CN103007259A

CN103007259A - Bromelain capsule for burn wound repair debridement and preparation method of same

Info

Publication number: CN103007259A
Application number: CN2012105505592A
Authority: CN
Inventors: 李正
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-12-19
Filing date: 2012-12-19
Publication date: 2013-04-03

Abstract

The invention discloses a bromelain capsule for burn wound repair debridement and a preparation method of the bromelain capsule. The preparation method of a product with a debridement function comprises the following steps: (1), taking crude enzyme extracted from a pineapple fruit as a raw material; (2), dissolving the crude enzyme with 0.002 mol/L of phosphate buffer with the pH of 6.0, centrifuging for removing insoluble impurities, and collecting supernatant; (3), dialyzing the supernatant with the 0.002 mol/L of phosphate buffer with the pH of 6.0; and (4), after dialyzing, centrifuging again and collecting the supernatant again so as to obtain the product. When the prepared product is used for therapy, in the whole therapeutic process, anesthesia and blood preparation are not required; the fee is low; surgical pains can be avoided; the debridement is very thorough; and the product has a greater significance for burn treatment of functional parts.

Description

A kind of be used to the bromelain capsule of molten crust wound repair and preparation method thereof of burning

Technical field

The present invention relates to a kind of be used to the bromelain capsule of molten crust wound repair and preparation method thereof of burning.

Background technology

The most popular method of dispelling clinically up to now the burn slough remains operation and cuts crust.But operation method is except fast effectively, and weak point is: one, operation wound clearing often closes on normal structure as cost take sacrifice thoroughly, causes patient's secondary painful thereby cause new wound surface to form; Two, to cut crust mainly be that the supervisor who depends on the doctor judges in operation, when normal structure during without remarkable boundary line, has increased particularly functional part of operating difficulty and risk with slough; Three, general operation is cut crust and is all caused relatively large hemorrhagely, surpasses 10% area and also need prepare 400---800 milliliters of whole bloods; Four, operation is cut crust wound healing convalescent period long and more cicatrix and is difficult to avoid.For the above-mentioned shortcoming of customer service, the doctor is seeking to use safety operation to make things convenient for the definite molten crust medicine of drug effect always in the industry.

Molten crust medicine comprises Chinese medicine, chemicals and biochemical enzymes preparation three major types.Molten crust 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 without bacteriostasis, can make traumatic infection heavy, and seldom be used.Chemical solution crust medicine, i.e. acidic drug diffusant, acid diffusant commonly used has salicylic acid, acetone acid, succinic acid and citric acid etc., but brings the reasons such as painful because of its zest to patient, is not used already clinically.At present the most frequently used debridement mode is still operation wound clearing and the debridement of Chinese medicine medicine clinically, and effect depends on that surgical doctor is to the judgement of wound surface ischemia, infection and slough boundary.Postoperative wound infection is not thorough because of debridement, and thoroughly debridement easily causes normal adjacent tissue impaired, gangrenous, damaged, healing delay occurs, even organ dysfunction occurs.The operation decrustation exists various risks, causes inevitably hemorrhagely, shows in many cases certain limitation.Operation causes the tremulous pulse ischemic ulcer further to increase the weight of because wound makes infection and necrosis.The mechanicalness debridement damages the wound that fragile granulation tissue growth is arranged.Bromelain debridement safety, efficient, normal tissue be without corrosiveness,

