CN1041170C - Bromelin decrustation preparation and its preparing method - Google Patents

Abstract

The present invention relates to a medicine for decrustation of burn, and a preparation method and an application method thereof. The present invention has the technical scheme that coarse enzyme extracted from pineapples is used as a raw material processed by the steps of lixiviating, centrifugation (filtration), ion exchange, saltingout, dialysis, drying, etc. to be preared into a bromelin mixture in a refining mode, wherein the bromelin mixture mainly comprises two peptidases with the formula weight of 27000 to 30000 dalton. The prepared decrustation preparation can be used for the decrustation of deep second-degree burn and deep third-degree burn within three to four hours. The present invention has the advantages that anesthetization and standby blood do not needed by the whole process, the cost is low, the operations pain does not exist, and the decrustation effect is complete. Thereby, the decrustation preparation of the present invention has a great significance of treating the burn of functional parts.

Description

Bromelin decrustation preparation and preparation method thereof

The present invention relates to biochemical enzymes decrustation after burn medicine and preparation thereof and application process.

The eschar (being decrustation) of removing each burn generation is a crucial link in the burn treating, and means the most commonly used at present remain the operation decrustation, but modus operandi has following deficiency: one, cause operation painful to patient easily; Its two, need to be equipped with fresh blood, in case urgent need during severe loss of blood; Its three, the situation that injures biological tissue often takes place, the particularly damage at key function position, consequence is more serious; Its four, the normal difficulty of side effect risk of anaesthetic treatment is avoided.In order to overcome the above-mentioned weak point of operation decrustation, people promptly seek the medicine decrustation means since six the seventies.But medical circle is thought the decrustation medicine of having succeeded in developing, and is all not ideal enough.

The decrustation medicine comprises Chinese medicine, chemicals and biochemical enzymes preparation three major types.Decrustation Chinese medicine is commonly used has and more creates No. ten, No. one, east and ointment for treating hot water and fire scald, because of these medicines do not have bacteriostastis, can make traumatic infection heavy, and seldom is used.Chemistry decrustation medicine, i.e. acidic drug diffusant, acid diffusant commonly used has salicylic acid, acetone acid, succinic acid and citric acid etc., but brings reason such as misery to patient because of its zest, is not used clinically.Biochemical enzymes decrustation agent is considered to comparatively promising decrustation medicine, and it comprises plant rennet, bacterial enzyme and animal ferment three class biochemical enzymes again; But have only the bacterial enzyme of a kind of being called " Travase " a small amount of application to be arranged clinically so far at mankind's burn.(" the burn treating handbook third edition, volumes such as the poplar of side, Science and Technology of Shanghai document publishing house in 1987; The volumes such as poplar of " burn theory and practice " side, Liaoning science and technology publishing house in 1989).In plant rennet, more with bromelain (Bromelain), ficin (Dericin) and papain (Papain) research, the present invention is directly relevant with bromelain, discloses a kind of new bromelin decrustation preparation and the method for preparation thereof.

