CN1065680A - Purifying method for agkistrodon acutus venin defibering enzyme - Google Patents
Purifying method for agkistrodon acutus venin defibering enzyme Download PDFInfo
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- CN1065680A CN1065680A CN 92102645 CN92102645A CN1065680A CN 1065680 A CN1065680 A CN 1065680A CN 92102645 CN92102645 CN 92102645 CN 92102645 A CN92102645 A CN 92102645A CN 1065680 A CN1065680 A CN 1065680A
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Abstract
The purification process of agkistrodon acutus venin defibering enzyme, it comprises that snake venom dissolving, the rough segmentation of column chromatography wash-out get the thrombin-like enzyme component, must make with extra care thrombin-like enzyme stoste through column chromatography wash-out purifying again, get defibrase stoste through desalination again, until making technologies such as injection defibrase finished product.This purification process is easy, is easy to grasp, and the productive rate height, the medicine source is abundant, low price.Having the effect of fine and molten fibre simultaneously by the made defibrase of this law, is a kind of new zymin of treatment vascular occlusive disease.Show that through clinical verification it has the curative effect height, short treating period, easy to use, safer characteristics.
Description
The invention belongs to a kind of method for preparing medicine, relate in particular to a kind of method that obtains high biological activity material-agkistrodon acutus venin defibering fibrillarin protoenzyme (abbreviation defibrase) with the ion-exchange chromatography separation and purification.
In the prior art as Taiwan Ouyang, (Thromb.Diath Haemorrth such as C., 26: 224 1971) with agkistrodon acutus poisons DEAE-Sephadex A-50 column chromatography for separation, get 12 protein peaks, peak 10 has thrombin-like enzyme (being called for short TLE) vigor, and it is got single TLE through twice purifying of Sephadex G-200 again, molecular weight is 33500.Ouyang is earlier with 800ml, 0.005M, PH8.0 ammonium acetate buffer wash-out, and then carry out salt concn and PH gradient elution with 1000ml, 0.25M, PH6.0 and 1000ml, 1.0M, PH5.4 ammonium acetate buffer, separable is 12 protein peaks, and peak 10 is for containing the TLE component.Then peak 10 is separated through Sephadex G-200 again, use the distilled water wash-out, repeat once to get the TLE of purifying again.By this method, its TLE yield is low, is about 0.5%.This operation simultaneously must be carried out under 5 ℃, i.e. operation at low temperatures.The main drawback of this method is the too low 5.4-6.0 of being of pH value, and TLE activity under this pH value is lower, even most loss of activity.Therefore, a peak 10 shows the TLE activity, and because ph value of buffer solution near the iso-electric point of TLE, both had been difficult to separate also being easy to precipitation, thereby most TLE does not separate and obtains or do not demonstrate its enzyme activity and abandoned.Another shortcoming is exactly must be in low temperature (5 ℃) operation down, and this not only increases equipment and cost is also made troubles to producers.This method also is not used for producing only in laboratory applications in addition.
Though abroad have by red mouthful of Pallas pit viper, spearhead Pallas pit viper and east water chestnut spot crotalin and produce TLE's, but the productive rate of its TLE is low, and China does not have this three kinds of snakes, promptly lack general medicine source, the said products Ancrod, Defibrase, Crotalus only are 70% to the curative effect of vascular occlusive disease simultaneously, and hematostaxis is about 5%.Solve problem, at first seek the new high source new drugs of the TLE amount that contains.Snake venom TLE is mainly from the Crotalinae snake venom.China Crotalinae poisonous snake is more has pallas pit viper, point kiss Pallas pit viper, green bamboo snake, flatiron first-class.Point kiss Pallas pit viper (Agkistrodon acutus) snake (Agkistrodon, Hundred-pace pit viper) main product is in China, be distributed in the Changjiang river and on the south the each province, according to incomplete statistics, at present more than annual desirable ahylysantinfarctase dried frozen aquatic products 4000 grams of China, and the part of just catching, actual reserve is quite abundant.China has more than the ahylysantinfarctase stock on hand 10kg at present.After testing, the TLE content in the ahylysantinfarctase is higher, accounts for 20% of full poison, and agkistrodon halyx pallas venom output is more, but TLE content is lower, and contains neurotoxin and be difficult for removing; Trimeresurus stejnegeri snake venom TLE content is higher, but output very little, does not go up 100 grams every year; Thereby ahylysantinfarctase is ideal TLE medicine source comparatively.Ahylysantinfarctase is the same with agkistrodon halyx pallas venom, and its hemorrhagin toxin content is also higher, thereby will obtain medicinal TLE, and its production technology (technology) promptly becomes vital problem.
Task of the present invention is to utilize new medicine source and a kind of productive rate height is provided, its product curative effect height, the purification process of the agkistrodon acutus venin defibering enzyme of few side effects.
