CN104771750A - Compound montmorillonite lysozyme powder as well as preparation method and application thereof - Google Patents
Compound montmorillonite lysozyme powder as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to compound montmorillonite lysozyme powder for treating skin ulcer, a preparation method and application thereof. The compound montmorillonite lysozyme powder is prepared from the following raw materials in parts by weight: 50-120 parts of montmorillonite, 5-10 parts of lysozyme, and 20-50 parts of hydroxy propyl cellulose. According to the invention, the montmorillonite and the lysozyme are used as the effective components and the pharmaceutic adjuvant of the compound montmorillonite lysozyme powder disclosed by the invention; and the compound montmorillonite lysozyme powder is prepared into a drug through the processes, such as a montmorillonite dry heat sterilization process, a lysozyme ultraviolet sterilization process and a hydroxy propyl cellulose ethylene oxide process. The invention further provides application of the preparation in preparation of a drug for treating skin ulcer. Due to drug delivery for skin external use, the compound montmorillonite lysozyme powder has better effects on skin ulcer in different periods.
Description
Technical field
The present invention relates to a kind of compound recipe Montmorillonitum lysozyme powder for the treatment of skin ulcer and its preparation method and application, belong to medical art.
Background technology
Chronic skin ulcer, also known as intractable ulcer, is more common in old people, especially more common in the elderly population having diabetes, vasculitis and veins of lower extremity backflow obstacle.Along with the aging trend of population and increasing of the sickness rate such as diabetes, cardiovascular disease, the sickness rate of chronic skin ulcer also rises to some extent.The skin ulcer caused because of atherosclerotic occlusive disease, diabetes, cardiovascular and cerebrovascular disease and wound clinically gets more and more, and accounts for about 3% of Surgical Inpatients.Because skin ulcer occurs in body surface, there is the features such as the course of disease is long, large and complication is many to appearance effects, great harm is caused to the quality of life of patient.
At present, the treatment of decubital ulcer skin ulcer is divided into constitutional treatment and topical therapeutic.Whole body supporting treatment, supplements high energy dietary, anemia and hypoproteinemia.Because long-term nutrition is bad, cause skin resistance low, skin elasticity weakens, and fat deposit is thinner, and immunity of organisms reduces, therefore diet nursing targetedly will be carried out to patient, give patient sufficient reasonably nutrient, meet the demand of patient to nutrition, high protein, high heat, homovitamin, rich cellulose diet be given, enhancing body resistance, the generation of pre-counteracting bedsores.Topical treatment is treated wound face, according to decubital ulcer different clinical period, takes corresponding remedy measures.
Eighties of last century eighties, medical worker from raising local immunity power, directly sterilization and antibacterial, antiinflammatory, improve local blood circulation and promote the aspects such as local organization regeneration, large quantifier elimination has been done to its short more mechanism.External used medicine Promotive union has the effects such as acidify wound surface, nutrition wound surface, immunomodulating, blood circulation promoting, bactericidal and the regeneration of promotion local organization.The mid-90 in 20th century, the further investigated mechanism of external medication intractable ulcer in capillary permeability, macrophage function, immunologically competent cell oxidative metabolism function and the dynamic studies of fibrin in wound healing etc. from wound healing, external used medicine can increase wound surface capillary permeability, promote the mutual adjustment between cell and between cell and body fluid, increase the non-specific immunity of wound surface and the repair ability of tissue, thus accelerate wound healing.The later stage nineties 20th century, molecular biological Theories and methods is used to inquire into the mechanism of external used medicine wound healing further, research shows that medicine for external use has a certain impact to some the somatomedin tool in wound tissue, and have developed the series of physical therapy such as ultraviolet irradiation treatment, laser therapy, negative aeroion and hyperbaric oxygen therapy, ultrasound therapy, galvanoiontophoresis therapy, electrical stimulating therapy.Review the past, by macroscopic view, microcosmic is deep into the research of skin ulcer, enter into molecular level by general horizontal, cellular level, move towards innovative research by confirmatory study.In new century, in fields such as Pathophysiology, cure mechanism, active dressing, new method, new thinking, new ideas, new theory will be proposed for the research of skin ulcer.
Chronic skin ulcer is also known as intractable ulcer, and be a kind of common refractory disease, it comprises vascular ulcers, pressure ulcer, radiation ulcer and infective ulcer etc.From the human body with bring great misery to patient mentally, bring huge misery and financial burden often to patient and family thereof.Therefore the extensive concern of scholars is caused.
Generally adopting the method for traditional treatment decubital ulcer to be at present clinically keeps wound dry as much as possible, to prevent bacteriological infection, absorb and get rid of wound exudate, but these methods also existing defects, wound dehydrates, incrustation can be made, in crust, be mixed with some epidermis cell, these cells are forced to shift to depths under dry crust, thus extend the agglutination of decubital ulcer.
In a word, all there is defect in various degree in above Therapeutic Method, and clinical practice is very limited.For meeting the clinical demand of extensive patients, therefore, those skilled in the art are seeking one for many years always and can develop safe and efficient, the external used medicine for the treatment of skin ulcer easy to use, i.e. compound recipe Montmorillonitum lysozyme powder, to enrich clinical application.
According to Chinese Pharmacopoeia 2010 editions and European Pharmacopoeia, American Pharmacopeia for burning or ointment, ointment, the gel of severe trauma, it should be sterile preparation.Unsterilised or the sterilizing methods of preparation crude drug, adjuvant and working condition used is talked about improperly, and easily produce secondary microbiological contamination clinically, cause the more difficult healing of wound face, therapeutic effect is very undesirable or produce larger harm to ulcer surface.In addition, be exclusively used in the compound recipe Montmorillonitum lysozyme powder agent medicine for the treatment of skin ulcer (after decubital ulcer, scald ulcer, calf ulcer, with shape herpes ulcer, diabetic ulcer, postoperative ulcer), particularly there is not been reported for compound recipe Montmorillonitum lysozyme Powder preparation.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide a kind of compound recipe Montmorillonitum lysozyme powder for the treatment of skin ulcer, the present invention also provides the preparation method and application of said preparation.
