circumference No. 10 building, No. 1 circumference apartment, road 3 single 605[summaries] the invention provides a kind of cultural method of Hirudinaria manillensis and the extracting method of r-hirudin, be by introducing fine provenance or the open-air Hirudinaria manillensis that captures stalwartness, carry out artificial hierarchical segmented taming and dociling, feed, carry out artificial propagation, wait to raise to a certain degree, adopt chemical substance to stimulate fresh and alive Hirudinaria manillensis to make it secrete saliva, then the further separating and filtering of its saliva is obtained the r-hirudin crude product, again the r-hirudin crude product is carried out deep processing and obtain meeting medical r-hirudin, this Hirudinaria manillensis contains anticoagulative substance than other kind Hirudinaria manillensis content height, medicinal effect is good, it is simple to extract the r-hirudin method, after extracting r-hirudin Hirudinaria manillensis is put back to the pond breed, be separated by one the week or can extract again again in 10 days, every fresh and alive Hirudinaria manillensis can extract 6-7 time repeatedly, can extract about r-hirudin crude product 6-7 milliliter at every turn, good product quality, economize on resources again, improved the utility value of Hirudinaria manillensis.The cultural method of claims 1, a kind of Hirudinaria manillensis and the extractive technique of r-hirudin, it is characterized in that: capture healthy and strong Hirudinaria manillensis, be placed on water body clean water pond or pond by introduction fine provenance or field, artificial hierarchical segmented domestication, feed, keep suitable temperature, carry out artificial propagation, wait to raise to a certain degree, adopt inductive substance to stimulate fresh and alive Hirudinaria manillensis to make it secrete saliva, just can obtain natural hirudin to its saliva through further purifies and separates then, again the r-hirudin crude product be carried out deep processing and obtain meeting medical r-hirudin.
5, life science institute of the bright Hu Qiangqian Min [mechanism] of the extraction of [autograph] natural hirudin and purifying [author] Liu Chen Xi East China Normal University, [periodical name] East China Normal University's journal: natural science edition .2004 (2) .-104-106[digest] r-hirudin is a kind of Acid polypeptide class material that is present in the leech saliva, be the strongest in the world up to now thrombin inhibitors. in various thrombotic diseases of treatment and disseminated inravascular coagulation, excellent prevention and result of treatment are arranged all, and be free from side effects, thereby it more has superiority than heparin.Studies show that r-hirudin can also prevent the transfer of tumour cell, with r-hirudin combined with chemotherapy and radiotherapy, can promote the blood flow in the tumour and heighten the effect of a treatment, thereby have broad potential applicability in clinical practice.
6, the Zhongshan Medical College biology teaching and research room of chemical environment [author] Tan Enguang [mechanism] Zhongshan University of [autograph] Guangdong Hirudinaria manillensis life water body, [periodical name] herbal medicine .2005,36 (10) .-1561-1563[digests] leeches is commonly called as leech, is traditional Chinese medicine.Compendium of Material Medica has been done record more comprehensively to pharmacology, curative effect, the using method of leech.The modern pharmacology experiment shows that the leech injection liquid can make tumour cell necrosis, disappearance, has enhancement to reticuloendothelial cell.Compare with the nonbloodsucking leeches, the Hirudinaria manillensis sialisterium of sucking blood contains more rich resisting blood coagulation r-hirudin, and important value is pharmaceutically being arranged.The relevant leeches propagating and breeding research of sucking blood,
7, [autograph] two kinds of novel antithrombotic reagent-r-hirudins and HIRULOG [author] king are in outstanding Chen Jing spring [mechanism] Beijing University of Chemical Technology, [periodical name] scientific and technological information exploitation and economic .2000,10 (5) .-60-61[digests] r-hirudin is the strongest natural thrombin inhibitors of finding so far, HIRULOG is the interaction mechanism of imitation r-hirudin and zymoplasm and synthetic peptides, this paper has introduced their mechanism of action and clinical study thereof, narrated outside superior that they compare with heparin, thought that r-hirudin and HIRULOG are the very promising new anti-embolism medicines of two classes.