Relevant bromelain prepares the non-patent literature report of molten crust medicine first appeared in earlier 1970s, and patent literature then sees first 1974 the earliest.According to the result for retrieval 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), in published patent, there are six pieces of trainings (wherein two pieces identical) and the present invention that in various degree relevant arranged, Gerold.K.V.Klein and John.C, Houck is at the United States Patent (USP) 4 of application in 1979,226,854, name is called water solution enzyme product and carries out in devitalized tissue's debridement (DEBRUDENEBT OF DEVITALIZED TISSUE WITHHYDROLYTIC ENZYME PRODUCT) patent, having described an isoelectric point, IP is 6, at least (each molecular weight subunit is 14 to contain two subunits, the key property of enzymatic mixture 300-15,000 dalton) and carry out the method detailed of molten crust with this enzymatic 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 enzymatic mixture (ENZYME MIXTURE), and this patent proposes, the concept of molten crust enzyme (Escharase), and the molecular weight that proposes enzymatic mixture on the basis of a upper patent is 30,000-50,000 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 (METHOD OF ENZYMEDEBRIDEMENT), this patent is described 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, and 0.5-5 collagen protein is active.December in 1991 Klein on the 3rd and Houck have applied for again a new United States Patent (USP), and applied for international monopoly (PCT/US92/10395) in December in 1992 2 days by PCT, name is called: contain the proteolytic enzyme mixt of molten crust enzyme and the separation method of this material (PROTEOLYTIC MIXTURE CONTAING ESCHARASE AND METHOD OF ISOLATING SAME).This patent described a kind of entirely different in before separation method, namely with ascorbic acid (Vc) as antioxidant, (NH4) 2SO4 is as the separation method of 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 (NH4) 2SO4 saturated solution (4 * 10 ℃) that filters or centrifuging is removed insoluble matter → 5. make 40% precipitation → the 6. centrifugal or filtration of spending the night, 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 lyophilizing is weighed and is the proteolytic enzyme mixt that contains molten crust enzyme with Amicon DC-30 ultrafiltration system, the molten crust enzyme (Escharase) that contains 1%-1.5% in this mixture, the aminoacid that the inventor gives molten crust enzyme forms, and because this azymia proline and tyrosine, the inventor thinks that it is a kind of list-albumen (Unique protein), this enzyme is 15 by three molecular weight, 000 daltonian subunit forms, total molecular weight is 45,000 dalton.The inventor thinks that this patent is at yield (25%), the aspects such as stability of the content of molten crust enzyme (1%-1.5%), molten crust time (4 hours) and mixture all significantly are better than 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 less.And can obtain purer molten crust enzyme by polydextran gel SephadexG-75 or isoelectric focusing method as required.Above-mentioned several patents in separation method or condition so that in the description to molten crust enzyme though difference is arranged, following some be identical: 1. used thick enzyme all extracts from pineapple stem; 2. the molecular weight of enzymatic mixture is all at 30,000-50, in 000 dalton's scope; 3. the high activity of enzymatic mixture is 35 units of every mg enzyme preparation (under the cysteine existence condition); 4. isoelectric point, IP is generally all about 6; 5. the molten crust time is 4 hours the soonest.

Summary of the invention

The purpose of this invention is to provide a kind of product with molten crust function and preparation method thereof.

Preparation provided by the present invention has the method for the product of molten crust function, comprises the steps:

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

(2) dissolve thick enzyme with pH6.0,0.002mol/L phosphate buffer, centrifugal removal insoluble impurities after the dissolving is collected supernatant;

(3) with the 0.002mol/L phosphate buffer dialysis of supernatant with pH6.0,

(4) recentrifuge after the dialysis is collected supernatant, and be get final product.

In the said method, in the step (2), pH6.0,0.002mol/L phosphate buffer are 5ml: 1g with the ratio of thick enzyme.

In the said method, in the step (2), the method for described dissolving is that 4-10 ℃ of condition lower magnetic force stirred 1 hour.

In the said method, in the step (3), described dialysis time is 2-3 hour.

In the said method, in the step (3), molecular cut off is 3500 in the described dialysis.

In the said method, the described thick enzyme that extracts from pineapple fruit obtains as follows:

(1) the pineapple fruit pulverizing is squeezed the juice;

(2) fruit juice being transferred to pH value is 4.0;

(3) to add mass percentage concentration be 10% tanning solution 10ml for every liter of fruit juice;

(4) with the solution stirring that configures 15 minutes;

(5) standing demix;

(6) separate also collecting precipitation;

(7) drying is pulverized, and obtains thick enzyme.

The molten crust product that is prepared by said method is the present invention's product with molten crust function required for protection.

Contain Fructus Ananadis comosi PEPA A and Fructus Ananadis comosi PEPB B in the said goods, the molecular weight of Fructus Ananadis comosi PEPA A and Fructus Ananadis comosi PEPB B is respectively 27,000 and 30,000 dalton; The isoelectric point, IP of Fructus Ananadis comosi PEPA A is 9.4;

The content of Fructus Ananadis comosi PEPA A is 1524.70mg/100ml in the said goods, and the content of Fructus Ananadis comosi PEPB B is 952.60mg/100ml in the described product.

The present invention also provides a kind of capsule with molten crust function.

Capsule with molten crust function provided by the present invention is the product drying that has molten crust function with above-mentioned, and desciccate is mixed with vitamin C, and desciccate and ascorbic mass ratio are 85-90: 15-10, and get final product.

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 than prior art---and 5 times, the every gram of enzyme activity is about 600,000 units:

(2) has higher yield, generally about 70%.