Term illustrates:
Skin ulcer of the present invention, refer to decubitus ulcer, scald ulcer, calf ulcer, ulcer, diabetic ulcer, postoperative ulcer after with shape herpes.
Lysozyme, English name Lysozyme hydrochloride, CAS accession number 9066-59-5, market can be purchased.
Technical solution of the present invention is as follows:
Treat a compound recipe Montmorillonitum lysozyme powder for skin ulcer, be made up by mass parts of following raw material:
Montmorillonitum 50 ~ 120 parts
Lysozyme 5 ~ 10 parts
Hydroxypropyl cellulose 20 ~ 50 parts;
According to the present invention, preferably, a kind of compound recipe Montmorillonitum lysozyme powder for the treatment of skin ulcer, be made up by mass parts of following raw material:
Montmorillonitum 50 ~ 100 parts
Lysozyme 5 ~ 8 parts
Hydroxypropyl cellulose 20 ~ 40 parts;
According to the present invention, further preferably;
Treat a compound recipe Montmorillonitum lysozyme powder for skin ulcer, be made up by mass parts of following raw material:
Montmorillonitum 50 parts
Lysozyme 5 parts
Hydroxypropyl cellulose 20 parts;
Or, a kind of compound recipe Montmorillonitum lysozyme powder for the treatment of skin ulcer, be made up by mass parts of following raw material:
Montmorillonitum 100 parts
Lysozyme 8 parts
Hydroxypropyl cellulose 40 parts;
Or, a kind of compound recipe Montmorillonitum lysozyme powder for the treatment of skin ulcer, be made up by mass parts of following raw material:
Montmorillonitum 75 parts
Lysozyme 7 parts
Hydroxypropyl cellulose 30 parts.
According to the present invention, the preparation method of described compound recipe Montmorillonitum lysozyme powder, comprises the steps:
(1) take Montmorillonitum by proportioning, lysozyme, each component of hydroxypropyl cellulose are for subsequent use;
(2) Montmorillonitum is put into dry heat sterilization cabinet, temperature is 250 ~ 350 DEG C of sterilizings, and sterilization time is 120min, and it is for subsequent use to be down to room temperature;
(3) hydroxypropyl cellulose is placed on ethylene oxide sterilizing cabinet and carries out sterilizing, temperature is 40 ~ 60 DEG C, and pressure is 35 ~ 50Kpa, and sterilization time is 3 ~ 6h, for subsequent use;
(4) lysozyme is put into ultraviolet disinfection cabinet and carry out sterilizing, ultra-violet bands controls in UV-C 250 ~ 270nm scope, and sterilization time is 20 ~ 30min, for subsequent use;
(5) aseptically, by the mixing of materials that step (2), (3), (4) obtain, stir;
(6) the said goods is carried out aseptic packaging, inspection, warehouse-in, to obtain final product.
It should be noted that, in above preparation method, all pipelines for mass transport must pass through sterilization treatment in advance.The equipment used in preparation method all conventionally.
The present invention also provides the application of compound recipe Montmorillonitum lysozyme powder in the medicine of preparation treatment skin ulcer.Described skin ulcer, comprise decubitus ulcer, scald ulcer, calf ulcer, ulcer, diabetic ulcer, postoperative ulcer after with shape herpes.
With regard to preparation method, be more than the preferred method of the present invention, but be not limited thereto.The knowledge that those skilled in the art knows according to it, can suitably be adjusted.For what be particularly limited in the above compound recipe Montmorillonitum lysozyme Powder preparation preparation method, can refer to prior art.
In preparation method of the present invention, compound recipe Montmorillonitum lysozyme Powder preparation quality standard is following but be not limited thereto.
Compound recipe Montmorillonitum lysozyme powder quality standard
This product should be 97.0% ~ 110.0% of labelled amount containing Montmorillonitum; Lysozyme should be 94.0% ~ 110.0% of labelled amount
[shape] this product is off-white color powder
[discriminating]
(1) get this product appropriate (being equivalent to 2mg lysozyme), the 5ml that adds water makes dissolving, adds 10% (w/w) sodium hydroxide solution 5 and 10% (w/w) copper-bath 1, displaing amaranth after mixing.
(2) get this product appropriate, (get anhydrous sodium acetate 6.7g, add water about 900ml, and jolting makes dissolving, by acetic acid adjust ph to 5.4, is diluted with water to 1000ml, shakes up to add Acetic acid-sodium acetate buffer.) make the solution of lysozyme 0.4mg in every 1ml, measure according to spectrophotography " Chinese Pharmacopoeia version in 2010 two annex IV A ", have absorption maximum at the wavelength place of 280nm, trap should be 0.39 ~ 0.49.
(3) 4g Montmorillonitum externally-applied soft ointment is got, after the 50ml that adds water stirs, filter, get filter cake 300ml water and repeatedly rinse three times, get filter cake 105 DEG C of oven dry, the above-mentioned this product that obtains is got in right amount, (exsiccator filled sodium chloride saturated solution in about 12 hours to put into exsiccator, relative humidity about 78% when 20 DEG C), then above-mentioned test sample is measured according to x-ray powder diffraction (Chinese Pharmacopoeia version in 2000 two annex Ⅸ F), record collection of illustrative plates, the X-ray diffracting spectrum of test sample should be consistent with reference substance collection of illustrative plates.