8, the Journal of Natural Science of Hunan Normal University .1998 of the purifying of [autograph] anticoagulation medicine r-hirudin and department of biology of evaluation [author] Huang Renhuai Shu Qin [mechanism] Hunan Normal University [periodical name], 21 (1) .-56-59, the 65[digest] produce Hirudo japonica crop Digestive system as raw material with the Hunan, with EDTA-cellulose chromatography single step purification, can obtain r-hirudin in batches through the Tricholroacetic Acid post precipitation after the reversed-phase HPLC desalination.R-hirudin to purifying has carried out the SDS-polyacrylamide gel electrophoresis, mass spectrum, N end sequence analysis and antithrombin activity analysis.
9, Chinese patent, application number: 200610019531.0 applyings date: 2006.06.29 title: a kind of is the method and application application (patent right) people: Zhou Weihai of feedstock production r-hirudin with the hirudinaria manillensis, address: mansion Room 2013,38-2 Tai'an, Minzu Road ,Nanning City ,Guangxi Zhuang Autonomousnationality District, summary: the invention discloses a kind of is the method and the application of feedstock production r-hirudin with the hirudinaria manillensis, this preparation method is characterised in that and comprises hirudinaria manillensis slurrying, preparation suspension liquid and dry three steps, resulting powder, it has preserved topmost effective active composition one natural hirudin of hirudinaria manillensis.Characteristics such as the present invention has that technology is simple, production cost is lower, the product long preservative period, and can adopt Markwardt zymoplasm direct titrimetric method to detect natural hirudin content, thereby makes steady quality, and the product curative effect is guaranteed.R-hirudin has wide range of applications, can make capsule, soft capsule, granule, tablet use separately by the method for routine, or make compound preparation with other animal and plant medicine, be used to prevent and treat cardiovascular and cerebrovascular diseases such as cerebral apoplexy, hypertension, hyperlipidaemia, coronary heart disease.
10, Chinese patent, animal watery blood leech hydrolysis method and Hirudohydrolysis blood thereof,＜application number〉95105650＜applicant〉Hu Yuanhua Chang Jianbo＜address〉Hubei Province's institute for drug control＜digest〉animal watery blood leech hydrolysis method and Hirudohydrolysis blood thereof, it is the new method of utilizing about animal blood, make blood-eating hirudo fully draw animal blood, with physics or chemical process blood is flowed out in the leech body after several minutes to a few hours, collect effusive orange red blood body.The Hirudohydrolysis blood that this method obtains have contain in the animal blood of small molecular weight protein or peptide, also contain the Taka-proteinase (former), Unidasa of r-hirudin, digestion blood and other and be difficult to the useful leech secretory product measured at present.Can effectively degrade animal blood and make it to contain the leech secretory product such as r-hirudin, Unidasa of medical value of this law, leech also can repeatedly be used, and improves its utilization ratio.＜claim〉animal watery blood leech hydrolysis method, it is characterized in that making blood-eating hirudo fully to draw animal blood, with physics or chemical process blood is flowed out in the leech body after several minutes to a few hours, collect effusive orange red blood body.
11, Chinese patent, application number: 200410037695.7 applyings date: 2004.04.29 title: a kind of r-hirudin freeze-dried powder and preparation method thereof application (patent right) people: address, Qianluchun Science and Technology Co., Ltd., Beijing: 1 1801 Room, No. 1 building, Lotus Residential Quarter, Haidian District, Beijing City, invention (design) people: Ni Dashi; Zhang Zhengsheng summary: the invention discloses a kind of r-hirudin freeze-dried powder and preparation method thereof, it is characterized in that extracting r-hirudin, add behind the purifying or do not add medicinal auxiliary material, be prepared into the r-hirudin freeze-dried powder from bright leech; Its feature is that also this preparation method does not adopt any organic solvent, adopt method purifying such as ultrafiltration, ion-exchange chromatography, reverse osmosis concentration, obtain the higher r-hirudin of purity, add or do not add medicinal auxiliary material and be prepared into lyophilized injectable powder, The pharmacological results shows that lyophilized injectable powder of the present invention has great pharmacological effects.
