CN101690733A - Leech enzymatic acetone powder enteric preparation and preparation method and application thereof - Google Patents
Leech enzymatic acetone powder enteric preparation and preparation method and application thereof Download PDFInfo
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- CN101690733A CN101690733A CN200910018600A CN200910018600A CN101690733A CN 101690733 A CN101690733 A CN 101690733A CN 200910018600 A CN200910018600 A CN 200910018600A CN 200910018600 A CN200910018600 A CN 200910018600A CN 101690733 A CN101690733 A CN 101690733A
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Abstract
The invention discloses a leech enzymatic acetone powder enteric preparation which takes dry leech whole bodies or fresh leeches as the raw materials and is obtained by the following preparation method: 1) taking leeches and homogenizing the leeches; 2) taking homogenate, regulating pH to 7-9, adding trypsase or complex enzyme with the trypsase as the main component according to the proportion of 2000-20000U enzyme to 1g of leech dry drug, controlling the temperature between 40 DEG C and 60 DEG C and carrying out enzymolysis for 2-12h; 3) after enzymolysis, cooling enzymolysis liquid to room temperature, adding acetone with volume 1-5 times that of the enzymolysis liquid into the enzymolysis liquid, precipitating for 4-12h by refrigerating, filtering and drying filtrate under reduced pressure to obtain the leech enzymatic acetone powder; and 4) adding pharmaceutic adjuvants into the leech enzymatic acetone powder to prepare the enteric preparation. The enteric preparation can be used as the drug for treating/preventing apoplexy.
Description
Technical field
The present invention relates to a kind of leech enzymatic acetone powder enteric agent and preparation method thereof and application.
Background technology
Apoplexy (apoplexy), be with fall in a swoon suddenly, syncope, companion distortion of commissure, dysphonia, hemiplegia, or, hemiplegia only askew with mouth without falling forward dusk is the disease of clinical primary symptom.Hurried because of falling ill, disease is seen multiterminal, and change of illness state is rapid, and to become characteristics similar to the benefaction number of wind, so an apoplexy, apoplexy.The primary disease M ﹠ M is higher, often leaves sequela; Sickness rate constantly increases in recent years, and age of onset also tends to rejuvenation, therefore, is to threaten human life and the great illness of quality of life.
Modern study shows that China's apoplexy mortality rate has leapt to the 2nd in the world, and on the rise.Apoplexy medical expense height, cure rate is low, and is very big to individual, family and social danger.Therefore prevention of stroke takes place bigger than treatment apoplexy meaning.This institute as Zhong Jing says: " go up worker's preventing diseases instead for the treatment of diseases.”
Hirudo (Leech) is that the property of medicine is gentle simply, and blood stasis dispelling power is hindered positive drug for invigorating blood circulation and eliminating stasis by force and not, has the various active composition.The Hirudo beginning is stated from Shennong's Herbal: " main by stagnant blood, blood stasis, the moon closes, and the removing blood stasis abdominal mass is gathered, loss of fecundity, dredging water passages." Compendium of Material Medica: " salty flavor acting on blood, the bitter blood that wins.The salty hardship of Hirudo to remove blood-retention, is a Liver Channel blood system medicine, so can lead to the poly-blood of Liver Channel.”
Modern times are used single Hirudo or compatibility compound recipe clinically, use dosage forms such as decoct, powder, capsule, and are evident in efficacy by drug treatment cardiovascular and cerebrovascular disease for oral administration.But traditional process of preparing Chinese medicine extraction, preparation technology's method make the part composition decompose and maybe can't extract, and cause loss of effective components, drug effect fluctuation, demand researching and developing stable and active high Hirudo preparation urgently to satisfy the modern medical service demand.
Summary of the invention
At above-mentioned prior art, the invention provides a kind of leech enzymatic acetone powder enteric agent, its good stability, active high, the loss of effective components of Hirudo is few, has farthest retained the medical value of Hirudo.The present invention also provides its preparation method, and discloses its application in the medicine of preparation treatment/prevention of stroke.