The characteristic spectral line of Montmorillonitum is at about 1.5nm and 0.45nm wavelength place, and in collection of illustrative plates, other Impurity Absorption peak intensities must not higher than second of a Montmorillonitum characteristic peak (about 0.45nm).
[inspection] granularity checks according to granularity and particle size distribution method " Chinese Pharmacopoeia version annex Ⅸ E first method in 2010 ", all must not detect the particle being greater than 180 μm.
Aseptic according to Sterility Test " Chinese Pharmacopoeia version annex Ⅺ H in 2010 " inspection, should conform with the regulations.
Other requirements under other this product should meet " Chinese Pharmacopoeia version annex I F in 2010 " ointment item.
[lysozyme content]
The preparation of need testing solution takes powder appropriate (being equivalent to lysozyme 25mg), puts in test tube, after the 20ml that adds water stirs, filter, get filter cake 80ml water and repeatedly rinse three times, filtrate is incorporated in 100 milliliters of measuring bottles, determines scale by pH6.2 phosphate buffered solution.Precision pipettes 2mL and puts in 50ml volumetric flask, adds pH6.2 phosphate buffer to scale, for subsequent use as need testing solution.
Standard solution is prepared precision and is taken lysozyme standard product 25 milligrams, puts in 100ml volumetric flask, adds phosphate buffered solution and dissolves, be settled to scale.Precision pipettes 1mL, 2mL solution and puts in 50ml volumetric flask respectively, adds pH6.2 phosphate buffered solution to scale, as standard solution (1) and standard solution (2).Sample solution, standard solution (1) standard solution (2) is put in ice bath and is preserved.
The preparation of substrate suspension takes lyase micrococcus 15 ~ 20mg, add phosphate buffer (pH6.2) 0.5 ~ 1ml, grind 3 minutes in mortar, add phosphate buffer (pH6.2) more appropriate, cumulative volume is made to be about 50ml, make suspension in 25 ± 0.1 DEG C time, the trap recorded at the wavelength place of 450nm is 0.70 ± 0.05 (prepared before use).
Algoscopy precision measures lysozyme substrate test solution 4ml, heat 5 minutes in 35 DEG C of water-baths, precision pipettes the sample solution 100 μ l heated in advance in water-bath 3 minutes, add in substrate test solution, and heat in 35 DEG C of water-baths after 10 minutes, the accurate hydrochloric acid solution 0.5mL measuring 1mol/L adds the end wherein again, shakes up.With water as blank, at wavelength 640nm place, the absorption value Ast of UV-VIS spectrophotometry sample solution.Standard solution (1), standard solution (2) measure absorbance A s by above method
1, As
2.
Ws: the lysozyme standard product amount of taking.
Wt: the sample amount of taking × specification.
As
1: standard solution (1) trap.
As
2: standard solution (2) trap.
[smectite content]
Get 4g compound recipe Montmorillonitum lysozyme powder, after the 50ml stirring and dissolving that adds water, filter, get filter cake 1000ml water and repeatedly rinse three times, get filter cake and be dried to constant weight at 105 DEG C, obtain the weight of contained Montmorillonitum in test sample, calculate, to obtain final product.
The compound recipe Montmorillonitum lysozyme powder for the treatment of skin ulcer of the present invention, its employing Montmorillonitum and lysozyme are the effective ingredient of medicine of the present invention, medicament is made with pharmaceutic adjuvant, through external preparation for skin drug administration, to the decubital ulcer of different times still to the treatment of the skin ulcer that diabetic complication causes, all show it and have the effect that height oozes, sterilizes, wherein possessed feature is as follows:
1, the lysozyme in compound recipe Montmorillonitum lysozyme powder has and promotes the proliferation function of fibrous bud cell, promotes tentative Wound Healing, promotes the pharmacological actions such as connective fiber formation effect.Active component lysozyme in compound recipe Montmorillonitum lysozyme powder is a kind of mucopolysaccharide lyase, its by hydrolysis destroy composition bacteria cell wall peptide glycan molecule by the β between-acetylmuramic acid and 2-Acetamido-2-deoxy-D-glucose ammonia-(1,4) glycosidic bond, cell is broken because osmotic pressure imbalance causes, thus causes somatic cells wall dissolve and kill antibacterial.
2, the Montmorillonitum in compound recipe Montmorillonitum lysozyme powder is a kind of safety non-toxic natural drug, after medication, is not less readily available for absorption by the skin, do not enter blood circulation, and Montmorillonitum is partially formed elecrtonegativity, then positively charged between layers, CHARGE DISTRIBUTION is polymorphic micro phase separation structure.Like this, by electrostatic interaction, Montmorillonitum can produce and interact with charging property biomolecule and antibacterial, virus, toxin, is fixed, removes; Reach putrefaction removing, granulation promoting, promotion ulcer healing.And spread on wound surface outside Montmorillonitum, by Montmorillonitum in the outer adsorption moisture effect of edematous tissue, change the osmotic pressure of local, thus by excessive moisture sucking-off in edematous tissue, topical wounds edema degree can be alleviated, accelerate granulation tissue and generate, healing acceleration, can reach the object of convergence granulation promoting.
3, Montmorillonitum associating lysozyme treatment skin ulcer, plays synergism.Be characterized in that antibacterial power is strong, tasteless, stable performance, nonirritant, toxicity be low, can anti-bacteria, virus, fungus, spore and protozoon etc., to the noiseless effect of wound healing, application surpasses other any one Wound care products.Add Montmorillonitum in invention formulation, play stability action on the one hand to lysozyme, persistence slow releasing lysozyme is to reach the object extending drug effect on the other hand.The two use in conjunction thus promote the quickly-healing of decubital ulcer.Be made into combined dosage form and meet the feature of drug medication and the feature of clinical treatment completely.