12, application number: 200510122784.6 applyings date: 2005.12.02 title: Iirudo-seu-whitmania essence inhalant and preparation method thereof application (patent right) people: Nanjing University of Traditional Chinese Medicine, address: No. 282, Hanzhong Road, Nanjing City, Jiangsu Province, summary: the invention discloses a kind of Iirudo-seu-whitmania essence inhalant and preparation method thereof, this inhalation adds the mixture micro mist that suitable auxiliary material is made by the pure product dry powder of r-hirudin or by Iirudo-seu-whitmania essence, its grain size of micropowder 0.1-1000 micron, the weight ratio scope of r-hirudin and auxiliary material is 1: 0-1: 000.The preparation method of Iirudo-seu-whitmania essence inhalant after at first r-hirudin or r-hirudin being added suitable auxiliary material, makes the micro mist of particle diameter at the 0.1-100 micron by pulverizing, spraying drying, crystallization or polymerization process; Secondly combine with the micro mist reunion or with granules of accessories and make the re-dispersable particles of particle diameter between the 1-1000 micron, at last micro mist or granule packaging are gone in capsule, bubble-cap or the Diskus.When using, this Foradil Aerolizer formoterol fumarate sucks by respiratory tract.Lung absorbs, and it has easy to use, easy preservation, advantage that bioavailability is high.
13, Chinese patent, application number: 03140487.1 applying date: 2003.09.12 title: Hirudo extract cosmetics and preparation method thereof application (patent right) people: Zhou Weihai, address: Xinzu Road ,Nanning City ,Guangxi Zhuang Autonomousnationality District 33-1 number 4 Unit 1 201 invention (design) people: Zhou Weihai, summary: the invention discloses a kind of cosmetics, it comprises Hirudo extract, grease, fatty acid ester, organic solvent, spices, nutrient substance and Purified Water, the preparation method is a certain proportion of oils, fatty acid ester, after organic solvent and an amount of Purified Water mix, be heated to 60-90 ℃, make its fusion, add Hirudo extract and an amount of essence when treating its cool to room temperature again, fully stir then and make it fully evenly to obtain product, cosmetics of the present invention have removing beverage, anti-acne, skin moisten, tender skin, whiten, the prevention skin aging, strengthen the effect of skin elasticity, for efficiently, green cosmetics.
14, Chinese patent, application number: 200510036509.2 applyings date: 2005.08.16 title: face-beautifying skin-caring lotion with leech extract and preparation method thereof application (patent right) people: week is tieed up location, Haiti: No. 13 invention of 38-2 Tai'an, Minzu Road ,Nanning City ,Guangxi Zhuang Autonomousnationality District mansion 18 floor (design) people: Zhou Weihai; Zhou Weiguan, summary: the invention discloses a kind of skincare liquid, it comprises Hirudo extract, auxiliary material and clean water, the preparation method is the Hirudo extract of certain live vol and the complete the finished product of the present invention that promptly obtain soluble in water of auxiliary material, it has two characteristics: the one, and autonomous infiltration, skin absorption fast and anti-inflammatory and detumescence all have unique effect to prevention skin aging, elimination color spot, acne and new and old scar; Two are to use conveniently, are more suitable for using when hot season, more suitable to the crowd of oily skin.
Recognize that from the above-mentioned documents and materials that retrieve the method that r-hirudin is extracted in present domestic research is leech to be rubbed or dry back water, ethanol, ultrafiltration or chromatography extraction.The r-hirudin extracting method that document 4 and document 10 are mentioned is to make blood-eating hirudo fully draw animal blood, with physics or chemical process (inductive substance) blood is flowed out in the leech body after several minutes to a few hours, collect effusive orange red blood body, be further purified separation and just can obtain natural hirudin.But aforesaid method also has a lot of weak points, the effective active composition is not high after main the extraction, the foreign matter content height, be not suitable for injecting drug use, the method leaching process that has is slower, the method that has is not suitable for suitability for industrialized production, so at present the extracting method of r-hirudin also has a lot of people in research, tries hard to find efficient height, cost is low, activeconstituents is high extracting method.
Summary of the invention
The purpose of this invention is to provide that a kind of method is simple, cost is low, foreign matter content is few, good product quality, extract the back obtain activeconstituents high extract the method for r-hirudin from hirudinaria manillensis.
