CN105233248A - Application of uroacitides to preparation of cicatrix treatment medicines - Google Patents
Application of uroacitides to preparation of cicatrix treatment medicines Download PDFInfo
- Publication number
- CN105233248A CN105233248A CN201510562475.4A CN201510562475A CN105233248A CN 105233248 A CN105233248 A CN 105233248A CN 201510562475 A CN201510562475 A CN 201510562475A CN 105233248 A CN105233248 A CN 105233248A
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- CN
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- Prior art keywords
- peptide
- acid
- uropoly acid
- uropoly
- cicatrix
- Prior art date
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the field of medicines, in particular to application of uroacitides and provides application of the uroacitides to preparation of cicatrix treatment medicines. The uroacitides can induce apoptosis of fibroblasts and inhibit proliferation of the fibroblasts, and fibroblast inhibition degree is increased along with increase of concentration of the uroacitides. According to experiments, fibroblast proliferation inhibition rate of the uroacitides is up to 88.45%, and skin cicatrices can be effectively treated by the uroacitides.
Description
Technical field
The present invention relates to medical art, be specifically related to the novelty teabag of a kind of Uropoly acid-peptide for the preparation of the medicine for the treatment of cicatrix of skin.
Background technology
Uropoly acid-peptide (CDA-II) is the biological preparation of the non-cell toxicity of a kind of active skull cap components composition of separating-purifying from Healthy People freshly voided urine, the analysis found that its main component has hippuric acid, phenylacetyl amido croak pyridine diketone, phenylacetic acid, heteroauxing, phenylacetylglutamine, heteroauxing and small-molecular peptides etc.
Main and the chemotherapy combined of current Uropoly acid-peptide is applied, and can be used for the auxiliary treatment of advanced breast cancer, nonsmall-cell lung cancer.CN99122049 discloses a kind of Uropoly acid-peptide composition and uses thereof, is mainly used in the recurrence treating and prevent cancer; CN200410000754.3 relates to a kind of Uropoly acid-peptide composition equally and is used for the treatment of and prevents the application of cancer return; CN200910305445.X relates to a kind of preparation method of Uropoly acid-peptide.
Cicatrix is the connective tissue Tumor like hyperplasia of local after dermis of skin damage and dysfunction and the pathologic structure that formed, and its histological characteristic is a large amount of collagen in extracellular matrix, and proteoglycan and glycoprotein deposit, collagen arrangement disorder.He is after wound, the complication that especially burnt degree is common.The cicatrix of hyperplasia often can destroy the integrity of human body surface or the dysfunction with various degree, brings the dual misery on mind & body to patient.Modern cell biology and molecular biology research think fibroblastic a large amount of Proliferation and apoptosis suppress and extracellular matrix synthetics and degradation unbalance, a large amount of generations of Some cytokines constitute the biological mechanism of cicatrix.
The treatment of current case cicatrix is mainly through surgical resection, mechanical pressure and laser therapy, radiation, the freezing and therapy such as injection of hormone and medicine.The medicine being used for the treatment of cicatrix mainly contains steroid hormone medicine, this categories of drugs is mainly through suppressing PDEF gene expression inhibition cell proliferation, thus suppress the synthesis of I, type III collagen, cause c-myc and P53 in scar tissue to raise, the apoptosis of final induced fibroblast; In addition some medicines relevant to cytokine, antfhistamine medicine is also had.Chinese medicine achieves significant progress in treatment cicatrix in recent years, and scar treatment is taken the course of its own.
The record about cicatrix is just had period as far back as the Western Han Dynastry, as " 52 pathological changes ", Holy Benevolent Prescriptions, " Prescriptions for Universal Relief " etc., the traditional Chinese medical science is mainly summarised as stagnation of QI-blood to the understanding of cicatrix, obstruction of meridians and collaterals, phlegm-damp fight knot, also the Chinese medicines such as blood circulation promoting and blood stasis dispelling, hard masses softening and resolving, removing obstruction in the collateral to relieve pain are taked clinically, by inside and outside method for the treatment of or the treatment of the two combined techniques.At present the experimentation object for the treatment of by Chinese herbs cicatrix is mainly concentrated on the blood circulation promoting and blood stasis dispelling and Chinese herbal medicine extract and effective ingredient thereof commonly used.