The compound recipe Montmorillonitum lysozyme powder sterile preparation of the external used medicine for the treatment of skin ulcer of the present invention (after decubital ulcer, scald ulcer, calf ulcer, with shape herpes ulcer, diabetic ulcer, postoperative ulcer), employing Montmorillonitum, lysozyme are the effective ingredient of medicine of the present invention, with selected pharmaceutic adjuvant, by Montmorillonitum dry heat sterilization, lysozyme ultraviolet disinfection, hydroxypropyl cellulose ethylene oxide sterilizing, make medicament etc. technique.The present invention also provides the application of said preparation in preparation treatment skin ulcer medicine.Through external preparation for skin drug administration, to the skin ulcer of different times, all there is good effect.Effect experimental specifically will be explained in experimental example.
Below in conjunction with experimental example, the present invention is described further, but be not limited thereto.
Experimental example 1: Montmorillonitum sterile test
In the present invention, active component Montmorillonitum is the layer mineral be made up of the hydrous alumino silicates that granule is superfine, is formed by basic igneous rock air slaking in alkaline environment, also has plenty of the product after the volcanic ash decomposition of sea-bottom deposit.According to Chinese Pharmacopoeia 2010 version (annex IF) regulation, during skin for large-area burns or severe trauma, aseptic requirement should be reached.Therefore, this preparation supplementary material needs sterilizing, and obtained sterile preparation in gnotobasis, for Clinical practice.Montmorillonitum sterilizing methods in the market all adopts Co
60method of radiating, the relevant Co of health ministry
60in irradiation sterilization dosage standard " defend medicine and send out 1997 No. 38 " notice, Co
60irradiation sterilization maximal dose is 6KGY.Because Montmorillonitum is a kind of mineral medicine, Co
60irradiation dose 6KGY cannot penetrate Montmorillonitum, causes sterilization effect poor, and is only improved Co
60irradiation dose is 15KGY, considerably beyond the 6KGY maximum dose level that health ministry specifies.Research Montmorillonitum sterilizing methods is under guaranteeing that montmorillonite medicine meets the prerequisite of quality standard, ensures that montmorillonite medicine can be used safely, has great strategic structural.
Different dry heat temperature and xeothermic time to the comparison of Montmorillonitum dry heat sterilization effect, in table 1:
Table 1 Montmorillonitum dry heat sterilization effectiveness comparison
Draw by analyzing in table 1:
1; Dry heat temperature is at 160 ~ 170 DEG C, 170 ~ 180 DEG C, and drying time, Montmorillonitum X-ray diffracting spectrum met national standard WS under 120 ~ 240min condition
1-(X-165)-2004Z specifies, shows xeothermicly with this understanding not destroy Montmorillonitum chemical constitution, but sterility test, against regulation.
2; Dry heat temperature is at 250 ~ 350 DEG C, and the xeothermic time, sterility test, conformed with the regulations at 180 ~ 240min, but destroyed Montmorillonitum chemical constitution, referred to Fig. 1 Montmorillonitum X-ray diffracting spectrum.
3; When dry heat temperature is more than 350 DEG C, the xeothermic time, sterility test, conformed with the regulations at 120min, but destroyed Montmorillonitum chemical constitution.
4; Dry heat temperature is at 250 ~ 350 DEG C, and the xeothermic time, Montmorillonitum X-ray diffracting spectrum met national standard WS at 120min
1-(X-165)-2004Z specifies, shows xeothermicly with this understanding not destroy Montmorillonitum chemical constitution, and sterility test, conforms with the regulations, and refers to Fig. 2 Montmorillonitum X-ray diffracting spectrum.
Experimental example 2: hydroxypropyl cellulose sterile test
Different ethylene oxide sterilizing pressure and time to the comparison of hydroxypropyl cellulose sterilization effect, in table 2.
Table 2 hydroxypropyl cellulose ethylene oxide sterilizing effectiveness comparison
Can analyze by table 3:
1. ethylene oxide sterilizing pressure is at 10 ~ 20Kpa, the time under 3 ~ 6h condition, sterility test, against regulation.
2. ethylene oxide sterilizing pressure is at 20 ~ 30Kpa, the time under 3 ~ 4h condition, sterility test, against regulation.
3. ethylene oxide sterilizing pressure is at 20 ~ 30Kpa, and the time, sterility test, conformed with the regulations under 6h condition.
4. ethylene oxide sterilizing pressure is at 35 ~ 50Kpa, and the time, sterility test, conformed with the regulations under 3 ~ 6h condition.
Experimental example 3: lysozyme sterile test
Different ultraviolet disinfection wave bands and time to the comparison of lysozyme sterilization effect, in table 3.
Table 3 lysozyme ultraviolet disinfection effectiveness comparison
1. lysozyme sterilizing wave band is at A band 320 ~ 400nm, the time under 10 ~ 30min condition, sterility test, against regulation.
2. lysozyme sterilizing wave band is at B wave band 275 ~ 320nm, the time under 10 ~ 30min condition, sterility test, against regulation.
3. lysozyme sterilizing wave band is at C-band 200 ~ 275nm, the time under 10min condition, sterility test, against regulation.
4. lysozyme sterilizing wave band is at C-band 200 ~ 275nm, and the time, sterility test, conformed with the regulations under 20 ~ 30min condition.