Method of the present invention is achieved in that
The method of extracting r-hirudin from Hirudinaria manillensis is the Hirudinaria manillensis live body of at first will sucking blood, water (pure water or distilled water) cleans up the back and adds by leech total mass content 2-5 doubly, the aqueous ethanolic solution of mass content 15～25% (can with the high concentration ethanol dilute with water) soaked after 30-50 hour, filter, get the filtrate 1 and the dregs of a decoction; The Hirudinaria manillensis dregs of a decoction are rubbed, add by dregs of a decoction mass content 3-4 again and doubly measure, mass content is that 15～25% aqueous acetone solution (can with high density acetone dilute with water) soaks, and soaks after 30-50 hour, filters, must filtrate 2.After respectively filtrate 1 and filtrate 2 vacuum being reclaimed ethanol and acetone, merging filtrate, conventional vacuum lyophilization obtains meeting pharmaceutical natural hirudin (being the Hirudinaria manillensis element) polypeptides freeze-dry powder extracting substance (product).
The above-mentioned principle of extracting r-hirudin from Hirudinaria manillensis is at first to use alcohol immersion leech live body, the effective constituent that is dissolved in low-concentration ethanol can be extracted earlier, then filter residue is rubbed the extracts active ingredients that is dissolved in acetone again, just can obtain all activeconstituentss substantially, product purity is higher, foreign matter content is low, meets medical.
Compared with prior art, outstanding substantive distinguishing features of the present invention and obvious improvement are:
1. technology is simple, with low cost; Reduce the input of equipment, helped production management; Yield is higher, good product quality, long preservative period, and whole process of production does not have " three wastes " discharging.
2. adopt the antithrombin activity analytical method to carry out quality monitoring, show the specific activity height of product.Therefrom separate purification again after extruding blood plasma the leech of method employing mechanical means after inhaling full blood with the document introduction, the result is than living about 2AT-U/mg, reach more than the 5AT-U/mg and ratio of the present invention is alive, foreign matter content is low, can be used to prepare oral pharmaceutical, also can be used to prepare injectable drug.
3. with existing domestic and international correlation technique relatively, technology advanced person is applicable to suitability for industrialized production.
4. need not high performance liquid chromatography or reversed phase chromatography, preserve its effective active composition to greatest extent.
5. product has the huge scientific research and the prospect of marketing as the up-to-date raw material in medicine, beauty treatment, protective foods three big fields.
Embodiment
Embodiment 1
Earlier with 10 kilograms of Hirudinaria manillensis live bodies of sucking blood, after cleaning up aqueous ethanolic solution (medical alcohol thin up) that the back adds 20 kilograms of content 20% by the leech total mass and soak 50 hours with pure water, filter, the filtrate 1 and the dregs of a decoction; The Hirudinaria manillensis dregs of a decoction are rubbed, and the aqueous acetone solution (can with the pure acetone dilute with water) that adds 3 times of amounts 20% by dregs of a decoction amount soaks again, after 35 hours, filter, filtrate 2; After respectively filtrate 1 and filtrate 2 vacuum being reclaimed ethanol and acetone, merging filtrate, conventional vacuum lyophilization obtains meeting the plain polypeptides freeze-dry powder extracting substance of pharmaceutical Hirudinaria manillensis.
Embodiment 2
Earlier with 10 kilograms of Hirudinaria manillensis live bodies of sucking blood, clean up alcohol immersion that the back adds 30 kilograms of content 25% by the leech total mass after 30 hours with distilled water, filter, the filtrate 1 and the dregs of a decoction; The Hirudinaria manillensis dregs of a decoction are rubbed, add the aqueous acetone solution immersion of 4 times of amount purity 25% again by dregs of a decoction amount, after 30 hours, filter, get filtrate 2; After respectively filtrate 1 and filtrate 2 vacuum being reclaimed ethanol and acetone, merging filtrate, conventional vacuum lyophilization obtains meeting the plain polypeptides freeze-dry powder extracting substance of pharmaceutical Hirudinaria manillensis.