Drug for invigorating blood circulation and eliminating stasis such as Radix Salviae Miltiorrhizae, Rhizoma Chuanxiong, Radix Angelicae Sinensis etc. are the common drugs of traditional scar treatment, after measured vitro human fibroblasts from hyperplastic scar
3under H-thymidine absorption value and mirror, dyeing calculates cell division index, discovery Radix Salviae Miltiorrhizae 1.25mg/ml, ligustrazine 125 μ g/ml have obvious inhibitory action to scar fibroblast proliferation, and under higher concentration and long period effect, the growth inhibited being applied cell is irreversibility.
The research of Chinese medicine extract to treatment cicatrix in recent years day by day increases, and mainly through suppressing fibroblast growth, works on the impact of cytokine and the accumulation etc. of T suppression cell epimatrix; Liu Dewu etc. use the tetrandrine extracted from Chinese medicine Radix Stephaniae Tetrandrae to observe the impact grown the human skin fibroblast of In vitro culture, and find that tetrandrine can be suppressed to fibroblast growth, inhibitory action and concentration, time are proportionate; The identical research of Yao finds that the extract-Radix Notoginseng total glucosides of Radix Notoginseng can suppress the growth cycle of fibroblasts from hypertrophic scars, and S phase cell is increased than number.
Hu little Long etc. find the propagation of curcumin Inhibiting proliferation fibroblasts from hypertrophic scars after adopting the curcumin handler fibroblast of variable concentrations, and along with drug level increase, the apoptosis rate of cell is also raising gradually.Centella triterpenes cream is the medicine of present stage clinical conventional treatment cicatrix, and Zhao Wenlu etc. find that the effective ingredient Asiaticoside of centella triterpenes cream can reduce the mrna expression amount of TGF-β 1, reach the effect suppressing cicatrix.In the forming process of cicatrix, extracellular matrix metabolism disorder is also the key factor that it is formed, in extracellular matrix, increase based on the synthesis of collagen again, skin collagen has tens kinds, wherein I, type III collagen is its main component, type III collagen content is higher, collagen fiber are thinner, cicatrix is lighter, Fang Quan etc. are collagen I in Radix et Caulis Opuntiae Dillenii extract is to rabbit ear hypertrophic scar tissue block, confirm in the experiment of III and Expression of MMP-1 impact that Radix et Caulis Opuntiae Dillenii extract has the collagen I reduced in rabbit ear veutro scar tissue block and expresses, promote that collagen I II expresses, thus lessen scar formation hypertrophy, promote that scar tissue block softens.Nearest discovery retinoic acid can disturb fibroblastic DNA to synthesize, and suppresses it to breed, and degraded type i collagen, has certain therapeutical effect to the cicatrix of rabbit scar model, thus for clinical treatment hypertrophic cicatrix provides certain theoretical foundation.But this kind of medicine is because easily recurrence, and the inconvenience of long term injections administration and limit its clinical practice.
Also not yet find at present about Uropoly acid-peptide (CDA-II) is for the preparation of the report of the application of the medicine for the treatment of cicatrix.
Summary of the invention
For the above state of the art, the invention provides the application of a kind of Uropoly acid-peptide in the medicine of preparation treatment cicatrix.
Uropoly acid-peptide of the present invention is in the application of the medicine of preparation treatment cicatrix, as one of embodiment, described Uropoly acid-peptide can be the Uropoly acid-peptide in any source, this area, it includes but not limited to be derived from commercial, also can be prepared according to means known in the art or general knowledge by those skilled in the art, but described Uropoly acid-peptide should to meet premised on the relevant regulations in national standard or industry standard.
