CN1131159A - Bromelin decrustation preparation and its preparing method - Google Patents

Abstract

The present invention relates to a decrustation medicine for burn and its preparation method and application. It is a refined bromelain mixture prepared by using crude bromelain extracted from pineapple fruit as raw material and making it pass through such processes of extracting, centrifugal separation (or filtering), ion-exchange, salting-out, dialyzing and drying, etc.. It mainly contains two peptidases their molecular weight is 27000-30000 daltons. For treatment of second-degree and third-degree burns, it can implement decrustation within 3-4 hours.

Description

Bromelin decrustation preparation and preparation method thereof

The present invention relates to biochemical enzyme decrustation after burn medicine and preparation thereof and application method.

The eschar (being decrustation) of removing each burn generation is a crucial link in the burn treatment, 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 enzyme 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 be used.Chemistry decrustation medicine, i.e. acidic drug diffusant, acid diffusant commonly used has Whitfield's ointment, pyruvic acid, succsinic acid and citric acid etc., but brings reason such as misery to patient because of its pungency, is not used clinically.The biochemical enzyme decrustation preparation is considered to more promising decrustation medicine, and it comprises plant protease, bacterial enzyme and animal ferment three class biochemical enzyme 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 treatment handbook third edition, volumes such as the poplar of side, Science and Technology of Shanghai document press in 1987; Work such as the poplar of " burn theory and practice " side, Liaoning science and technology press in 1989).In plant protease, more with bromeline (Bromelain), ficin (Dericin) and papoid (Papain) research.The present invention is directly relevant with bromeline, discloses a kind of new bromelin decrustation preparation and the method for preparation thereof.

The non-patent literature report beginning that relevant bromeline prepares enzyme decrustation medicine sees 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 Patent Office of the People's Republic of China's World Intellectual Property Organization's database (WIPOData Base of Chinese 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 OF DEVITALIZED TISSUE WITH HYDROLYTICENZYME PRODUCT) patent, having described an iso-electric point is 6, at least (molecular weight of each subunit is 14 to contain two subunits, the main characteristic of enzyme mixture 300-15,000 dalton) and the detailed method of carrying out decrustation thereof with this enzyme mixture water soluble preparation.On August 25th, 1980, Klein and Houck two people applied for that again (patent No. is 4 to a patent, 307,081), name is called enzyme mixture (ENZYME MIXTURE), and this patent proposes, the notion of decrustation enzyme (escharase), and the molecular weight that proposes enzyme mixture on the basis of a last patent is 30,000-50,000 dalton has carried a treatment prescription (proportioning that is enzyme mixture and physiological saline is 1: 1) simultaneously.William Galbraith has proposed a new patent application (United States Patent (USP) 4 on May 5th, 1981,61,551), name is called enzyme wound excision (METHOD OF ENZYME DEBRIDEMENT), this patent is described the separation method and the characteristic thereof of enzyme mixture in detail: the protease activity under the halfcystine existence condition is every mg15-35 active casein, 0.5-5 collagen protein activity.On December 3rd, 1991, Klein and Houck applied for a new United States Patent (USP) again, and applied for international monopoly (PCT/US92/10395) by PCT on December 2nd, 92, 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 ADNMETHOD OF ISOLATING SAME) described a kind of entirely different in before separation method, promptly with xitix (Vc) as antioxidant, (NH ₄) ₂SO ₄Separation method as protein precipitant.Concrete steps are: 1. thick enzyme is dissolved in the solution that contains 1%Vc (4-10 ℃) → 2. and transfers pH to 3.5-3.9 with HCl; → 3. under 4-10 ℃ of condition, stir 18 hours → 4. with filtering or centrifuging removal insolubles → 5. make 40% (NH ₄) ₂SO ₄Spend the night precipitation → 6. centrifugal or filter of saturated solution (4-10 ℃), collecting precipitation → 7. precipitation is dissolved in (pH3.4, low temperature) in the 0.3M acetum that contains 0.1%Vc; → 8. filtration → 9. the clear liquid freeze-drying is weighed and is the proteolytic enzyme mixt that contains the decrustation enzyme with Amicon DC-30 ultrafiltration system, contain the decrustation enzyme (escharase) of 1-1.5%, the amino acid composition that the contriver gives the decrustation enzyme in this mixture, and because this enzymatic defect proline(Pro) and tyrosine, the contriver thinks that it is a kind of single albumen (Uniqueprotein), this enzyme is 15 by three molecular weight, 000 daltonian subunit is formed, and total molecular weight is 45,000 dalton.The contriver thinks that this patent is at yield (25%), the aspects such as stability of the content of decrustation enzyme (1-1.5%), decrustation time (4 hours) and mixture all significantly are better than previous patent in the lytic enzyme mixture, can produce better skin-grafting bed (grafting bed), the remaining eschar in center (central escharremaing) is also littler.And can obtain purer decrustation enzyme by dextrane 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 enzyme mixture is all 30, and 000-50 is in 000 dalton's scope; 3. the high reactivity of enzyme mixture is 35 units of every mg protein (under the halfcystine existence condition); 4. iso-electric point 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 lytic enzyme 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 Mierocrystalline cellulose DE52 to carry out column chromatography as ion-exchanger) by technologies such as lixiviate, centrifugal (or filtration), concentrate dryings.