The purification process of the said snake venom defibrase of the present invention, it comprises: (1) snake venom dissolves with damping fluid, and centrifugation gets the thrombin-like enzyme component through the anionite column chromatography with the buffer solution elution rough segmentation; (2) with gained thrombin-like enzyme component again respectively through the anionite column chromatography, purifying gets the purified thrombin-like enzyme; (3) will make with extra care thrombin-like enzyme stoste and get defibrase stoste through desalination again; (4), filtration, packing, freezing, dry, sealing by fusing, promptly get injection defibrase finished product with the former liquid formula of gained defibrase; It is characterized in that:
A, snake venom dissolve with the damping fluid of PH7.8-8.6, through the anionite column chromatography, get the thrombin-like enzyme component with the rough segmentation of PH7.8-8.6 damping fluid sodium-chlor straight line gradient elution;
B, with gained thrombin-like enzyme component in order or divide two groups through twice above column chromatography of anionite must make with extra care thrombin-like enzyme stoste with twice above wash-out purifying of PH7.8-8.6 damping fluid sodium-chlor straight line gradient;
Gained thrombin-like enzyme stoste is without lyophilize or concentrate and can directly go up sample in c, rough segmentation gained thrombin-like enzyme component and the repurity, and process operations is carried out in the aseptic or sterilisable chamber with second at 10 °～20 ℃ entirely.
The used damping fluid of dissolving snake venom is 0.05～0.10M Tris-HCl damping fluid or glycine-NaOH damping fluid, the used anionite of rough segmentation is DEAE-Sephadex, A-50 or QAE-Sephadex, A-25, the used sodium-chlor gradient of rough segmentation is 0～1.2M, purification process, it is characterized in that the used anionite of twice column chromatography of purifying is DEAE-Mierocrystalline cellulose DE52 or DE32 and DEAE-SephadexA-50 or QAE-Sephadex, A-25, the used sodium-chlor gradient of twice wash-out of purifying is 0.1～0.7M, 0.3～0.9M.Advantage of the present invention is:
1, this law yield is that yield is the highest in all snake venom TLE production methods, is 15～20%, external 6～8 times, is 30～40 times of OuyangC.Separated the obtaining of TLE70～100% in the thick poison, the result shows that this method advanced person is feasible.
2, in general column chromatography, when secondarily purified, its last time gained stoste need through frost drying or concentrate to go up sample.This rule does not need such step, but will need the repurity component solution directly to go up sample, 4 * 110cm
2Post can be gone up 200～300ml solution, and the effect no significant difference of sample is gone up in its separating effect and freeze-drying or concentrated back.Like this, promptly reduce reduction of erection time equipment while more than 3 times.
3, DEAE-Sephadex, DEAE-Cellulose, we adopt acid-alkali processing for the first time, secondary alkaline purification, three acid treatment the regeneration of Sephadex ... six alkali-acid treatment, circulate with this, this operates in beaker or the big Buchner funnel and carries out, and this method regeneration is complete, and prolong work-ing life, available 15～20 times or more, and generally can only be with 10 times less than promptly losing efficacy or separating not good.
4, gained finished product 0.05U can make 4mg/ml fibrinogen solution 0.3ml condense in 5 minutes; 0.5U/Kg body weight dosage can make the former content of rabbit fibrin reduce more than 50%; Big white mouse is subcutaneous hemorrhage spot not occur 1.0U be injected in.Show product low toxicity that this method produces, efficient thus, reliable in quality.
5, purification process of the present invention has improved the TLE productive rate of ahylysantinfarctase greatly, become a reality for China produces defibrase with ahylysantinfarctase, and economic benefit is very high, launches as calculated than being 4.
6, the defibrase of producing with this law shows through the 333 routine patient's clinical trials of the whole nation 22 tame hospitals, to cerebral thrombosis, vascular occlusive diseases such as retina Tipping Center vein obstruction, Acute Myocardial Infarction, strand artery and vein thrombus have curative effect preferably, and total effective rate is 86.7%, and obvious effective rate is 60%.Think through expert statement: the agkistrodon acutus venin defibering enzyme vein has fine and molten fine two kinds of effects with injection, is a kind of new zymin of treatment occlusive vascular disease, and is evident in efficacy, and short treating period is easy to use, safer.Through routine patient's clinical applications up to ten thousand, be more than 92% to cerebral thrombus forming patient's treatment total effective rate, produce effects 62%; Treatment total effective rate to the central retinal vein occlusion patient is 80%, produce effects 40%, and with urokinase (daily output) contrast, the urokinase total effective rate is 46.7%, produce effects 6.7%, two is compared and significantly is better than urokinase.This medicine also is used for the treatment of the concurrent renal failure of glomerulonephritis, has now made 80 many cases, and total effective rate is 86.7%, produce effects 60%.Treatment to comprehensive ephrosis has also obtained better curative effect.In addition, Chaoyang, Beijing institute, Beijing Tongren Hospital use defibrase treatment pulmonary heart disease and have also obtained curative effect preferably.Be worth special propose be, intracardiac professors Huang Daxian of section of PLA General Hospital etc.s use defibrase treatment Acute Myocardial Infarction, through the contrast examination recanalization rate be 10/10, to contrast be 3/10, now made 40 examples, recanalization rate is 90%.And internationally recognized its recanalization rate of thrombolysis good medicine t-PA is 70%, thinks that tentatively defibrase might be best at present thrombolysis new drug.