The present invention is achieved by the following technical solutions:
A kind of leech enzymatic acetone powder enteric agent is to be raw material with the dry all or fresh and alive Hirudo of Hirudo, obtains by following preparation method:
1) water intaking trematodiasis drying all is ground into 20-150 order powder, adds the water of 6-16 times of weight, homogenate;
Perhaps: the water intaking trematodiasis is fresh and alive all, adds the water of 1-6 times of weight, homogenate;
2) getting above-mentioned homogenate, regulate pH to 7-9, is the compound enzyme of main component in the ratio adding trypsin of the dried medical material adding of every 1g Hirudo 2000-20000U enzyme or with the trypsin, 40-60 ℃ of control temperature, enzymolysis 2-12 hour;
3) after enzymolysis finished, the cooling enzymolysis solution added the acetone of 1-5 times of volume to room temperature in enzymolysis solution, and cold preservation precipitation 4-12 hour is filtered, and the filtrate decompression drying gets leech enzymatic acetone powder;
4) in above-mentioned leech enzymatic acetone powder, add pharmaceutic adjuvant, be prepared into enteric coated preparation.
Described enteric coated preparation is any in enteric coated tablet, enteric coated capsule, the enteric coated drop pill.
Acetone substitutes with organic solvents such as ethanol, methanol or ethyl acetate in the described step (3).
Described step (4) is specially: after leech enzymatic acetone powder and pharmaceutic adjuvant are prepared into granule or tablet, re-use enteric material and carry out coating, obtain enteric coated preparation.
Described leech enzymatic acetone powder enteric agent can be used for preparing the medicine of treatment/prevention of stroke.
The present inventor is on the basis of Chinese scholars to Hirudo chemistry, pharmacology and clinical research in recent years, obtain higher degree anticoagulation HIRULOG (AHT) by extraction, separation and purification, and to its external free radical resisting Oxidation, preliminary study has been carried out in the effect of cerebral ischemia pharmacological effect in the body, and has applied for patent of invention: a kind of Hirudo extract and preparation method thereof and application (number of patent application: 200810138936.5).But in the later stage research process, find that this Hirudo extract active treatment activity reduces on the contrary with the raising of purity, and activity reduces significantly also after pepsin.Therefore just use trypsin treatment, make the leech enzymatic acetone powder enteric agent, studies show that by experiment leech enzymatic acetone powder is the highest in the prevention of stroke effect.
The specific embodiment
The present invention is further illustrated below in conjunction with embodiment:
Embodiment 1: the preparation of leech enzymatic acetone powder
Water intaking trematodiasis drying all is ground into 80 order powder, adds the water of 10 times of weight, homogenate; Get homogenate, regulate pH to 8.2, the ratio that adds the 8000U enzyme in the dried medical material of every 1g Hirudo adds trypsin, 50 ℃ of control temperature, and enzymolysis 5 hours is cooled to room temperature; After enzymolysis was intact, the acetone cold preservation that adds 4 times of volumes in above-mentioned enzymolysis solution precipitated 6 hours, filtered, and the filtrate decompression drying gets leech enzymatic acetone powder.
Embodiment 2: the capsular preparation of leech enzymatic acetone powder enteric
Behind water intaking trematodiasis enzymatic acetone powder, starch, the dextrin mix homogeneously, add an amount of concentration ethanol, make soft material, 18 mesh sieves are granulated, 40 ℃ of dryings, and 16 mesh sieve granulate, behind the adding magnesium stearate mix homogeneously, the enteric coated capsule of packing into promptly gets the leech enzymatic acetone powder enteric capsule.
Embodiment 3: the preparation of leech enzymatic acetone powder enteric tablet
Water intaking trematodiasis enzymatic acetone powder 100g behind 10g starch mix homogeneously, adopts 5% starch slurry to make soft material, and 18 mesh sieves are granulated, 40 ℃ of dryings, and 16 mesh sieve granulate, behind the adding Sodium Hydroxymethyl Stalcs mix homogeneously, tabletting.
Polyvinyl acetate phthalic acid ester, diethyl phthalate, Pulvis Talci are joined in proper amount of acetone/alcohol mixeding liquid, airtight, stirred 2 hours, then above-mentioned tablet is carried out coating, after coating is finished, in 40 ℃ of dryings 2 hours, the leech enzymatic acetone powder enteric tablet.