The non-patent literature report that relevant bromelain prepares enzyme decrustation medicine starts from the beginning of the seventies, and the patent documentation report then sees 74 years the earliest.According to result for retrieval, in disclosed patent, there are six pieces of patents (wherein two pieces identical) and the present invention that in various degree relevant arranged with the data base of World Intellectual Property Organization of Patent Office of the People's Republic of China (WIPO Data Base ofChinese Patent Office).Gerold.K.V.Klein and John.C.Houck are at the United States Patent (USP) 4 of application in 1979,226,854, name is called water and separates enzyme product and carry out in devitalized tissue's debridement (DEBRUDENEBT OFDEVITALIZED TISSUE WITH HYDROLYTIC ENZYME PRODUCT) patent, having described an isoelectric point, IP is 6, at least (molecular weight of each subunit is 14 to contain two subunits, the principal character of enzymatic mixture 300-15,000 dalton) and the detailed method of carrying out decrustation thereof with this enzymatic mixture water soluble preparation.On August 25th, 1980, Klein and Houck two people applied for that again (patent No. is 4307 to a patent, 081), name is called enzymatic mixture (ENZYME MIXTURE), this patent proposes, the notion of decrustation enzyme (escharase), and the molecular weight of proposition enzymatic mixture is 30 on the basis of a last patent, 000-50,000 dalton has proposed a treatment prescription (proportioning that is enzymatic mixture and normal saline is 1: 1) simultaneously.William Galbraith has proposed a new patent application (United States Patent (USP) 4 on May 5th, 1981,361,551), name is called enzyme wound excision (METHODOF ENZYME DEBRIDEMENT), this patent is described the separation method and the characteristic thereof of enzymatic mixture in detail: the proteinase activity under the cysteine existence condition is every mg15-35 active casein, 0.5-5 the active .1991 December of individual collagen protein Klein on the 3rd and Houck have applied for a new United States Patent (USP) again, and applied for international monopoly (PCT/US92/10395) in 2, PCT of 92 on Decembers, the name be called: contain this patent of the proteolytic enzyme mixt of decrustation enzyme and the separation method of this material (PROTEOLYTIC MIXTURE CONTAING ESCHARASE AND METHODOF ISOLATING SAME) described a kind of entirely different in before separation method, promptly with ascorbic acid (Vc) as antioxidant, (NH ₄) ₂SO ₂Separation method as protein precipitant.1, thick enzyme is dissolved in (4-10 ℃) → 2 in the solution that contains 1%Vc, with HCl transfer pH to 3.5-3.9 → 3, under 4-10 ℃ of condition, stir 18 hours → 4, with filtering or centrifuging removal insoluble matter → 5, making 40% (NH ₄) ₂SO ₂Saturated solution (4-10 ℃) precipitation → 6 of spending the night, centrifugal or filter, are dissolved in precipitation (pH3.4, low temperature) in the 0.3M acetum that contains 0.1%Vc at collecting precipitation → 7; → 8, filter with Amicon DC-30 ultrafiltration system; → 9, the clear liquid lyophilizing is weighed and is the proteolytic enzyme mixt that contains the decrustation enzyme, contain the decrustation enzyme (escharase) of 1-1.5%, the aminoacid composition that the inventor gives the decrustation enzyme in this mixture, and because this azymia proline and tyrosine, the inventor thinks that it is a kind of single albumen (Unique protein), this enzyme is 15 by three molecular weight, 000 daltonian subunit is formed, and total molecular weight is 45,000 dalton.The inventor thinks that this patent is at yield (25%), the aspects such as stability of decrustation enzyme content (1-1.5%), decrustation time (4 hours) and mixture are all significantly because previous patent in the hydrolytic enzyme mixture, can produce better skin-grafting bed (grafting bed), the remaining eschar in center (central eschar remaing) is also littler.And can obtain purer decrustation enzyme by polydextran gel SephadexG-75 or isoelectrofocusing method as required.Above-mentioned several patents in separation method or condition so that on to the description of decrustation enzyme though difference is arranged all, following some be identical: 1, used thick enzyme all extracts from pineapple stem; 2, the molecular weight of enzymatic mixture is all 30, and 000-50 is in 000 dalton's scope; 3, the high activity of enzymatic mixture is 35 units of every mg protein (under the conditions that the acid of half Guang saddle exists); 4, isoelectric point, IP is generally all about 6; 5, the decrustation time is 4 hours the soonest.

The present invention's purpose is to seek a kind of decrustation better effects if, shorter enzyme decrustation preparation of time.This goal of the invention is achieved by technical scheme described below.

The refining bromelain that the present invention describes is to adopt the thick enzyme that extracts from pineapple fruit as extracting raw material, obtains (more highly purified highly finished product can obtain by adopting DEAE cellulose DE52 to carry out column chromatography as ion-exchanger) by technologies such as lixiviate, centrifugal (or filtration), concentrate dryings.