In sum, produce defibrase with this law from method kiss agkistrodon halyx pallas venom, productive rate height not only, and be easy to promote the use of shows this method advanced person, easy row.The defibrase of producing might be present best thrombolytics aspect thrombolysis through the curative effect height and the few side effects of its curative effect of clinical verification than Ancrod, the Reptilase etc. that produce with other snake venom, and its price is again far below similar medicine.Therefore this law not only is fit to China's national situation, and will bring good social benefit and economic benefit.
Fig. 1 is the production scheme of the purification process of the said agkistrodon acutus venin defibering enzyme of the present invention.
The invention will be further described below by embodiment:
Embodiment one,
Ahylysantinfarctase 2.5g is dissolved in 0.05M, among the PH8.0 Tris-HCl damping fluid 8ml dissolving after 2000 rev/mins centrifugal 10 minutes, get the mixed rotary liquid upper prop, precipitation stirs evenly mixed rotary liquid upper prop on the centrifuging and taking with the above-mentioned damping fluid of 3ml more again, to use 13 times DEAE-Sephadex A-50 to handle through acid-alkali (0.2M acetic acid, 2% ammoniacal liquor), be washed till neutrality with distilled water, use 0.05M, PH8.2Tris-HCl damping fluid balance again to PH8.2, dress post (4 * 110Cm
2) reequilibrate, last sample.With above-mentioned damping fluid 1500ml in the gradient bottle, damping fluid 1500ml adds Nacl to 1.2M concentration in the storage bottle, connect the gradient elution bottle and carry out the straight line gradient elution one time, earlier without Fraction Collector, with collecting every pipe 10ml, per hour 70～80ml again with automatic Fraction Collector behind the about 800ml of beaker collection, collect liquid and under 280nm, measure absorbance value, determine peak number according to absorbance value and pipe number.Then by the collection liquid of a protein peak merge the rough segmentation component, it is contained in port grinding bottle, puts refrigerator and preserve to detect, compile by peak number with TLE vigor be 1,2,3,4 ... etc. group, 1,2 merge 3,4 merging then, this batch 1,2 merged into 210ml, contains protein 37 4mg.Be washed till neutrality after will handling with 0.5M HCl with the DEAE-Ce-llulos DE52 that crosses 8 times, use above-mentioned damping fluid balance to PH8.2 again, dress post reequilibrate is with above-mentioned gained 1,2 peak 210ml solution upper prop (2.5 * 110cm
2), the above-mentioned damping fluid of gradient bottle 600ml, Nacl 0.2M, the above-mentioned damping fluid 600ml of reservoir bottle, NaCl 0.7M, the straight line gradient elution, every pipe 10ml, per hour 60ml measures absorbance value down in 280nm, be separated into 3 protein peaks, 1 peak is the TLE peak, and 230ml contains protein 24 0mg.3,4 merge 260ml, contain albumen 540mg, and by last method DEAE-Cellulos DE52 column chromatography, damping fluid is the same, NaCl0.4～0.9M straight line gradient elution, and 1 peak is the TLE peak, 240ml contains protein 31 0mg.With the DEAE-Sephadex A～50 dress post (3 * 110Cm that handle
2), with 1, the 21 peak 230ml upper props that get, with above-mentioned Tris-Hcl buffering, gradient bottle 800ml, Nacl 0.2M, reservoir bottle 800ml, NaCl 0.7M, a straight line gradient elution gets two peaks, and peak 1 is the TLE peak, and 120ml contains enzyme amount 220mg; Peak 1 with 3,4,240ml are splined on DEAE-SephadeA-50 post (3 * 110Cm
2), the above-mentioned damping fluid 500ml of gradient bottle, NaCl 0.4M, reservoir bottle damping fluid 800ml, NaCl 0.9M, one time the straight line gradient elution gets three peaks, and peak 1 is the TLE peak, and 210ml contains enzyme amount 220mg.Through hemorrhage poison, the vigor that condenses etc. is up to the standards the 120ml(220mg with 1,2 with this product of twice) add the 210ml(220mg at 3.4 peaks 1) merge desalination; 11 times Sephaded G-25(was homemade with using) soaked 30 minutes with 2% ammoniacal liquor, be washed till neutrality with distilled water, boil 20 minutes cooling back dress posts (4.5 * 110Cm) again, with above-mentioned amalgamation liquid 330ml(440mg enzyme amount) upper prop, use the sterile distilled water wash-out, get 1 peak, 440ml, the enzyme amount 399mg(rate of recovery is 93%).Through prescription, it is 1880 that filtration, packing and frost drying, sealing by fusing get qualified product, and average every gram is 752, total digit rate 15.1%.