Uropoly acid-peptide of the present invention is in the application of the medicine of preparation treatment cicatrix, as one of embodiment, when experimenter use Uropoly acid-peptide be used for the treatment of cicatrix time, described dosage scope comprise be 80 ~ 8000mg/ every day/everyone, preferred 200-6000mg/ every day/everyone, the explanation of property as an example, can be such as 80, 100, 150, 200, 250, 300, 350, 400, 450, 500...1000, 1050...1500, 1550...2000, 2050...3000, 3050...3500, 3550...4500, 4550...5000, 5050...5500, 5550...5950 or 6000mg/ every day/everyone dosage, wherein suitable change can be carried out according to the difference of administering mode in administration number of times and interval, in usual one day each administration time and secondary between time period be identical, the explanation of such as exemplarily property, as one day three times, normally the morning, noon and afternoon are each once.
Uropoly acid-peptide of the present invention is in the application of the medicine of preparation treatment cicatrix, and the administering mode of described Uropoly acid-peptide can be the conventional administering mode in this area, the present invention includes but be not limited to inject, oral or external.
Uropoly acid-peptide of the present invention is in the application of the medicine of preparation treatment cicatrix, as one of embodiment, when injecting Uropoly acid-peptide to experimenter and being used for the treatment of cicatrix, the amount using described Uropoly acid-peptide to patient comprise 80 ~ 3500mg/ every day/everyone, the exemplarily explanation of property can be such as 80,100,150,200,250,300,350,400,450,500...1000,1050...1500,1550...2000,2050...3000,3050...3450 or 3500mg/ every day/everyone dosage.Administration number of times comprises and is not limited to 1 ~ 3 time/every day; The exemplarily explanation of property, as once a day, twice or three times.
Time in the present invention's application with injection system administration, made preparation includes but not limited to injection, lyophilized formulations or powder injection formulation etc., can be determined in conjunction with the present invention and this area general knowledge of being correlated with by those skilled in the art containing the amount of Uropoly acid-peptide in wherein said ejection preparation, as one of embodiment, in described unit preparation, the content of Uropoly acid-peptide includes but not limited to such as 35 ~ 60mg/ml.
Uropoly acid-peptide of the present invention is in the application of the medicine of preparation treatment cicatrix, as one of embodiment, when Uropoly acid-peptide oral to experimenter, described dosage scope be 80 ~ 8000mg/ every day/everyone, the exemplarily explanation of property, can be such as 80, 100, 150, 200, 250, 300, 350, 400, 450, 500...1000, 1050...1500, 1550...2000, 2050...3000, 3050...3500, 3550...4500, 4550...5000, 5050...5500, 5550...5950, 6000...7000, 7050...7500, 7550...7950, 8000mg/ every day/everyone dosage, administration number of times comprises and is not limited to 1 ~ 3 time/every day, the exemplarily explanation of property, as once a day, twice or three times.
Time in the present invention's application with oral administration, made oral formulations includes but not limited to tablet, capsule or soft capsule etc., can be determined in conjunction with the present invention and this area general knowledge of being correlated with by those skilled in the art containing the amount of Uropoly acid-peptide in oral formulations of the present invention, as one of embodiment, in described unit preparation, the content of Uropoly acid-peptide includes but not limited to such as 50-500mg.
Uropoly acid-peptide of the present invention is in the application of the medicine of preparation treatment cicatrix, as one of embodiment, when being used for the treatment of cicatrix to experimenter's external Uropoly acid-peptide, described dosage scope comprise for 80-8000mg/ every day/explanation of everyone exemplarily property, can be such as 80, 100, 150, 200, 250, 300, 350, 400, 450, 500...1000, 1050...1500, 1550...2000, 2050...3000, 3050...3500, 3550...4500, 4550...5000, 5050...5500, 5550...5950, 6000...7000, 7050...7500, 7550...7950, 8000mg/ every day/everyone dosage, administration number of times comprises and is not limited to 1 ~ 6 time/every day, preferably 1 ~ 4 time/every day, the exemplarily explanation of property, as once a day, twice, three times, four times, five times or six times etc.