(3) be conducive to commercialization production after the work simplification, product quality is more easy to control, and has reduced production cost, for the patient reduces the heavy burdens.

(4) the molten crust time short, the general 2-3 of second degree burn hour, only need 60 minutes the soonest the most clinically, and molten crust is thorough.

(5) any toxic and side effects is not found in clinical practice, and is fool proof.

(6) patient is admitted to hospital, and molten crust can be removed focus as early as possible immediately, is beneficial to hinder face and heal early.

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

(8) be beneficial to the burn degree Accurate Diagnosis, this burn treating to the extremity functional part has profound significance.

(9) convenient transportation is preserved effect duration long, 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%.

The specific embodiment

Employed experimental technique is conventional method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

The enzyme activity determination method is as follows among the following embodiment:

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

Embodiment

One, the preparation of bromelain

1, the preparation of thick enzyme

Take pineapple fruit as raw material, extract enzyme, concrete steps are as follows:

(1) pineapple fruit is pulverized and is squeezed the juice;

(2) fruit juice being transferred to pH value is 4.0;

(4) with the solution stirring that configures 15 minutes;

(5) standing demix;

(6) separate also collecting precipitation;

(7) drying is pulverized, and obtains thick enzyme.

2, the preparation of smart enzyme

1. the thick enzyme product of step 1 gained is mixed with pH6.0,0.002mol/L phosphate buffer, pH6.0,0.002mol/L phosphate buffer are 5ml: 1g with the ratio of thick enzyme product, and 4-10 ℃ of condition lower magnetic force stirred 10 1 hour, 000 rev/min centrifugal 20 minutes, collect supernatant;

2. supernatant was contained in the bag filter (molecular cut off 3500), with pH6., the dialysis of 0.002mol/L phosphate buffer 2-3 hour;

3. 10,000 rev/mins of solution after the dialysis are centrifugal 20 minutes, get supernatant;

4. the supernatant lyophilizing gets final product to get the bromelain highly finished product, and yield is about 70%.

When enzymatic solution is made lyophilized powder, adopt lyophilization or concentrated solution with cold acetone precipitation, centrifugal again low temperature natural drying;

Two, the property analysis of smart enzyme

The effective ingredient of essence enzyme is proteolytic enzyme, measure through SDS-PAGE, wherein contain two aminosal compositions, be referred to as respectively Fructus Ananadis comosi PEPA A and Fructus Ananadis comosi PEPB B, the molecular weight that SDS-PAM gel electrophoresis is measured PEPA A and PEPB B is respectively 27,000 and 30,000 dalton.The isoelectric point, IP 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 aminoacid of Fructus Ananadis comosi PEPA A and Fructus Ananadis comosi PEPB B forms

The bromelain hydrolytic enzyme highly finished product that said method obtains, hydrolytic enzyme vigor are every gram enzyme preparation 600,000 units.

Three, the preparation of capsule

Fixed according to the Chinese Pharmacopoeia two-shift system, specific as follows:

Smart enzyme is mixed with vitamin C, and smart enzyme and ascorbic mass ratio are 85-90: 15-10, and mill is mixed even, and packing is molten crust preparation dry powder finished product, and the moistureproof preservation of low temperature is stand-by.Wherein vitamin C is as activator.

Step 1 products obtained therefrom is distributed into the specification of every gram 400,000 units: 0.25g/ grain (1000); 0.2g * 10 * 2 plates/box.

Four, effect experiment---with molten crust enzyme capsule treatment tissue of burn

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

1. remove the dirt hinder on the face with Oleum Terebinthinae, clean with normal saline and hinder face, tear off and treat that molten crust hinders the epidermis on the face, such as top layer carbonization, available scalpel etc. strike off it.

2. use 1% lignocaine solution or medical PH7.0, the 0.0002mol/L phosphate buffer is reduced into pasty state with above-mentioned molten crust enzyme capsule room temperature, it is coated in equably hinders on the face, approximately 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 petechia, and with a large amount of sepages, prove that molten crust is effective, need the molten crust time short.Otherwise the molten crust time is slightly long.

4. the aseptic kpetrolatum gauze of lid layer outside molten crust medicine again, it is outer to cover 6-9 layer sterile gauze again, and the outermost layer binder is fastened.