The effective ingredient of this product is a proteolytic enzyme, measure through polyacrylamide gel electrophoresis, wherein contain two and have the aminosal composition, be referred to as bromelain A and bromelain B respectively, the molecular weight that the SDS-polyacrylamide gel electrophoresis is measured protease A and protease B is respectively 27,000 and 30,000 dalton.The isoelectric point, IP that isoelectric focusing electrophoresis is measured protease A is 9.4.The amino acid composition of protease A and protease B is as follows:

The aminoacid of bromelain A and bromelain B is formed

	Mg/100ml in the sample		Each aminoacid accounts for total amount (%)
					Protease A	Protease B	Protease A	Protease B
	Aspartic acid threonine serine glutamic acid glycine alanine cystine valine methionine isoleucine leucine tyrosine phenylalanine arginine lysine histidine proline tryptophan	146.57 70.07 106.59 142.90 98.33 104.40 78.95 93.08 22.64 97.82 67.78 126.74 61.20 120.36 10.76 65.64 110.87 do not survey	104.25 45.70 71.86 102.72 63.90 61.30 42.03 61.84 19.80 63.12 47.90 105.08 40.52 41.74 5.60 35.88 39.36 do not survey	9.61 4.60 6.99 9.37 6.45 6.85 5.18 6.10 1.49 6.42 4.45 8.31 4.02 7.90 0.71 4.28 7.27 do not survey					10.94 4.80 7.54 10.78 6.71 6.44 4.41 6.49 2.08 6.63 5.03 11.03 4.25 4.38 0.59 3.77 4.13 survey
Summation					1524.70	952.60

Refining enzyme adds a certain amount of cysteine and is enzyme decrustation preparation dry powder as the activator mix homogeneously, 3-4 hour decrustation time, the shortest need 1 hour.

The preparation technology of bromelin decrustation preparation

(1) bromelain highly finished product technology

1, get a certain amount of fruit bromelain semifinished product, use pH6.0-pH7.2, the phosphoric acid of 0.02-0.05M is slow

Towards the liquid lixiviate, the ratio of buffer and crude protein enzyme is 15-20: 1 (V/W), 4-10 ℃ of condition lower magnetic force stirs

2-4 hour, 4,000-10,000 rev/min centrifugal about 10 minutes, collect supernatant; Or go with Filtration

Remove the insoluble impurity in the lixiviating solution;

2, collection liquid precipitates with the sulphuric acid amine of 45% saturation, and precipitation process is finished very soon; The centrifugal supernatant that removes

Liquid;

3, sulphuric acid amine is sloughed in precipitate reuse distill water dialysis desalination (1: 100), obtains concentrated solution;

4, the concentrated solution lyophilizing can obtain the bromelain highly finished product, yield 20-34% (because of thick enzyme source difference).

The also available cold acetone precipitation of the dialysis concentrated solution that step 3 obtained, centrifugal 10 minutes of reuse tool lid centrifuge tube refrigerated centrifuger, precipitate natural low temperature drying can obtain purer proteolytic enzyme mixt dry powder.

The bromelain hydrolytic enzyme highly finished product that said method obtains, hydrolytic enzyme vigor are every gram 15-18 ten thousand units; Protein content 16-20%.

If need the more highly purified mixed enzyme goods of preparation, then lixiviating solution can be carried out ion-exchange chromatography with DEAE-cellulose DE-52, collect two absworption peaks that have proteinase activity at the 280nm place, collect the part of optical density value more than 0.5.Collection liquid carries out lyophilization and can obtain the higher highly finished product of purity.

(2) decrustation preparation preparation

The exquisiteness product that make with said method add the cysteine of 10-20% and make activator, and mill is mixed even, packing, promptly

Be decrustation preparation dry powder finished product, the moistureproof preservation of low temperature is stand-by.