Time in the present invention's application with the administration of external mode, made preparation includes but not limited to gel, ointment, spray or transdermal patch etc., can be determined in conjunction with the present invention and this area general knowledge of being correlated with by those skilled in the art containing the amount of Uropoly acid-peptide in described external preparation, as one of embodiment, the percentage ratio that described external preparation accounts for described preparation gross mass containing the amount of Uropoly acid-peptide includes but not limited to be 1% ~ 48%, exemplarily property illustrates, such as 1%, 2%, 4%, 8%, 16%, 32% or 48%; As one of embodiment, when for gel or ointment, the exemplarily explanation of property, amount containing Uropoly acid-peptide in described unit gel or ointment can be 0.2 ~ 12.8mg/ml, the exemplarily explanation of property can be the Uropoly acid-peptide as 0.2,0.4,0.8,1.6,3.2,6.4 or 12.8mg/ml.
In the application of Uropoly acid-peptide preparation treatment cicatrix medicine of the present invention, described Uropoly acid-peptide can be the Uropoly acid-peptide in any source, this area, as one of embodiment, described Uropoly acid-peptide comprises hippuric acid, phenylacetylglutamine, 4-hydroxyl phenylacetic acid and 5-HIAA; As one of embodiment, in Uropoly acid-peptide of the present invention, hippuric acid, phenylacetylglutamine, 4-hydroxyl phenylacetic acid and 5-HIAA amount account for the percentage ratio of gross mass is 28.5% ~ 37.75%; As one of embodiment, containing hippuric acid 0.10mg ~ 0.15mg, phenylacetylglutamine 0.15mg ~ 0.20mg, 4-hydroxyl phenylacetic acid 10 μ g ~ 20 μ g, 5-HIAA 2.5 μ g ~ 7.5 μ g in 1mg Uropoly acid-peptide of the present invention.
The method of this area routine can be adopted to prepare Uropoly acid-peptide of the present invention, and as one of embodiment, described method includes but not limited to prepared by following method:
1) get freshly voided urine, add HCl adjust ph, carry out ultrafiltration, collect the filtrate of molecular weight lower than 10kD; Then by filtrate with XAD-8 silicagel column on 1.5l/min flow velocity, first use 20L distilled water with 3l/min adsorption column afterwards, then add in XAD-8 post with 15L ethanol with the flow velocity of 1.0l/min, collect the eluent of coloured moiety; Vacuum drying treatment, is slowly warming up to 50 DEG C, until solvent all evaporates; After this carry out lyophilization, obtain the dry product of Uropoly acid-peptide composition;
2) dissolving dry product to concentration with distilled water is 40mg/ml, uses NaOH adjust ph, obtains Uropoly acid-peptide crude product solution; Crude product solution is put 4 DEG C of cryogenic conditions 120hr, get supernatant and carry out filtering with microporous membrane, filtrate uses ultrafilter membrane ultrafiltration, removing thermal source and virus.
Uropoly acid-peptide of the present invention in the application of the medicine of preparation treatment cicatrix, as one of embodiment, described step 1) in pH value be 1.5-3.0.
Uropoly acid-peptide of the present invention in the application of the medicine of preparation treatment cicatrix, as one of embodiment, described step 2) in pH value be 6.5-7.5.