Experiment 1: the experiment of leech enzymatic acetone powder enteric capsule prevention of stroke
1.1 medicine and reagent
Be subjected to reagent: according to the leech enzymatic acetone powder of method preparation provided by the invention
Positive control drug: XUESAITONG ZHUSHEYE, specification: 100mg:2ml, Zhenbao Island, Heilungkiang pharmaceutical Co. Ltd, lot number: 20080702;
Reagent: 2,3,5-triphenyltetrazolium chloride (TTC), chloral hydrate, paraformaldehyde, heparin is sigma import packing;
Protein quantification (Coomassie brilliant blue method) test kit, the MDA test kit; The SOD test kit; GSH reagent;
ATPase (Na-K-ATPase; Na
+-Mg
2+-ATPase) enzyme reagent kit, the NO test kit; The LDH test kit;
Above test kit all builds up bio-engineering research institute available from Nanjing; Lot number is: 20090502;
The HE staining kit, the green skies produce.
1.2 animal
180 of Wistar rats, male and female half and half, body weight 250-280g is provided by Shandong University's Experimental Animal Center, quality certification numbering: SCXK (Shandong)-2003004.
1.3 experimental technique
1.3.1 animal grouping
Get 60 of Wistar rats, be divided into 6 groups at random, sham operated rats, model group, XUESAITONG 20mgkg
-1Group, leech enzymatic acetone powder 16mgkg
-1, 8mgkg
-1, 4mgkg
-1Group, 10 every group, male and female half and half.
1.3.2 animal modeling and administration
Test the 1st to 10d, all the other 5 groups of rats adopt compound modeling methods such as hunger, fatigue, cold-damp, alarmed melancholy are feared, high fat diet to make syndrome of blood stasis due to qi deficiency rat apoplexy model except that sham operated rats; Test 11d, all the other 5 groups of rats adopt bilateral ligation methods to make acute full property cerebral ischemic model except that sham operated rats, and wherein sham operated rats is only separated bilateral common carotid arteries and sew up wound but do not carried out ligation; All the other 5 groups of rats close bilateral common carotid arteries with miniature harmless bulldog clamp folder after separating bilateral common carotid arteries except that sham operated rats, unclamp bulldog clamp behind the 3h and make its tremulous pulse recover blood flow, sew up wound sterilization then.
Wherein the prophylactic treatment group is given XUESAITONG or leech enzymatic acetone powder intraperitoneal injection in modeling 9:00 every day, no longer administration behind the operation on neck.
1.3.3 brain tissue homogenate's liquid preparation
Rat is poured into 24h again, sacrificed by decapitation, on ice bath, take out cerebral cortex immediately, take by weighing the preceding cerebral tissue of 0.5g, placing the 4.5ml pre-cooling is 4 ℃ fully homogenate of normal saline, 4 ℃ of centrifugal 10min of 3000rpm, and supernatant is 10% brain tissue homogenate's liquid, supernatant is measured protein content with the Coomassie brilliant blue method, stores to be measured according to following surface condition after the packing.
1.3.4 the mensuration of superoxide dismutase (SOD) vigor
Produce ultra-oxygen anion free radical by xanthine and xanthine oxidase response system, latter's oxidation azanol forms nitrite, presents aubergine under the effect of developer, surveys its absorbance with visible spectrophotometer.When containing SOD in the sample, then ultra-oxygen anion free radical there is narrow spectrum inhibitory action, the nitrite of formation is reduced, and the absorbance of measuring pipe during colorimetric is lower than the absorbance of control tube, calculates the SOD vigor that can obtain in the sample by formula.
1.3.5 malonaldehyde (MDA) Determination on content
MDA in the lipid peroxide catabolite can with the thiobarbituricacid condensation, form red product, at the 532nm place maximum absorption band is arranged, can calculate MDA content by formula.
1.3.6 lactic acid dehydrogenase (LDH) Determination on content
LDH can generate acetone acid by catalysis lactic acid, and acetone acid and 2,4 dinitrophenyl hydrazine reaction generate the acetone acid dinitrophenylhydrazone, are brownish red in alkaline solution, and 440nm surveys its absorbance can calculate LDH content.