Experimental example 4, the acute toxicity test of compound recipe Montmorillonitum lysozyme powder rabbit dialogue rabbit skin
1 material
1.2 reagents: compound recipe Montmorillonitum lysozyme powder; Prepared by embodiment 1; Blank: hydroxypropyl cellulose;
1.3 animals: female Japan large ear rabbit (rabbit) 12, body weight 2.0 ~ 2.5kg, purchased from Shandong University's experimental animal center;
1.2 method
1.2.1 12 white rabbits are divided into 3 groups by skin sensibiligen experiment at random, often organize 4.Experiment condition is temperature 18 ~ 29 DEG C, relative humidity 40% ~ 70%, first back depilation 50cm × 50cm on the right side of white rabbit, after 24h, A group is in depilation district's coating compound recipe Montmorillonitum lysozyme powder 0.1g (2g/kg), B group smears compound recipe Montmorillonitum lysozyme powder 0.2g (4g/kg) in depilation district, and C group (matched group) smears hydroxypropyl cellulose 0.1 (2g/kg) in depilation district; All immobilization with adhesive tape is used after coating.Remove the depilation left drug in district and hydroxypropyl cellulose with clear water after coating 24h, continuous 7d observes each group of skin, hair, oral mucosa, diet, eyes and changes of body mass, and extremity are movable and central nervous system with or without poisoning manifestations, with or without death.As to intact skin non-toxic reaction, then on the left of white rabbit, depilation district in back causes scratch, makes skin occur intensive petechia, and at scratch site coating, its dosage and observation index the same.
1.2.3 evaluation methodology
Skin sensibiligen is tested: the behavioral activity of main detection rabbit, skin, diet, weight and death condition.
1.2.4 statistical method adopts SPSS160 statistical software, and measurement data represents with x ± s, compares and check with t between group; Enumeration data percentage ratio represents, compares and use χ between group
2inspection.There is statistical significance P≤0.05 for difference.
2 results
After 2.1 skin sensibiligen result coatings 1 week, 3 groups of white rabbit diet, oral mucosa, eyes, skin and hair, extremity activity are all without exception, changes of body mass little (see table 4), without central nervous system's Novel presentation and systemic toxicity profiles reaction, without dead.
Table 43 groups of white rabbit medication forebody-afterbody mass change compare (kg,
)
Note: compare before and after the medication of A, B, C group, p > 0.05
Result shows: compound recipe Montmorillonitum lysozyme powder low dose (2g/kg), heavy dose of (4g/kg) spread upon on the complete or damaged skin of white rabbit, all do not cause acute toxic reaction; The safety of pointing out compound recipe Montmorillonitum lysozyme powder to be used for the treatment of skin ulcer is high, untoward reaction is little, has good application prospect.
Experimental example 5: compound recipe Montmorillonitum lysozyme powder local irritation test
1 materials and methods
1.1 material compound recipe Montmorillonitum lysozyme powders, prepared by embodiment 1, room temperature is preserved; Blank: hydroxypropyl cellulose;
Rabbit 4 is used in 1.2 laboratory animal health experiments, and 1.5 ~ 2.0kg, is provided by Shandong University's Experimental Animal Center, and single cage is raised, and adapts to 3d before test in experimental animal room environment, and conventional feed is fed, and drinking-water does not limit.24h before 1.3 medication tests, cut by hair spinal column both sides, laboratory animal back, unhairing scope is 2cmx3cm.Get compound recipe Montmorillonitum lysozyme powder 0.5g, even application is on left side depilation skin, hydroxypropyl cellulose is applied in contrast in right side, drug of topical application area 2.5cmx2.5cm, then two layers of gauze (2.5cm × 2.5cm) and one deck cellophane or analog are used to cover, fixed with nonirritant adhesive plaster and binder, adopt blocking test, the drug of topical application time is 4h again.
1.4 evaluation of result
(1) skin wound repair scoring: after drug of topical application 4h, with warm saline cleaning drug of topical application position, both sides, respectively at remove 1 after tested material, 24,48,72h, observe the reaction of both sides drug of topical application area skin.Carry out dermoreaction scoring by table 5, calculate each observation time point top.
Table 5 skin wound repair is marked
| Dermoreaction | Integration |
| Erythema and eschar are formed | |
| Without erythema | 0 |
| Slight erythema (visible reluctantly) | 1 |
| Obvious erythema | 2 |
| Moderate ~ severe erythema | 3 |
| Severe erythema (aubergine) is formed to slight eschar | 4 |
| Edema is formed | |
| Without edema | 0 |
| Slight edema (visible reluctantly) | 1 |
| Mild edema (cutaneous protuberance profile is clear) | 2 |
| Intermediate edema (cutaneous protuberance is about 1mm) | 3 |
| Severe edema's (cutaneous protuberance more than 1mm, expanded range) | 4 |
| Top | 8 |
(2) every day every animal average integral: by skin irritation strength grading.Nonirritant: <0.5; Subexcite: 0.5 ~ 2.0; Moderate stimulation: 2.0 ~ 6.0; Strong and stimulating: >6.0.Integration is by following formulae discovery.Every animal is put down at every turn
1.5 this test statistics of statistical method data all with building database form typing SPSS 13.0 statistical software, test data with
represent, adopt t inspection, inspection level ɑ=0.05.
2 results
Result of the test shows, after left side drug of topical application compound recipe Montmorillonitum lysozyme powder man rabbit back depilation skin 4h, left side skin wound repair integration compares with right side, no matter be at every turn every animal integration, or every animal integral mean value, difference that there are no significant (P>0.05), therefore thinks that compound recipe Montmorillonitum lysozyme powder is for the no significant difference (P=0.670) compared with hydroxypropyl cellulose of its zest during family's rabbit back, safe and reliable, in table 6.
Table 6 compound recipe Montmorillonitum lysozyme powder is to rabbit skin wound repair integration result of the test
The response situation that when this test is long by observation rabbit intact skin, the external application of contact compound recipe Montmorillonitum lysozyme powder produces illustrates the skin wound repair degree of compound recipe Montmorillonitum lysozyme powder.Result shows, and rabbit no abnormality seen changes, drug of topical application position does not also occur, edema, petechia, pigmentation and pachylosis etc.The test no significant difference (P>0.05) compared with hydroxypropyl cellulose.When compound recipe Montmorillonitum lysozyme powder is used for family's rabbit back, its zest, safe and reliable, compound recipe Montmorillonitum lysozyme powder clinical practice are the good external preparation of a kind of safety.