Uropoly acid-peptide of the present invention is in the application of the medicine of preparation treatment cicatrix, and as one of embodiment, described Uropoly acid-peptide is adopted and prepared with the following method:
1) get freshly voided urine, add 1mol/lHCl and regulate its pH to 2.0, by nylon cloth collecting by filtration urine, ultrafiltration apparatus carries out ultrafiltration, in ultrafiltration apparatus, add deionized water, washes bubble ultrafilter membrane, the urine of collection is poured in charging bucket, opens nanofiltration, collect the filtrate of molecular weight lower than 10kD; Get XAD-8 silica gel, use 95% soak with ethanol, load in bag to be positioned over and mould in bucket, make adsorption column, with 95% alcohol flushing pillar, use the deionized water wash adsorption column of same volume afterwards; Then the urine after filtering is added in post with the flow velocity of 1.5l/min, add and first add adsorption column with distilled water with 3l/min flow velocity afterwards, then add in XAD-8 post with 95% ethanol with the flow velocity of 1.0l/min, collection coloured moiety eluent; Through vacuum drying treatment, slowly heating up and controlling feed temperature is 50 DEG C and all evaporates to solvent; Lyophilization, obtains the dry product of Uropoly acid-peptide composition;
2) dried residue is dissolved with distilled water, its concentration is made to be 40g/ml, after regulating the pH value of Uropoly acid-peptide crude product to 7.4 with 2mol/lNaOH, 120hr is placed in 4 DEG C of cryogenic conditions, get supernatant, use the filtering with microporous membrane of 1 μm and 0.45 μm successively, then use the ultrafilter membrane ultrafiltration of 10000 molecular weight, removing thermal source, then through UltiporVF
tMdV50 (PALL company) filter, except virus, to obtain final product.
The application of Uropoly acid-peptide treatment cicatrix of the present invention, effectively can treat cicatrix, solve the inapparent problem of most of scar treatment curative effect of medication in the market.
Through human dermal fibroblast cell experiment in vitro and zoopery, Uropoly acid-peptide has obvious apoptosis-induced effect to cicatrix of skin fibroblast, and evident in efficacy to rabbit ear scar treatment, thus may be used for the medicine preparing treatment cicatrix of skin.In vitro cell experiment and zoopery confirm, Uropoly acid-peptide is except having antitumaous effect, can also induced fibroblast apoptosis, suppress it to breed, suppression degree increases with the increase of Uropoly acid-peptide concentration, become obvious dose-effect relationship, and suppression ratio is up to 88.45%, and the highest suppression ratio of the effective ingredient-Asiaticoside of current clinical treatment cicatrix common drug, curcumin and retinoic acid several drugs is 52.99% ~ 75.69%.
In addition, zoopery also shows, medicine containing Uropoly acid-peptide is applied in rabbit ear cicatrix place, after 28 days, cicatrix obviously reduces, scar hyperplasia block growth inhibition ratio reaches 89.28%, and conventional medicine centella triterpenes cream and compound haparin sodium allantoin gel and the ointment containing Asiaticoside, curcumin, retinoic acid and allantoin are 50.46% ~ 76.68% to scar hyperplasia block suppression ratio, in cicatrix, the ratio of collagen fiber surface density and collagen fiber I and III obviously increases compared with other several drugses.
Compare the medicine of other treatment cicatrix, Uropoly acid-peptide curative effect of the present invention is more remarkable, will be for one of good medicine of clinical treatment cicatrix of skin.
Accompanying drawing explanation
Fig. 1: for testing 5 kinds of medicines used with drug level to people's scar fibroblast proliferation suppression ratio change curve, A: asiaticoside is to people's scar fibroblast proliferation suppression ratio change curve; B: curcumin is to people's scar fibroblast proliferation suppression ratio change curve; C: retinoic acid is to people's scar fibroblast proliferation suppression ratio change curve; D: allantoin is to people's scar fibroblast proliferation suppression ratio change curve; E: Uropoly acid-peptide is to people's scar fibroblast proliferation suppression ratio change curve;
Fig. 2 be cell proliferation inhibition rate maximum time, 5 kinds of medicines suppression ratio significance of difference separately analyzes block diagram; (wherein * P < 0.5, * * P < 0.01, n=3);
Fig. 3 tests 5 kinds of medicines used with medicament contg to rabbit ear scar hyperplasia block suppression ratio change curve, A: asiaticoside is to rabbit ear scar hyperplasia block suppression ratio change curve; B: curcumin is to rabbit ear scar hyperplasia block suppression ratio change curve; C: retinoic acid is to rabbit ear scar hyperplasia block suppression ratio change curve; D: allantoin is to rabbit ear scar hyperplasia block suppression ratio change curve; E: Uropoly acid-peptide is to rabbit ear scar hyperplasia block suppression ratio change curve;
Fig. 4 be rabbit ear scar hyperplasia block suppression ratio maximum time, 8 kinds of medicines significance of difference separately analyzes block diagram.(wherein * P < 0.5, * * P < 0.01, * * P < 0.001, n=4).