Embodiment two
Get ahylysantinfarctase 2.5 grams and be dissolved in 0.05M, among the PH8.3Tri-HCl buffering 8ml, other operation just is changed to PH8.3 with damping fluid with example one, and DEAE-Sephadex A-50, Sephadex G-25, DEAE-Cellulose, DE52 use after regenerating for exhausted among the embodiment one.Get enzyme liquid 460ml after separation and purification and desalination, enzyme amount 528mg gets 2580 of finished products at last, and average every gram is 1032, and total yield is 20.64%.
More than two batches all do not have significant difference through drug inspection and clinical application.
Claims (6)
1, the purification process of agkistrodon acutus venin defibering enzyme, it comprises: (1) snake venom dissolves with damping fluid, and centrifugation gets the thrombin-like enzyme component through the anionite column chromatography with the buffer solution elution rough segmentation; (2) with gained thrombin-like enzyme component again respectively through the anionite column chromatography, purifying gets the purified thrombin-like enzyme; (3) will make with extra care thrombin-like enzyme stoste and get defibrase stoste through desalination again; (4), filtration, packing, freezing, dry, sealing by fusing, promptly get injection defibrase finished product with the former liquid formula of gained defibrase; It is characterized in that:
A, snake venom dissolve with the damping fluid of PH7.8-8.6, through the anionite column chromatography, get the thrombin-like enzyme component with the rough segmentation of PH7.8-8.6 damping fluid sodium-chlor straight line gradient elution;
B, with gained thrombin-like enzyme component in order or divide two groups through twice above column chromatography of anionite must make with extra care thrombin-like enzyme stoste with twice above wash-out purifying of PH7.8-8.6 damping fluid sodium-chlor straight line gradient;
Gained thrombin-like enzyme stoste can directly go up sample in c, rough segmentation gained thrombin-like enzyme component and the repurity, and full process operations is carried out in 10 ℃～20 ℃ half aseptic or sterilisable chamber.
2, purification process according to claim 1, it is characterized in that dissolving the used damping fluid of snake venom is 0.05～0.10M Tris-HCl damping fluid or glycine-NaOH damping fluid.
3,, it is characterized in that the used anionite of rough segmentation is DEAE-Sephadex A-50 or QAE-Sepha-dex A-25 according to the said purification process of claim 1.
4, purification process according to claim 1 is characterized in that the used sodium-chlor gradient of rough segmentation is 0～1.2M.
5, purification process according to claim 1 is characterized in that twice used anionite of above purification column chromatography is DEAE-Mierocrystalline cellulose DE52 or DE32 and DEAE-Sephadex A-50 or QAE-Sephadex A-25.
6, purification process according to claim 1 is characterized in that the twice used damping fluid sodium-chlor of above column chromatography purification wash-out gradient is 0.1～0.7M, 0.3～0.9M.
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| CN 92102645 CN1065680A (en) | 1992-04-09 | 1992-04-09 | Purifying method for agkistrodon acutus venin defibering enzyme |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100336903C (en) * | 2004-07-27 | 2007-09-12 | 沈阳斯佳科技发展有限公司 | Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase |
| CN102757948A (en) * | 2012-05-28 | 2012-10-31 | 广东瑞昇药业有限公司 | Haemocoagulase agkistrodon and separation method thereof |
| CN110791491A (en) * | 2019-12-11 | 2020-02-14 | 昆明龙津药业股份有限公司 | Method for extracting defibrase from snake venom |
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1992
- 1992-04-09 CN CN 92102645 patent/CN1065680A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100336903C (en) * | 2004-07-27 | 2007-09-12 | 沈阳斯佳科技发展有限公司 | Extracting norfibroase from snake venom and preparation process for liquid drugs injection of norfibroase |
| CN102757948A (en) * | 2012-05-28 | 2012-10-31 | 广东瑞昇药业有限公司 | Haemocoagulase agkistrodon and separation method thereof |
| CN102757948B (en) * | 2012-05-28 | 2014-02-05 | 广东瑞昇药业有限公司 | Haemocoagulase agkistrodon and separation method thereof |
| CN110791491A (en) * | 2019-12-11 | 2020-02-14 | 昆明龙津药业股份有限公司 | Method for extracting defibrase from snake venom |
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