Experimental example 6: the long-term stable experiment of compound recipe Montmorillonitum lysozyme powder
Compound recipe Montmorillonitum lysozyme powder embodiment 1 prepared carries out long-term stable experiment under the storage requirement of listing regulation, investigate the stability features of compound recipe Montmorillonitum lysozyme powder in transport, preservation, use procedure, thus as determining the foundation of effect duration and storage requirement.To aluminum-plastic packaged compound recipe Montmorillonitum lysozyme powder sample be adopted, be positioned over 25 DEG C ± 2 DEG C, the constant temperature of RH60% ± 10%, place 24 months in constant humidity cabinet, respectively at sampling in 0,3,6,9,12,18,24 month, detect every quality index.The results are shown in Table 7.
Table 7 compound recipe Montmorillonitum lysozyme powder long-term stable experiment
Result: through the long-time stability investigation of 24 months, result display compared indices without significant change with 0 month, show compound recipe Montmorillonitum lysozyme powder 25 DEG C ± 2 DEG C, RH60% ± 10% condition stability inferior is good, under the terms and conditions of regulation, production, packaging, storage, transport all can not have a negative impact to the quality of this product, can ensure that clinical drug safety is effective.
Experimental example 7: compound recipe Montmorillonitum lysozyme powder act as the pharmacological effect comparative test of index to the antibacterial action of staphylococcus aureus and wound healing
This test is the use of the guinea pig skin infection model of staphylococcus aureus, has carried out the therapeutic test acting as index with its antibacterial action and wound healing.
1. test material
(1) reagent: compound recipe Montmorillonitum lysozyme powder, prepared by embodiment 1 method.
(2) blank product (base): hydroxypropyl cellulose.
(3) contrast agents: compound recipe Montmorillonitum lysozyme ointment, Shandong Sibangde Pharmaceutical Co., Ltd. provides.
(4) staphylococcus aureus: Shandong Prov. Sanitation and Antiepidemic Station.
2. laboratory animal
Male guinea pig (body weight: 320 ~ 360g) 30; Shandong University's animal experimental center.
3. the formation of experimental group
Cavia porcellus is 30 of random selection, tests according to the constituted mode of following experimental group:
Grouped table tested by table 8
4. test method
Shave off with the hair of shaver by Cavia porcellus abdominal part in the previous day of test, then with depilation paraffin, hair is removed.Matched group (infecting contrast) and blank product are applied and smears group, a position is respectively selected in left side and right side, with contrast agents deposited group of smearing, a position, right side is then respectively selected for the deposited group of smearing of reagent agent, suddenly take off again after using the square adhesive tape of 2cm (NICHIBAN) to paste, and repeat 5 ~ 6 these operations.The inoculation staphylococcus aureus bacterium liquid above-mentioned skin abrasion position made being smeared separately 50 μ L is inoculated.By infection site pastille can adhesive bandage carry out 20 hours fixing infect process to make.Take the external transdermal skin administering mode same with clinical administration, 24th hour after inoculation bacterium, by a corpse or other object for laboratory examination and chemical testing twice daily each 0.1g smear 7 days continuously at skin surface.
5. result
Reagent agent is applied and smears group and contrast agents is deposited smears group, in Macroscopic and culture experiment, test negative rate, see Fig. 3, all can judge that two kinds of preparations are equal biologically.
Experimental example 8: the pharmacological effect comparative test acting as index cured by compound recipe Montmorillonitum lysozyme powder to the scald of Rat Scald Model
This test cures with the scald of the scalding model of rat the therapeutic test acting as index and carry out.
1. test material
(1) reagent: compound recipe Montmorillonitum lysozyme powder, prepared by embodiment 1 method.
(2) blank product: hydroxypropyl cellulose.
(3) contrast agents: compound recipe Montmorillonitum lysozyme ointment, Shandong Sibangde Pharmaceutical Co., Ltd. provides.
2. laboratory animal
Wistar class male rat 30, Shandong University's animal experimental center.
3. the formation of experimental group
By random for 30 animals be 1 group with 10, test according to the formation of following experimental group:
Grouped table tested by table 9
4. test method
At use (pentobarbital sodium, Shanghai Xinya Pharmaceutical Industry Co. Ltd., 400mg/kg, i.p.) carry out, when anaesthetizing, back part of animal skin is carried out defeathering, with rubbing alcohol by after skin degerming, use the flatiron burning heat on median line, make 1 place and scald (200 DEG C, 5s, diameter 1cm) disposal.In scald after 24 hours, use each 50mg of animal subject to apply continuously twice daily and smear 10 days, each time period of during this period the 0th, 1,3,5,7,10 and the 14th day, the area of scalding position is measured, takes the external transdermal skin administering mode same with clinical administration.
5. result
Reagent group compound recipe Montmorillonitum lysozyme powder is applied and smears group and contrast agents compound recipe Montmorillonitum lysozyme ointment is deposited smears group, in the healing effect calculated by scalding area and scald in site tissue at skin of back and detect on hydroxyproline content, all really can judge that two kinds of preparations are equivalent biologically.See Fig. 4, Fig. 5.