Detailed description of the invention
Following examples are used for setting forth the present invention further, but do not limit effective range of the present invention in any manner.
The preparation of embodiment 1, Uropoly acid-peptide and qualification
1. the preparation of Uropoly acid-peptide composition
Get 20L freshly voided urine, add 1L1mol/lHCl and regulate its pH to 2.0, by nylon cloth collecting by filtration urine.Ultrafiltration apparatus carries out ultrafiltration, in ultrafiltration apparatus, add deionized water, washes bubble ultrafilter membrane, pours in charging bucket by the urine of collection, opens nanofiltration, collects the filtrate of molecular weight lower than 10kD.
Get XAD-8 silica gel 10kg, with 25L95% soak with ethanol 20min, load in bag to be positioned over and mould in bucket, make adsorption column, rinse pillar with 95% ethanol (V/W=ethanol/silica gel=2), use the deionized water wash adsorption column 2 times of same volume afterwards, the ethanol in Ex-all pillar; Then the urine after filtration is added in post with the flow velocity of 1.5l/min, add and first add adsorption column with 20L distilled water with 3l/min flow velocity afterwards, add in XAD-8 post with 15L95% ethanol with the flow velocity of 1.0l/min again, the effective ingredient of absorption is disintegrated down, collects coloured moiety and be effective eluent; By the effective ingredient of collection through vacuum drying treatment, slowly heating up and control feed temperature is 50 DEG C, until solvent all evaporates; After this carry out lyophilization, obtain the dry product of Uropoly acid-peptide composition.
Dissolve dried residue with distilled water, make its concentration be 40mg/ml, after regulating the pH value of Uropoly acid-peptide crude product to 7.4 with 2mol/lNaOH, obtain Uropoly acid-peptide crude product solution; Crude product is put 4 DEG C of cryogenic conditions 120hr, remove the supernatant processing rear crude product, use the filtering with microporous membrane of 1 μm and 0.45 μm successively, filtrate is Uropoly acid-peptide solution; With the ultrafilter membrane ultrafiltration of 10000 molecular weight, removing thermal source, then through UltiporVF
tMdV50 (PALL company) filter is except virus, and the response rate is about 2.5% (volume ratio).
2. each constituent structure confirmation and content detection in Uropoly acid-peptide composition
The Uropoly acid-peptide composition extracted from healthy human urine in the present invention is containing four kinds of organic acid (hippuric acid, phenylacetylglutamine, 4-hydroxyl phenylacetic acid, 5-HIAA) and a series of small-molecular peptides isoreactivity composition, and structural identification test is mainly for above-mentioned two large class materials.Four kinds of organic acid high performance liquid chromatography.Application of sample is adopted to confirm and separation determination and mass spectrometric determination molecular weight; Small-molecular peptides adopts liquid-mass chromatography, measures the molecular weight ranges of peptide, and determines the sequence of wherein two main peptides.
Four kinds of organic acid structural identifications in 2.1 Uropoly acid-peptides
2.1.1 Instrumental Analysis type HPLC, Japanese Shimadzu Corporation LC-10AVP.Chromatographic column: DikMA company, Luna5 μ 18 (Z), 250 × 4.6mm (dm).
2.1.2 standard substance 4-hydroxyl phenylacetic acid; 5-HIAA; Hippuric acid; Phenylacetylglutamine all purchases Sigma company.
2.1.3 chromatographic condition mobile phase: methanol: water: glacial acetic acid=50: 15: 1; Flow velocity: 1ml/min; Sample size 20ul; Determined wavelength: 260nm.
2.1.4 the preparation of reference substance solution
Accurately respectively take 4-hydroxyl phenylacetic acid, 5-HIAA, hippuric acid, phenylacetylglutamine 45mg, 5.0mg, 45mg, 60mg put in the measuring bottle of 4 50ml, add water about 40ml, and ultrasonic 30min dissolves completely, and standardize solution is to scale afterwards, mixes.