1.3.7 the mensuration of glutathion-peroxidase (GSH) vigor
GSH and the effect of dithio dinitrobenzoic acid generate 5-sulfo-dinitrobenzoic acid anion and present stable yellow, and 412nm surveys its absorbance can calculate GSH content.
1.3.8 nitric oxide (NO) Determination on content
NO metabolism in vivo is converted into NO very soon
2-And NO
3-, and NO2-further is converted into NO
3-, utilize the nitrate reductase enzyme spcificity with NO
3-Be reduced to NO
2-, measure its concentration at 550nm place absorbance value by colour developing.
1.4 experimental result
1.4.1 leech enzymatic acetone powder is to the influence of the syndrome of blood stasis due to qi deficiency rat apoplexy model SOD of rat cerebral tissue
As shown in table 1, compare with sham operated rats, the SOD content of model group obviously reduces, and difference has statistical significance (P＜0.01), and cerebral ischemia re-pouring hindbrain tissue SOD content significantly reduces.Compare with model group, each the liquid SOD of dosage group brain tissue homogenate content of leech enzymatic acetone powder all raises to some extent, and the content of wherein middle and high dosage group has significant difference (P＜0.05 and 0.01), is dose dependent antagonism SOD and reduces.
Table 1 leech enzymatic acetone powder is to the active influence of the syndrome of blood stasis due to qi deficiency rat apoplexy model SOD of rat cerebral tissue
Compare * P＜0.05, * * P＜0.01 with model group.Compare △ △ P＜0.01 with sham operated rats
1.4.2 leech enzymatic acetone powder is to the influence of the syndrome of blood stasis due to qi deficiency rat apoplexy model MDA of rat cerebral tissue
The result is as shown in table 2, model group MDA content is significantly higher than sham operated rats (P＜0.01), the cerebral tissue lipid peroxidation is strong after showing ischemia-reperfusion, reduction with gathering of cerebral tissue free radical and activities of antioxidant enzymes, the middle and high dosage group of leech enzymatic acetone powder can obviously reduce MDA content, and comparing difference with model group has significance (P＜0.05 and 0.01).
Table 2 leech enzymatic acetone powder is to the syndrome of blood stasis due to qi deficiency rat apoplexy model MDA of rat cerebral tissue content influence
Compare * P＜0.05, * * P＜0.01 with model group.Compare △ △ P＜0.01 with sham operated rats
1.4.3 leech enzymatic acetone powder is to the influence of the syndrome of blood stasis due to qi deficiency rat apoplexy model LDH of rat cerebral tissue
The result is as shown in table 3, model group LDH content is significantly higher than sham operated rats (P＜0.01), brain tissue cell damaged degree is serious after showing ischemia-reperfusion, the basic, normal, high dosage group of leech enzymatic acetone powder can obviously reduce LDH content, and comparing difference with model group has significance (P＜0.05 and 0.01).
Table 3 leech enzymatic acetone powder is to the syndrome of blood stasis due to qi deficiency rat apoplexy model LDH of rat cerebral tissue content influence
Compare * P＜0.05, * * P＜0.01 with model group.Compare △ △ P＜0.01 with sham operated rats
1.4.4 leech enzymatic acetone powder is to the influence of the syndrome of blood stasis due to qi deficiency rat apoplexy model GSH of rat cerebral tissue
The result is as shown in table 4, the GSH of model group is active significantly to descend, there were significant differences (P＜0.01) with sham operated rats, show that ischemia-reperfusion hindbrain tissue GSH activity is subjected to very big influence, the leech enzymatic acetone powder high dose group GSH content in the cerebral tissue that can obviously raise, comparing difference with model group has significance (P＜0.05);
Table 4 leech enzymatic acetone powder is to the syndrome of blood stasis due to qi deficiency rat apoplexy model GSH of rat cerebral tissue content influence
Compare * P＜0.05, * * P＜0.01 with model group.Compare △ △ P＜0.01 with sham operated rats
1.4.5 leech enzymatic acetone powder is to the influence of the syndrome of blood stasis due to qi deficiency rat apoplexy model NO of rat cerebral tissue
The result is as shown in table 5, compares model group rat cerebral tissue content of nitric oxide significantly raise (P＜0.01) with sham operated rats; Compare with model group, three dosage groups of leech enzymatic acetone powder and positive drug XUESAITONG all can significantly reduce the cerebral tissue nitric oxide.