Accompanying drawing explanation
Fig. 1 be baking temperature 250 ~ 350 DEG C, drying time at 180 ~ 240min, Montmorillonitum X-ray diffracting spectrum; Wherein, abscissa is 2 θ (°), and vertical coordinate is intensity (arbitrary unit);
Fig. 2 be baking temperature 250 ~ 350 DEG C, drying time at 120min, Montmorillonitum X-ray diffracting spectrum; Wherein, abscissa is 2 θ (°), and vertical coordinate is intensity (arbitrary unit);
Fig. 3 is culture experiment negative rate figure in the test of the therapeutic effect of infection of staphylococcus aureus model for Cavia porcellus, and wherein abscissa is culture experiment negative rate %, vertical coordinate: 1 contrast agents group (contrast medicine), 2 reagent groups (by testing medicine), 3 blank product groups (base), 4 matched groups (infecting contrast);
Fig. 4 is for the effective percentage figure in the test of After Scalding In Rat model therapeutic effect, and wherein abscissa is treatment natural law, vertical coordinate effective percentage (%);
Fig. 5 scalds hydroxyproline content figure in position in the test of After Scalding In Rat model therapeutic effect, wherein abscissa be hydroxyproline content mg/g, vertical coordinate 1, contrast agents group (contrast medicine), 2, reagent group (by testing medicine), 3, blank product group (base).
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described further, but be not limited thereto.
Embodiment 1 ~ 3 supplementary material illustrates:
Device involved in embodiment and equipment are powder and produce common apparatus, and market can be purchased.Be described as follows:
Dry heat sterilization cabinet (model HDA500) yangtze river in nanjing Yao Ji Manufacturing Co., Ltd is on sale; Full automatic epoxy ethane cabinet: model (200L) Hua County red sun Medical Devices Co., Ltd. is on sale; Batch mixer (model SYH-800), ultraviolet disinfection cabinet (specification: 160L) Taizhou Hong Di medical equipment factory are on sale; Powder racking machine (model DXDC-F60) ten thousand moral Huifeng package packing machine company is on sale.
Embodiment 1, compound recipe Montmorillonitum lysozyme Powder preparation and preparation
1, raw and auxiliary material inventory proportioning (w/w):
Montmorillonitum 50 parts
Lysozyme 5 parts
Hydroxypropyl cellulose 20 parts
2, be prepared as follows:
(1) take Montmorillonitum by proportioning, lysozyme, each component of hydroxypropyl cellulose are for subsequent use;
(2) Montmorillonitum is put into dry heat sterilization cabinet, temperature is 250 DEG C of sterilizings, and sterilization time is 120min, and it is for subsequent use to be down to room temperature;
(3) hydroxypropyl cellulose is placed on ethylene oxide sterilizing cabinet and carries out sterilizing, temperature is 40 DEG C, and pressure is 50Kpa, and sterilization time is 3h, for subsequent use;
(4) lysozyme is put into ultraviolet disinfection cabinet and carry out sterilizing, ultra-violet bands controls in UV-C 253nm scope, and sterilization time is 20min, for subsequent use;
(5) aseptically, step (2) (3) (4) are mixed, stirs;
(6) the said goods is carried out aseptic packaging, inspection, warehouse-in.Obtained compound recipe Montmorillonitum lysozyme Powder preparation product.
Testing result:
1. differentiate: (1) Montmorillonitum, (2) lysozyme, be all positive.
2. content (w/w): Montmorillonitum 98.33%, lysozyme 98.65%.
3. other index of compound recipe Montmorillonitum lysozyme powder all conforms with the regulations.
Embodiment 2, compound recipe Montmorillonitum lysozyme Powder preparation and preparation
1, raw and auxiliary material inventory proportioning (w/w):
Montmorillonitum 100 parts
Lysozyme 8 parts
Hydroxypropyl cellulose 40 parts
2, be prepared as follows:
(1) take Montmorillonitum by proportioning, lysozyme, each component of hydroxypropyl cellulose are for subsequent use;
(2) Montmorillonitum is put into dry heat sterilization cabinet, temperature is 350 DEG C of sterilizings, and sterilization time is 120min, and it is for subsequent use to be down to room temperature;
(3) hydroxypropyl cellulose is placed on ethylene oxide sterilizing cabinet and carries out sterilizing, temperature is 60 DEG C, and pressure is 35Kpa, and sterilization time is 6h, for subsequent use;
(4) lysozyme is put into ultraviolet disinfection cabinet and carry out sterilizing, ultra-violet bands controls in UV-C 270nm scope, and sterilization time is 30min, for subsequent use;
(5) aseptically, step (2) (3) (4) are mixed, stirs;
(6) the said goods is carried out aseptic packaging, inspection, warehouse-in.Obtained compound recipe Montmorillonitum lysozyme Powder preparation product.
Testing result:
1. differentiate: (1) Montmorillonitum, (2) lysozyme, be all positive.
2. content (w/w): Montmorillonitum 98.81%, lysozyme 98.23%.
3. other index of compound recipe Montmorillonitum lysozyme powder all conforms with the regulations.
Embodiment 3, compound recipe Montmorillonitum lysozyme Powder preparation and preparation
1, raw and auxiliary material inventory proportioning (w/w):
Montmorillonitum 75 parts
Lysozyme 7 parts
Hydroxypropyl cellulose 30 parts
2, be prepared as follows:
(1) take Montmorillonitum by proportioning, lysozyme, each component of hydroxypropyl cellulose are for subsequent use;
(2) Montmorillonitum is put into dry heat sterilization cabinet, temperature is 300 DEG C of sterilizings, and sterilization time is 120min, and it is for subsequent use to be down to room temperature;
(3) hydroxypropyl cellulose is placed on ethylene oxide sterilizing cabinet and carries out sterilizing, temperature is 50 DEG C, and pressure is 40Kpa, and sterilization time is 4h, for subsequent use;
(4) lysozyme is put into ultraviolet disinfection cabinet and carry out sterilizing, ultra-violet bands controls in UV-C 260nm scope, and sterilization time is 30min, for subsequent use;
(5) aseptically, step (2) (3) (4) are mixed, stirs;
(6) the said goods is carried out aseptic packaging, inspection, warehouse-in.Obtained compound recipe Montmorillonitum lysozyme Powder preparation product.