2.1.5 the preparation of test sample solution
Get Uropoly acid-peptide crude product appropriate, make the solution containing 8mg in every 1ml.
2.1.6 assay method and measurement result
Get above-mentioned standard solution and need testing solution each 20ul injection chromatographic column, by external standard method with calculated by peak area 4 kinds of organic acid content.Measurement result is as follows:
Organic acid measurement result in table 1-1 Uropoly acid-peptide extract
Result shows that in test sample, each organic acid retention time and reference substance are basically identical, and provable this product is containing above four kinds of organic acid.According to four kinds of organic acid content in Peak area analysis Uropoly acid-peptide crude product test sample respectively: hippuric acid 1.01mg/ml; Phenylacetylglutamine 1.43mg/ml; 4-hydroxyl phenylacetic acid 0.12mg/ml; 5-HIAA 0.04mg/ml; Namely hippuric acid 0.126mg, phenylacetylglutamine 0.179mg, 4-hydroxyl phenylacetic acid 15 μ g, 5-HIAA 5 μ g is contained in 1mg Uropoly acid-peptide crude product.The percentage ratio that four kinds of organic acid gross masses account in Uropoly acid-peptide is 36%.
2.2 electrospray mass spectrographs measure peptide composition and partial peptide sequences measures
Testing conditions: cation detection mode, capillary voltage 3000V, Cone voltage 50V, Collision voltage 30V, Mep detect voltage 2000V.Peptide sequence analysis software is the Biolynx of Micromass company.Temperature: 22 DEG C; Detecting instrument title and model: Q-T0F2ESI-MS/MS (Micromass).
2.2.1 analytical method:
(1) sample pretreatment: take Uropoly acid-peptide crude product and be about 10.0mg in 10ml tool plug scale test tube, add 5.0ml chloroform, fully vibrate and have children outside the state plan extraction 3min, standing 10min, with the suction pipe of cleaning by careful for chloroform sucking-off; Add 5ml petroleum ether, same ultrasonic rear absorption; Add 5.0ml dehydrated alcohol again, with step above in triplicate, residue dries stand-by (about 40min) naturally.
(2) searching of peptide components and identification: the sample dissolution of above-mentioned process, in 5ml1% formic acid deionized water, is got 1ul and carried out capillary high performance liquid chromatography Nanoliter electrospray-level Four bar flight time tandem mass spectrum automated analysis after centrifugal.
(3) sequencing of partial peptide composition: the sample dissolution of above-mentioned process, in 5ml1% formic acid deionized water, is got 1ul and carried out Nanoliter electrospray-level Four bar flight time Tandem Mass Spectrometry Analysis after centrifugal, select 1-2 main peak to carry out sequencing.
2.2.2 result
(1) chromatography of ions figure shows I, II, III tri-region retention times and is respectively: 14 ~ 19min, 30 ~ 45min, 45 ~ 55min.
(2) total mass spectrum result in I district shows that relative amount the higher person molecular ion peak is respectively 1218.69,1240.67,1251.69,1322.74,1452.81 (molecular weight=molecular ion peaks-1.0078) about containing more than 20 peptide.
(3) total mass spectrum in II district does not find obvious quasi-molecular ions, and before and after the collection of illustrative plates display conversion after changing with special software MaxEnt3, mass spectrum is consistent, therefore can find out that the peptide composition in this district is little.
(4) total mass spectrum in III district shows the same with II district, and peptide composition is little, does not almost have.
2.2.3 the particle that selection two intensity are larger carries out Tandem Mass Spectrometry Analysis
The sequencing results sequence of m/z626.36 is: AVEGPSSALGPLCGP, single isotopic mass 1250.7043Da, and theoretical value is 1250.6506Da, and deviation is 0.05Da;
The sequencing results of m/z609.84 is: GPSTPGPPPNGGA, single isotopic mass 1217.6644Da, and theoretical value is 1217.6040Da, and deviation is 0.06Da.