5. observe the variation of wrapping gauze, the wrapping gauze is soaked into by courageous and upright sepage, proves molten crust success.Usually the shallow face of hindering only needs 2-3 hour, and the darker face time of hindering is slightly long, general 4-5 hour.It often is colourless sepage rather than courageous and upright thoroughly liquid that three degree are hindered face.6. open wrapping, scrape off medicine and slough with tweezers, thoroughly clean wound surface with 0.5%NaHCO3 solution again.Or dip in gauze with normal saline and clean and hinder face, expose the fresh face of hindering that is covered with active hemorrhage point, so far, molten crust process is finished, and does further treatment according to different situations and processes.

Example 1

The women, 19 years old, the electrician, clinical diagnosis is: mixed degree burn, wound surface area 4%, burned part: both hands (wherein left hand is dark II °), the cause of disease course of disease: the molten crust of electric arc burn distance 24 hours.

The molten crust process of medicine: wound surface is with the conventional method debridement, use 12 of molten crust capsules of bromelain (0.25g/ grain), add a small amount of normal saline furnishing pasty state, evenly smear and wound surface, after 1 minute oozing of blood is arranged, the gauze soak, molten crust finishes to remove dressing after 4 hours, strike off and dissolve slough, both reached the skin grafted wounds requirement with normal saline flushing.Both hands are clenched fist, are put down and stretch normally after 3 months, and the hand cicatrix obviously is less than operation and cuts crust.

Example 2

The male, 23 years old, the driver, clinical diagnosis: II °, III ° burn, the wound surface area: 12%, body part: right lower extremity, the cause of disease course of disease: the automobile of driving catches fire, and right lower extremity is admitted to hospital after 2 hours by the gasoline flame burn, the molten crust of injured distance 24 hours.The molten crust process of medicine: conventional method debridement face, with 24 bromelain capsules with a little normal saline mixing furnishing pasty state, be applied in affected part, 4 minutes blood oozing from the wound surface, dressing adds the gauze soak, and molten crust finishes to remove dressing after 5 hours, with normal saline flushing, remove dissolving thickness tissue with tongue scraper, through flushing, reached the skin grafted wounds requirement again.Leave hospital after 2 weeks, check after 3 months, the lower limb bending is stretched normally, and cicatrix obviously is less than operation.

Zhao, the man, people from Jiangxi, foot is scalded by boiled water, with still getting blister behind the ice compress, the long 10cm of wound, wide 2cm with the Scald Ointment of company's infirmary, worsened redness in three days afterwards, flow densely, Drug therapy of the present invention was used in a burst of twinge on the 4th day, took a turn for the better two days later decrustation recovery from illness after the week.

New certain, the woman, people from Zhejiang, the burn that catches fire, head and shoulder burn surface area reach 350cm ², have no improvement in Wenzhou City's hospitalize a couple of days, with medicine treatment of the present invention, only be clearly better with 2 days burn site, be used in conjunction this medicine and substantially fully recovered in 8 days, without cicatrix.

Pay certain, the woman lives people from Hubei, and long-term sick Lying bed does not rise, and boiled water is scalded, and area is very large, has two places to reach 20cm ²Above, with this medicine treatment recovery from illness in 15 days.

Wang Xiaohu, man, 3 years old.Because boiled water is scalded right lower limb, area 2%, is mixed, went to a doctor in rear 0.5 hour in scalding, covers plaster contg. tuber of hyacinth bletilla for treating burns through debridement with II degree, III degree, behind the enclosing cover sterile gauze, with bandaging, pain is namely ended, the 1st day, changed dressings in 12 hours 1 time, thereafter change dressings every day 1 time, stop to oozing out, change dressings every day 1 time, anti symptom treatment was fully recovered in 12 days simultaneously.

Zhang Yuan, woman, 48 years old.Facial area and cervical region boiled water are scalded, area 5%, the II degree is scalded, and goes to a doctor Dual-eyelid height swelling during first visit in rear 24 hours in burn, the vesicle that as seen facial area and cervical region differ in size, make a definite diagnosis eyeball section without after scalding, after debridement, cover plaster contg. tuber of hyacinth bletilla for treating burns, the eye anti symptom treatment, change dressings recovery from illness on the 4th every day 1 time.

Lang Riqin, woman, 63 years old.Thigh shank boiled water scald, II degree when having a bath, area 7%, medical in hindering rear 6 hours, two buttocks, the swelling of thigh and calf height during first visit, as seen the vesicle that differs in size, as seen low-grade infection covers plaster contg. tuber of hyacinth bletilla for treating burns, through anti symptom treatment such as anti-inflammations behind debridement, disinfection, change dressings recovery from illness in 8 days every day 1 time.