Testing result:
1. differentiate: (1) Montmorillonitum, (2) lysozyme, be all positive.
2. content (w/w): Montmorillonitum 98.81%, lysozyme 98.23%.
3. other index of compound recipe Montmorillonitum lysozyme powder all conforms with the regulations.
Claims (8)
1. treat a compound recipe Montmorillonitum lysozyme powder for skin ulcer, it is characterized in that, be made up by mass parts of following raw material:
Montmorillonitum 50 ~ 120 parts
Lysozyme 5 ~ 10 parts
Hydroxypropyl cellulose 20 ~ 50 parts.
2. compound recipe Montmorillonitum lysozyme powder as claimed in claim 1, is characterized in that, be made up of following raw material by mass parts:
Montmorillonitum 50 ~ 100 parts
Lysozyme 5 ~ 8 parts
Hydroxypropyl cellulose 20 ~ 40 parts.
3. compound recipe Montmorillonitum lysozyme powder as claimed in claim 2, is characterized in that, be made up of following raw material by mass parts:
Montmorillonitum 50 parts
Lysozyme 5 parts
Hydroxypropyl cellulose 20 parts.
4. compound recipe Montmorillonitum lysozyme powder as claimed in claim 2, is characterized in that, be made up of following raw material by mass parts:
Montmorillonitum 100 parts
Lysozyme 8 parts
Hydroxypropyl cellulose 40 parts.
5. compound recipe Montmorillonitum lysozyme powder as claimed in claim 2, is characterized in that, be made up of following raw material by mass parts:
Montmorillonitum 75 parts
Lysozyme 7 parts
Hydroxypropyl cellulose 30 parts.
6. the preparation method of the compound recipe Montmorillonitum lysozyme powder as described in as arbitrary in Claims 1 to 5, comprises the steps:
(1) take Montmorillonitum by proportioning, lysozyme, each component of hydroxypropyl cellulose are for subsequent use;
(2) Montmorillonitum is put into dry heat sterilization cabinet, temperature is 250 ~ 350 DEG C of sterilizings, and sterilization time is 120min, and it is for subsequent use to be down to room temperature;
(3) hydroxypropyl cellulose is placed on ethylene oxide sterilizing cabinet and carries out sterilizing, temperature is 40 ~ 60 DEG C, and pressure is 35 ~ 50Kpa, and sterilization time is 3 ~ 6h, for subsequent use;
(4) lysozyme is put into ultraviolet disinfection cabinet and carry out sterilizing, ultra-violet bands controls in UV-C 250 ~ 270nm scope, and sterilization time is 20 ~ 30min, for subsequent use;
(5) aseptically, by the mixing of materials obtained by step (2), (3), (4), stir;
(6) the said goods is carried out aseptic packaging, inspection, warehouse-in, to obtain final product.
7. the application of the compound recipe Montmorillonitum lysozyme powder as described in as arbitrary in Claims 1 to 5 in the medicine of preparation treatment skin ulcer.
8. apply as claimed in claim 7, wherein said skin ulcer comprise decubitus ulcer, scald ulcer, calf ulcer, ulcer, diabetic ulcer, postoperative ulcer after with shape herpes.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111686243A (en) * | 2020-07-16 | 2020-09-22 | 上海中华药业有限公司 | Composition for inhibiting oral pathogenic bacteria and application thereof |
| CN111840126A (en) * | 2020-07-16 | 2020-10-30 | 上海中华药业有限公司 | Oral care agent with antibacterial effect and preparation method thereof |
| CN113461575A (en) * | 2021-06-30 | 2021-10-01 | 万华化学集团股份有限公司 | Taurine crystallization and mother liquor sterilization method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1698890A (en) * | 2005-05-20 | 2005-11-23 | 张华� | Powder of human lysozyme, its production method and application |
| CN101449659A (en) * | 2007-12-03 | 2009-06-10 | 陈健雄 | Environment regulating agent and production method thereof |
| CN102178939A (en) * | 2011-05-06 | 2011-09-14 | 山东司邦得制药有限公司 | Muramidase hydrolase external sterile cream for treating skin ulcer and preparation method thereof |
-
2015
- 2015-04-01 CN CN201510152120.8A patent/CN104771750A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1698890A (en) * | 2005-05-20 | 2005-11-23 | 张华� | Powder of human lysozyme, its production method and application |
| CN101449659A (en) * | 2007-12-03 | 2009-06-10 | 陈健雄 | Environment regulating agent and production method thereof |
| CN102178939A (en) * | 2011-05-06 | 2011-09-14 | 山东司邦得制药有限公司 | Muramidase hydrolase external sterile cream for treating skin ulcer and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 泰安中敖918: "蒙脱石粉应用领域", 《百度文库》 * |
| 蒋卫健: "蒙脱石散治疗慢性皮肤溃疡", 《中外健康文摘》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111686243A (en) * | 2020-07-16 | 2020-09-22 | 上海中华药业有限公司 | Composition for inhibiting oral pathogenic bacteria and application thereof |
| CN111840126A (en) * | 2020-07-16 | 2020-10-30 | 上海中华药业有限公司 | Oral care agent with antibacterial effect and preparation method thereof |
| CN113461575A (en) * | 2021-06-30 | 2021-10-01 | 万华化学集团股份有限公司 | Taurine crystallization and mother liquor sterilization method |
| CN113461575B (en) * | 2021-06-30 | 2022-08-02 | 万华化学集团股份有限公司 | Taurine crystallization and mother liquor sterilization method |
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Application publication date: 20150715 |