(3) use the decrustation enzyme preparation to treat the method for tissue of burn

After burn patient is admitted to hospital, handle as follows:

1, remove the dirt of hindering on the face with Oleum Terebinthinae, the reuse normal saline cleans hinders face, tears the epidermis on the decrustation face for the treatment of off,

As top layer carbonization, available scalpel etc. strike off it.

2, with 1% lignocaine solution with above-mentioned decrustation enzyme dry powder furnishing pasty state, it is coated in equably hinders on the face,

About 1-2mm.

3, watch 5-10 minute quietly, change above observing, as it is many to hinder on the face little petechia, and, prove with a large amount of sepages

Decrustation is effective, needs the decrustation time short.Otherwise the decrustation time is long slightly.

4, the aseptic kpetrolatum gauze of lid layer outside the decrustation medicine again, its outer 6-8 layer sterile gauze, outermost layer of covering again

Be fastened with binder.

5, observe the variation of wrapping gauze, the wrapping gauze is soaked into by courageous and upright immersion, proves the success of decrustation.Common shallow wound

Face only needs 2-3 hour, and the darker face time of hindering is long slightly, general 4-5 hour.It often is colourless oozing that three degree are hindered face

Liquid rather than courageous and upright saturating liquid.

6, open wrapping, scrape off medicine and slough, reuse 0.5%NaHCO with tweezers ₃Solution thoroughly cleans wound surface

Or dip in gauze with normal saline and clean and hinder face, expose and be covered with above active hemorrhage point fresh, so far, decrustation

Process is finished, and does further treatment according to different situations and handles.

(4) enzyme activity determination

With the casein is substrate, contains in the 5ml reactant: 1mMEDTA; 5mM cysteine (new preparation); 1% casein, 20mM sodium phosphate buffer, pH7.2; 20-80 10.5 μ g enzyme preparations.Above solution is incubated 10 minutes at 35 ℃, add the 5ml10% trichloroacetic acid and stop enzyme reaction, filter with quantitative filter paper, measure the optical density value of filtrate at 275nm, the condition of experiment contrast and above identical, just adding trichloroacetic acid solution before adding the substrate insulation earlier makes enzyme deactivation (the prolease activity unit definition is: at pH7.2, per minute caseinhydrolysate generation optical density value equals the optical density value of 10.5 μ g/ml tyrosine under 275nm in the time of 35 ℃.)

The present invention compared with prior art has following good effect:

1, adopt the thick enzyme that extracts in the pineapple fruit to make raw material, the enzyme activity of refining enzymatic mixture exceeds 3-5 doubly than prior art, the every gram of enzyme activity 15-18 ten thousand units;

2, has higher efficient, generally at 20-34%.

3, decrustation time weak point, the general 2-3 of second degree burn hour, the fastest clinically only needed 60 minutes, and decrustation is thorough.

4, any toxic and side effects is not found in clinical practice, and is fool proof.

5, the patient is admitted to hospital, and decrustation immediately can be removed focus as early as possible, is beneficial to hinder face and heal early.

6, can overcome the painful and problem of losing blood of the operation of performing the operation when going crust, need not anaesthetize, expense is lower, and the patient takes like a shot.

7, be beneficial to the burn degree accurate diagnosis, this burn treating to the extremity functional part has profound significance.

8, convenient transportation, it is long to preserve effect duration, and the basic not loss of living of refrigerator cryopreservation 1 year, its enzyme was preserved 1 year under the room temperature, and its enzyme is lived and lost only is 5%

Below describing is four embodiments of the present invention

Example 1

1, take by weighing the thick enzyme 30mg of pineapple fruit, use pH7.2 available from Guangxi, the phosphate buffer 450ml lixiviate of 0.02M, 4 ℃ of condition magnetic agitation 2 hours, 4,000 rev/mins centrifugal 10 minutes, supernatant 400ml.

2, under the room temperature with said extracted liquid 400ml with 45% saturation ammonium sulfate (about 110 gram) precipitation, 4,000 rev/mins centrifugal 20 minutes, precipitate is with distill water dialysis desalination (1: 100), (4 ℃) desalination is 24 hours under the low temperature, during change water 4 times.Get concentrated solution 100ml.