The gold city, people from Hunan, man, 14 years old.The right leg boiled water is scalded, area 2%, and II degree burn was gone to a doctor in scalding, is covered plaster contg. tuber of hyacinth bletilla for treating burns through debridement in rear 1 hour, and behind the enclosing cover sterile gauze, with bandaging, pain is namely ended, and children's's sob stops.The 1st day, changed dressings 1 time in 12 hours, changed dressings 1 time in per two days thereafter, stop to oozing out, change dressings every day 1 time, anti symptom treatment was fully recovered in 7 days simultaneously.

Zhang Wanxin, people from Shandong, man, 53 years old.The I degree, 5% scalds right lower limb, covers plaster contg. tuber of hyacinth bletilla for treating burns through debridement because of boiled water in rear 4 hours of burn is medical, and behind the enclosing cover sterile gauze, with bandaging, pain is namely ended.The 1st day, changed dressings 1 time in 12 hours, change dressings thereafter recovery from illness in 3 days every day 1 time.

Hu wind, woman, 31 years old.II, III degree, breast, abdomen, lap, area 9%, medical in rear 24 hours of burn, breast during first visit.Abdomen.The vesicle that as seen lap differs in size and one's water breaks bleb skin, the surface forms shallow in ulcer, covers plaster contg. tuber of hyacinth bletilla for treating burns after debridement, covers with sterile gauze, changes dressings recovery from illness in 19 days every day 1 time.

From vertical celebrating, man, 66 years old.In rear 4 hours of burn medical scald right lower limb, area 4%, II degree because of boiled water and scald, in scald rear 1 hour medical, cover plaster contg. tuber of hyacinth bletilla for treating burns through debridement, behind the enclosing cover sterile gauze, with bandaging, pain is namely ended.The 1st day, changed dressings 1 time in 12 hours, change dressings every day thereafter 1 time, stop to oozing out, change dressings thereafter recovery from illness in 12 days every day 1 time.

Experimental results show that: use the patient convalescent period after the molten crust of bromelain capsule short, cicatrix obviously is less than operation and cuts crust.

In order to increase the drug safety Journal of Sex Research, the bromelain capsule has been done acute toxicity test, the result shows:

Through applying with the burn of Cmax, maximum volume behind the molten crust capsule for tested treated animal, within the observation period in a week, the animal of intact skin group and damaged skin group is compared with the blank treated animal, all without any poisoning symptom and the phenomena of mortality.

Skin allergy test is the result show: animal skin excites contact 6 as a child not find anaphylaxis through observing after repeating to contact molten crust capsule again.

Claims

1. a method for preparing the product with molten crust function comprises the steps:

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

(3) with the 0.002mol/L phosphate buffer dialysis of supernatant with pH6.0,

2. method according to claim 1 is characterized in that: in the step (2), pH6.0,0.002mol/L phosphate buffer are 5ml: 1g with the ratio of thick enzyme.

3. method according to claim 1 and 2 is characterized in that: in the step (2), the method for described dissolving is that 4-10 ℃ of condition lower magnetic force stirred 1 hour.

4. method according to claim 1 and 2, it is characterized in that: in the step (3), described dialysis time is 2-3 hour.

5. method according to claim 1 and 2, it is characterized in that: in the step (3), molecular cut off is 3500 in the described dialysis.

6. method according to claim 1 and 2, it is characterized in that: the described thick enzyme that extracts from pineapple fruit obtains as follows:

(1) the pineapple fruit pulverizing is squeezed the juice;

(2) fruit juice being transferred to pH value is 4.0;

(4) with the solution stirring that configures 15 minutes;

(5) standing demix;

(6) separate also collecting precipitation;

(7) drying is pulverized, and obtains thick enzyme.

7. the molten crust product that is made by arbitrary described method among the claim 1-6.

8. product according to claim 7, it is characterized in that: contain Fructus Ananadis comosi PEPA A and Fructus Ananadis comosi PEPB B in the described product, the molecular weight of Fructus Ananadis comosi PEPA A and Fructus Ananadis comosi PEPB B is respectively 27,000 and 30,000 dalton; The isoelectric point, IP of Fructus Ananadis comosi PEPA A is 9.4;

The content of Fructus Ananadis comosi PEPA A is 1524.70mg/100ml in the described product, and the content of Fructus Ananadis comosi PEPB B is 952.60mg/100ml in the described product.

9. the capsule with molten crust function is with the described product drying of claim 7, and desciccate is mixed with vitamin C, and desciccate and ascorbic mass ratio are 85-90: 15-10, and get final product.