Embodiment 3
Earlier with 10 kilograms of Hirudinaria manillensis live bodies of sucking blood, after cleaning up aqueous ethanolic solution that the back adds 28 kilograms of content 15% by the leech total mass and soak 40 hours with pure water, filter, the filtrate 1 and the dregs of a decoction; The Hirudinaria manillensis dregs of a decoction are rubbed, add the aqueous acetone solution immersion of 3 times of amount purity 25% again by dregs of a decoction amount, after 38 hours, filter, get filtrate 2; After respectively filtrate 1 and filtrate 2 vacuum being reclaimed ethanol and acetone, merging filtrate, conventional vacuum lyophilization obtains meeting pharmaceutical natural hirudin (being the Hirudinaria manillensis element) polypeptides freeze-dry powder extracting substance.
Embodiment 4
Earlier with 10 kilograms of Hirudinaria manillensis live bodies of sucking blood, clean up alcohol immersion that the back adds 20 kilograms of content 25% by the leech total mass after 35 hours with pure water, filter, the filtrate 1 and the dregs of a decoction; The Hirudinaria manillensis dregs of a decoction are rubbed, add the aqueous acetone solution immersion of 4 times of amount purity 20% again by dregs of a decoction amount, after 40 hours, filter, get filtrate 2; After respectively filtrate 1 and filtrate 2 vacuum being reclaimed ethanol and acetone, merging filtrate, conventional vacuum lyophilization obtains meeting the plain polypeptides freeze-dry powder extracting substance of pharmaceutical Hirudinaria manillensis.
Embodiment 5
Earlier with 10 kilograms of Hirudinaria manillensis live bodies of sucking blood, after cleaning up aqueous ethanolic solution that the back adds 35 kilograms of ethanol contents 20% by the leech total mass and soak 40 hours with distilled water, filter, the filtrate 1 and the dregs of a decoction; The Hirudinaria manillensis dregs of a decoction are rubbed, add the aqueous acetone solution immersion of 3.5 times of amount purity 25% again by dregs of a decoction amount, after 45 hours, filter, get filtrate 2; After respectively filtrate 1 and filtrate 2 vacuum being reclaimed ethanol and acetone, merging filtrate, conventional vacuum lyophilization obtains meeting the plain polypeptides freeze-dry powder extracting substance of pharmaceutical Hirudinaria manillensis.
Embodiment 6
Earlier with 10 kilograms of Hirudinaria manillensis live bodies of sucking blood, after cleaning up aqueous ethanolic solution that the back adds 50 kilograms of content 15% by the leech total mass and soak 45 hours with pure water, filter, the filtrate 1 and the dregs of a decoction; The Hirudinaria manillensis dregs of a decoction are rubbed, add the aqueous acetone solution of 4 times of amount purity more than 20% by dregs of a decoction amount again and soak, after 50 hours, filter, get filtrate 2; After respectively filtrate 1 and filtrate 2 vacuum being reclaimed ethanol and acetone, merging filtrate, conventional vacuum lyophilization obtains meeting the plain polypeptides freeze-dry powder extracting substance of pharmaceutical Hirudinaria manillensis.
The product that obtains for the present invention relatively and the activity of the mechanical extrusion method of documents, we have provided following form (table 1):
See that from table 1 product that the present invention obtains is than the specific activity height of mechanical extrusion method, the shelf time is long.