3, with above-mentioned 100ml concentrated solution freezer dryer lyophilization, obtain containing mixture highly finished product 9.6 grams of proteolytic enzyme, yield is 32%.Enzyme activity 170,000 units.Add cysteine 1.5 grams, mill is mixed even, packs, and cryopreservation is stand-by.

Example 2

1, take by weighing thick enzyme 30 grams of pineapple fruit available from Guangxi, with the identical method lixiviate of example 1, supernatant 400ml.

2, with above-mentioned lixiviating solution with 45% saturation ammonium sulfate precipitation, 5000 rev/mins centrifugal 20 minutes, precipitate is with 1: 100 distill water dialysis desalination 5 times, totally 24 hours (4 ℃), concentrated solution 100ml.

3, above-mentioned concentrated solution is precipitated with cold acetone, centrifugal 10 minutes of 8,000 rev/mins of apparatus lid centrifuge tube refrigerated centrifugers remove supernatant, and precipitate is natural drying at low temperatures, bromelain hydrolytic enzyme mixture highly finished product 7.3 grams, yield is 24.3%.Enzyme activity is 180,000 units.Add 1 gram cysteine, mill is mixed even, and it is stand-by to pack cryopreservation.

Example 3 (treatment case)

Patient: the male, 19 years old, the right upper arm second degree burn, area 1.3% is scalded and was admitted to hospital in back 27 hours.Conventional cleaning is hindered face, occurs little petechia after 5 minutes with decrustation preparation coating of the present invention, wraps up after 4 hours, and slough is softening, strikes off slough, cleans and hinders face, and decrustation is very thorough, hinders the face healing after routine was changed dressings 9 days.

Example 4 (treatment case)

Patient is the male, and because of the lonely light of the electricity chemical fibre clothes that ignites causes facial and two burn of upper limb, the gross area 10%, two upper limb are third degree burn, and area 6% is hindered back 10 hours with decrustation preparation decrustation of the present invention.Hindered face in 6 minutes after coating this product and little petechia occurs, after 3 hours 40 minutes, decrustation fully is above the place to go slough cleans, hinder the face substrate and still be corium, cover with the radiation Corii Sus domestica and hinder face, after 21 days, hinder face and heal substantially, be in hospital and do not have scar hyperplasia when leaving hospital in 41 days, the both hands function is normal.

Claims (4)

1, a kind of decrustation enzyme preparation that comprises refining bromelain hydrolytic enzyme is characterized in that:

(1) decrustation enzyme preparation is ground even the forming that be mixed by the cysteine that refining bromelain adds 10-20%;

(2) refining bromelain hydrolytic enzyme mixture mainly is made of its molecular weight branch protease A and protease B

Be not 27,000 and 30,000 dalton;

(3) hydrolase of proteolysis of refining enzymatic mixture is 15-18 ten thousand units.

2, the preparation of refining bromelain hydrolytic enzyme according to claim 1 adopt lixiviate, centrifugal or filter, concentrate, drying process, it is characterized in that:

(1) the refining enzyme of preparation is raw materials used is the thick enzyme that extracts from pineapple fruit;

(2) lixiviate pH6.0-7.2 phosphoric acid buffer clear liquid;

(3) with (NH of 45% saturation ₄) ₂SO ₂Precipitate, saltout then, dialysis desalting;

(4) dry lyophilization or the concentrated solution of adopting is with cold acetone precipitation, the centrifugal natural drying of low temperature again.

3, refining bromelain hydrolytic enzyme preparation method according to claim 2 is characterized in that the lixiviating solution precipitation adopts DEAE-cellulose DE-52 to carry out ion exchange and carries out drying again to obtain more highly purified refining enzymatic mixture.

4, refining bromelain hydrolytic enzyme preparation method according to claim 2 is characterized in that when carrying out anion exchange chromatography, under 280nm, collect optical density value 0.5 with top solution.