Table 5 leech enzymatic acetone powder is to the syndrome of blood stasis due to qi deficiency rat apoplexy model NO of rat cerebral tissue content influence
Compare * P＜0.05, * * P＜0.01 with model group.Compare △ △ P＜0.01 with sham operated rats
Above result of study shows, model group cerebral tissue MDA, and LDH content obviously increases than sham operated rats, and SOD significantly descends, and content of nitric oxide raises, and each dosage group of leech enzymatic acetone powder has preventive effect preferably to syndrome of blood stasis due to qi deficiency rat apoplexy model.
Claims (6)
1. a leech enzymatic acetone powder enteric agent is characterized in that, is to be raw material with the dry all or fresh and alive Hirudo of Hirudo, obtains by following preparation method:
1) water intaking trematodiasis drying all is ground into 20-150 order powder, adds the water of 6-16 times of weight, homogenate;
Perhaps: the water intaking trematodiasis is fresh and alive all, adds the water of 1-6 times of weight, homogenate;
2) getting above-mentioned homogenate, regulate pH to 7-9, is the compound enzyme of main component in the ratio adding trypsin of the dried medical material adding of every 1g Hirudo 2000-20000U enzyme or with the trypsin, 40-60 ℃ of control temperature, enzymolysis 2-12 hour;
3) after enzymolysis finished, the cooling enzymolysis solution added the acetone of 1-5 times of volume to room temperature in enzymolysis solution, and cold preservation precipitation 4-12 hour is filtered, and the filtrate decompression drying gets leech enzymatic acetone powder;
4) in above-mentioned leech enzymatic acetone powder, add pharmaceutic adjuvant, be prepared into enteric coated preparation.
2. a kind of leech enzymatic acetone powder enteric agent according to claim 1 is characterized in that: described enteric coated preparation is any in enteric coated tablet, enteric coated capsule, the enteric coated drop pill.
3. the preparation method of the described leech enzymatic acetone powder enteric agent of claim 1 is characterized in that, may further comprise the steps:
1) water intaking trematodiasis drying all is ground into 20-150 order powder, adds the water of 6-16 times of weight, homogenate;
Perhaps: the water intaking trematodiasis is fresh and alive all, adds the water of 1-6 times of weight, homogenate;
2) getting above-mentioned homogenate, regulate pH to 7-9, is the compound enzyme of main component in the ratio adding trypsin of the dried medical material adding of every 1g Hirudo 2000-20000U enzyme or with the trypsin, 40-60 ℃ of control temperature, enzymolysis 2-12 hour;
3) after enzymolysis finished, the cooling enzymolysis solution added the acetone of 1-5 times of volume to room temperature in enzymolysis solution, and cold preservation precipitation 4-12 hour is filtered, and the filtrate decompression drying gets leech enzymatic acetone powder;
4) in above-mentioned leech enzymatic acetone powder, add pharmaceutic adjuvant, be prepared into enteric coated preparation.
4. preparation method according to claim 3 is characterized in that: acetone substitutes with ethanol, methanol or ethyl acetate in the described step (3).
5. preparation method according to claim 3 is characterized in that: described step (4) is specially: after leech enzymatic acetone powder and pharmaceutic adjuvant are prepared into granule or tablet, re-use enteric material and carry out coating, obtain enteric coated preparation.
6. the application of the described leech enzymatic acetone powder enteric agent of claim 1 in the medicine of preparation treatment/prevention of stroke.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017849A (en) * | 2014-05-16 | 2014-09-03 | 山东大学（威海） | Leech fibrinolytic protein, preparation method and application thereof |
| CN113583092A (en) * | 2021-09-27 | 2021-11-02 | 渤海水产股份有限公司 | Leech peptide and application and product thereof |
-
2009
- 2009-10-10 CN CN200910018600XA patent/CN101690733B/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017849A (en) * | 2014-05-16 | 2014-09-03 | 山东大学（威海） | Leech fibrinolytic protein, preparation method and application thereof |
| CN113583092A (en) * | 2021-09-27 | 2021-11-02 | 渤海水产股份有限公司 | Leech peptide and application and product thereof |
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