| Project | Yield | Preservation period | Specific activity | |||||||||
| Yield for the first time | Yield for the second time | Yield for the third time | Three average yields | Extract for the first time preservation period | Extracting for the second time preservation period protects | Extract preservation period for the third time | Extract average preservation period three times | Extract for the first time specific activity | Extract for the second time specific activity | Extract specific activity for the third time | Extract the average specific activity three times | |
| The present invention | 3.2％ | 3.0％ | 3.4％ | 3.2％ | 3.5 year | 3.5 year | 3.5 year | 3.5 year | 4AT-U /mg | 6AT-U /mg | 5AT-U /mg | 5AT-U /mg |
| Therefrom separate purification again after extruding blood plasma the leech of mechanical means after inhaling full blood | 1.2％ | 1.3％ | 1％ | 1.2％ | 2.5 year | 2.5 year | 2.5 year | 2.5 year | 1.5AT-U /mg | 2AT-U /mg | 2.5AT-U /mg | 2AT-U /mg |
Table 1, the product that the present invention obtains and mechanical extrusion method contrast situation
Claims (1)
1. the extracting method of an active ingredient of natural leech essence is characterized in that: water cleans up the back and adds by leech total mass content 2-5 doubly, and the aqueous ethanolic solution of content 15～25% soaked after 30-50 hour, filter, the filtrate 1 and the dregs of a decoction; The Hirudinaria manillensis dregs of a decoction are rubbed, add again that doubly to measure purity by dregs of a decoction mass content 3-4 be that 15～25% aqueous acetone solution soaks, after 30-50 hour, filter, get filtrate 2, respectively with behind filtrate 1 and filtrate 2 vacuum recovery ethanol and the acetone, merging filtrate, conventional vacuum lyophilization obtains product.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101974085A (en) * | 2010-11-01 | 2011-02-16 | 南宁市金贝生物技术有限责任公司 | Method for extracting high-anticoagulation-activity hirudin from natural leeches |
| CN102286098A (en) * | 2011-08-18 | 2011-12-21 | 周维官 | Production method of natural hirudin |
| CN102603881A (en) * | 2011-01-20 | 2012-07-25 | 中国中医科学院中药研究所 | Anticoagulant active oligopeptide, and extraction and purification of anticoagulant active oligopeptide compound in Whitmania pigra |
| CN105153274A (en) * | 2015-08-31 | 2015-12-16 | 南宁市金贝生物技术有限责任公司 | Method for removing heavy metal by electrodialysis method during hirudin extraction production |
| CN105412154A (en) * | 2015-12-02 | 2016-03-23 | 云南永安制药有限公司 | Preparing method for fresh lumbricus extract |
| CN106581075A (en) * | 2015-10-19 | 2017-04-26 | 云南云域瑞达生物药业有限公司 | Application of flos carthami or extract thereof for removing fishy smell of medicinal material |
| CN113073093A (en) * | 2021-04-25 | 2021-07-06 | 山东中医药大学 | Leech fibrinolytic active protein and preparation method thereof |
| CN113520898A (en) * | 2021-09-17 | 2021-10-22 | 汇泰渤海水产有限责任公司 | Application of hirulog HE-D in skin care and whitening |
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2007
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101974085A (en) * | 2010-11-01 | 2011-02-16 | 南宁市金贝生物技术有限责任公司 | Method for extracting high-anticoagulation-activity hirudin from natural leeches |
| CN101974085B (en) * | 2010-11-01 | 2013-09-25 | 南宁市金贝生物技术有限责任公司 | Method for extracting high-anticoagulation-activity hirudin from natural leeches |
| CN102603881A (en) * | 2011-01-20 | 2012-07-25 | 中国中医科学院中药研究所 | Anticoagulant active oligopeptide, and extraction and purification of anticoagulant active oligopeptide compound in Whitmania pigra |
| CN102286098A (en) * | 2011-08-18 | 2011-12-21 | 周维官 | Production method of natural hirudin |
| CN102286098B (en) * | 2011-08-18 | 2013-09-04 | 周维官 | Production method of natural hirudin |
| CN105153274A (en) * | 2015-08-31 | 2015-12-16 | 南宁市金贝生物技术有限责任公司 | Method for removing heavy metal by electrodialysis method during hirudin extraction production |
| CN106581075A (en) * | 2015-10-19 | 2017-04-26 | 云南云域瑞达生物药业有限公司 | Application of flos carthami or extract thereof for removing fishy smell of medicinal material |
| CN105412154A (en) * | 2015-12-02 | 2016-03-23 | 云南永安制药有限公司 | Preparing method for fresh lumbricus extract |
| CN105412154B (en) * | 2015-12-02 | 2018-11-09 | 云南永安制药有限公司 | A kind of preparation method of fresh Pheretima extract |
| CN113073093A (en) * | 2021-04-25 | 2021-07-06 | 山东中医药大学 | Leech fibrinolytic active protein and preparation method thereof |
| CN113520898A (en) * | 2021-09-17 | 2021-10-22 | 汇泰渤海水产有限责任公司 | Application of hirulog HE-D in skin care and whitening |
| CN113520898B (en) * | 2021-09-17 | 2021-12-21 | 汇泰渤海水产有限责任公司 | Application of hirulog HE-D in skin care and whitening |
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