The effective constituent of this product is proteolytic ferment, measure through polyacrylamide gel electrophoresis, wherein contain two and have the protein hydrolysate composition, be referred to as pineapple PEPA A and pineapple PEPB B respectively, the molecular weight that the SDS-polyacrylamide gel electrophoresis is measured PEPA A and PEPB B is respectively 27,000 and 30,000 dalton.The iso-electric point that isoelectric focusing electrophoresis is measured PEPA A is 9.4.The amino acid composition of PEPA A and PEPB B is as follows:

The amino acid of pineapple PEPA A and pineapple PEPB B is formed

	Mg/100ml in the sample		Each amino acid accounts for total amount (%)
					PEPA A	PEPB B	PEPA A	PEPB 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 4.8 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 do not survey
Summation					???1524.70	????952.60

Refining enzyme adds a certain amount of halfcystine and mixes as activator and be enzyme decrustation preparation dry powder, 3-4 hour decrustation time, the shortest need 1 hour.The preparation technology of bromelin decrustation preparation

(1) bromeline highly finished product technology

1. get a certain amount of fruit bromeline raw product, use pH6.0-7.2,0.02-0.05M the phosphoric acid buffer lixiviate, the ratio of damping fluid and crude protein enzyme is 15-20: 1 (V/W), 4-10 ℃ of condition lower magnetic force stirred 2-4 hour, and 4,000-10,000 rev/min centrifugal about 10 minutes, collect supernatant liquor; Or remove insoluble impurity in the vat liquor with filtration method;

2. collect liquid and precipitate with the ammonium sulfate of 45% saturation ratio, precipitation process is finished very soon; The centrifugal supernatant liquor that removes;

3. throw out is used distill water dialysis desalination (1: 100) again, sloughs ammonium sulfate, gets concentrated solution;

4. the strong solution freeze-drying get final product the Traumanase highly finished product, yield 20-34% (because of thick enzyme source difference).

The also available cold acetone precipitation of the poly-dialysis concentrated solution that is 3. obtained of step, apparatus lid centrifuge tube refrigerated centrifuge is centrifugal 10 minutes again, and throw out natural low temperature drying can obtain purer proteolytic enzyme mixt dry powder.

The bromelain lytic enzyme highly finished product that aforesaid method obtains, lytic 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 vat liquor can be carried out ion-exchange chromatography with DEAE-Mierocrystalline cellulose DE-52, collect two absorption peaks that have protease activity at the 280nm place, collect the part of optical density value more than 0.5.Collection liquid carries out lyophilize and can obtain the higher highly finished product of purity.

(2) decrustation preparation preparation

The highly finished product that make with aforesaid method add the halfcystine of 10-20% and make activator, and mill is mixed even, and packing is decrustation preparation dry powder finished product, low temperature is moistureproof preserve stand-by

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

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

1. remove the dirt hinder on the face with turps, clean with physiological saline and hinder face, tear off and treat that decrustation hinders the epidermis on the face, as top layer charing, available scalpel etc. strike off it.

2. use 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. watched quietly 5-10 minute, and observed and to hinder face and change, as it is many to hinder on the face little blutpunkte, and, prove that decrustation is effective, need the decrustation time to lack with a large amount of sepages.Otherwise the decrustation time is long slightly.

4. the aseptic petrolatum gauze of lid layer outside the decrustation medicine again, it is outer to cover 6-8 layer sterile gauze again, and outermost layer is fastened with bandage.

5. observe the variation of wrapping gauze, the wrapping gauze is soaked into by courageous and upright sepage, proves the success of decrustation.Usually the shallow face of hindering only needs 2-3 hour, and the darker face time of hindering is long slightly, general 4-5 hour.It often is colourless sepage rather than courageous and upright saturating liquid that three degree are hindered face.

6. open wrapping, scrape off medicine and necrotic tissue, use 0.5%NaHCO again with tweezers ₃Solution thoroughly cleans the surface of a wound.Or clean with physiological saline libation at an ancient wedding ceremony gauze and to hinder face, expose the fresh face of hindering that is covered with active hemorrhage point, so far, the 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 reaction solution: 1mM EDTA; 5mM halfcystine (new preparation); 1% casein, 20mM sodium phosphate buffer, pH7.2; 20-80 μ g enzyme preparation.Above solution is incubated 10 minutes at 35 ℃, add 5ml 10% trichoroacetic acid(TCA) and stop enzyme reaction, filter with quantitative paper, measure the optical density value of filtrate at 275nm, the condition of experiment contrast and above identical, just adding trichoroacetic acid(TCA) solution before adding the substrate insulation earlier makes enzyme deactivation (the proteinase activity unit definition is: at pH7.2, per minute caseinhydrolysate generation optical density value equals the optical density value of 1 μ g/ml tyrosine under 275nm in the time of 35 ℃.)

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

(1) adopt the thick enzyme that extracts in the pineapple fruit to make raw material, the enzyme activity of refining enzyme mixture is than existing

Technology exceeds 3-5 doubly, and the every gram of enzyme activity is 15-18 ten thousand units;

(2) has higher yield, 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) face fiber crops and use, do not find any toxic side effect, fool proof.

(5) 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 scab, need not anaesthetize, expense is lower,

The patient takes like a shot.

(7) be beneficial to the burn degree accurate diagnosis, this burn treatment to the four limbs functional part has far-reaching

Meaning.

(8) convenient transportation, the preservation validity period is long, and refrigerator cryopreservation 1 year, its enzyme are lived and are not decreased substantially

Lose, preserved 1 year under the room temperature, its enzyme is lived and lost only is 5%.

Described below is four embodiment of the present invention

Example 1

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

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

3. with above-mentioned 100ml concentrated solution freeze drier lyophilize, obtain containing mixture highly finished product 9.6 grams of proteolytic ferment, yield is 32%.Enzyme activity 170,000 units.Add halfcystine 1.5 grams, mill is mixed even, sealing name dress, and cryopreservation is stand-by.

Example 2

1. take by weighing thick enzyme 30 grams of pineapple fruit,, get supernatant liquor 400ml with reality method lixiviate together available from Guangxi.

2. with above-mentioned vat liquor with 45% saturation ratio (NH ₄) ₂SO ₄Precipitation, 5000 rev/mins centrifugal 20 minutes, throw out is with 1: 100 distill water dialysis desalination 5 times, totally 24 hours (4 ℃) must 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 centrifuges remove supernatant liquor, and throw out is seasoning at low temperatures, bromelain lytic enzyme mixture highly finished product 7.3 grams, yield is 24.3%.Enzyme activity is 180,000 units.Add the half deamination acid of 1 gram, mill is mixed even, and it is stand-by to pack cryopreservation.

Example 3 (treatment case)

Patient: the male sex, 19 years old, the right upper arm deep second degree burn, area 1.3% is scalded and was admitted to hospital in back 27 hours.Conventional cleaning is hindered face, occurs little blutpunkte after 5 minutes with decrustation preparation coating of the present invention, wraps up after 4 hours, and necrotic tissue is softening, strikes off necrotic tissue, 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 sex, and because of the lonely light of the electricity chemical fibre clothes that ignites causes facial and two burn of upper limb, the total area 10%, two upper limbs 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 blutpunkte occurs, after 3 hours 40 minutes, decrustation is fully removed necrotic tissue and is cleaned and hinders face, hinder the face substrate and still be corium, cover with the radiation pigskin 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. one kind is raw material with the thick enzyme of bromelain, and the preparation method who adopt lixiviate, centrifugal (or filtration), concentrate, prepared such as drying is used for the refining proteolytic enzyme mixt of tissue of burn decrustation is characterized in that:

(1) using the thick enzyme that extracts from pineapple fruit is raw material;

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

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

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

2. method according to claim 1 is characterized in that the vat liquor precipitation adopts DEAE-Mierocrystalline cellulose DE-52 to carry out ion-exchange and carries out drying again to obtain more highly purified refining enzyme mixture.

3. 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.

4. one kind comprises the decrustation zymin made from extra care the bromelain lytic enzyme, it is characterized in that:

(1) decrustation zymin is ground even the forming that be mixed by the halfcystine that refining bromeline adds 10-20%;

(2) refining bromelain lytic enzyme mixture mainly is made of its molecular weight branch PEPA A and PEPB B

Be not 27,000 and 30,000 dalton;

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