The experiment of experimental example 1 Uropoly acid-peptide induction human hypertrophic scar fibroblast (hHSF) apoptosis
1, the fibroblastic preparation of human hypertrophic scar
Get 16-30 year patient skin hypertrophic cicatrix, aseptically remove epidermis and subcutaneous tissue, after normal saline rinses repeatedly, cut into 0.5-1.0mm
3piece of tissue, appropriate FBS (calf serum) cyclic washing piece of tissue, be inoculated on batch cultur bottle wall afterwards, piece of tissue spacing controls at 0.3-0.5mm, add the DMEM culture fluid (containing penicillin 100U/ml, streptomycin 100U/ml) of 5ml containing 20%FBS, in 37 DEG C, 5%CO
2cultivate in cell culture incubator, change weekly liquid 2 times, when primary cell growth is fused into sheet substantially, draw culture fluid, 0.25% trypsinization 1min is added after PBS rinsing 2 times, to be seen to intercellular substance increase, and when seeing that kytoplasm bounces back under microscope, pancreatin is abandoned in suction, the DMEM culture medium added containing serum stops digestion, blowing and beating cell gently makes in cell suspension, be transferred in 15ml centrifuge tube, in the centrifugal 3min of 1000r/min, abandon supernatant and add DMEM culture medium re-suspended cell containing 10%FBS again, go down to posterity in 1: 3 ratio, 3-6 is used to carry out following experiment for cell.
2, Uropoly acid-peptide is on the impact of people's fibroblasts from hypertrophic scars vigor
MTT (3-(4,5)-dimethylthiahiazo (-2-y1)-3,5-di-phenytetrazoliumromide) colorimetry detects cell survival and grows conventional method, succinate dehydrogenase in living cells mitochondrion can make MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation, and dead cell can not, DMSO (dimethyl sulfoxide) can first a ceremonial jade-ladle, used in libation in dissolved cell, and having maximum absorption band at 490nm place, this absorption value reflects cell viability indirectly.
The present invention compares Asiaticoside (Asiaticoside, C
48h
78o
19, the main component of centella triterpenes cream, purchased from Sigma-Aldrich, article No.: 43191), curcumin (Curcumin, purchased from Sigma-Aldrich, 08511), retinoic acid (Tretinoin, C article No.:
20h
28o
2, purchased from Sigma-Aldrich, article No.: R2625), allantoin (Allantoin, C
4h
6n
4o
3the main component of compound haparin sodium allantoin gel, purchased from Sigma-Aldrich, article No.: 93791) and Uropoly acid-peptide (CDA-II) (obtaining by the embodiment of the present invention 1 method) these four kinds of crude drug on the impact of people's fibroblasts from hypertrophic scars vigor.Cell viability assay method is as follows:
The DMEM culture fluid of the people fibroblasts from hypertrophic scars 10%FBS of exponential phase is made 5 × 10
4the cell suspension of individual/ml, is inoculated in 3 96 well culture plates respectively with every hole 200ul, and edge hole Du Shi phosphate buffer (DPBS) is filled; 37 DEG C, 5%CO
224hr is cultivated in incubator.By above medicine according to the diluent being mixed with variable concentrations shown in table 1-1.Wherein curcumin and retinoic acid water insoluble, therefore first configure 10mg/ml mother solution respectively with DMSO, afterwards with DMEM culture medium stepwise dilution to showing the concentration shown in 1-1.Abandon supernatant culture fluid after 24hr, add the medicine of 200ul variable concentrations respectively, matched group adds the DMEM culture fluid of 200ul containing 10%FBS, and each concentration of often kind of medicine does 4 multiple holes, in 37 DEG C, and 5%CO
2add 20ulMTT (5mg/ml, i.e. 0.5%MTT) after cultivating 48hr in incubator, continue to cultivate 4hr, inhale and abandon culture fluid, every hole adds 150ulDMSO cessation reaction, measures and inhale brightness A after vibration 15min on enzyme immunoassay instrument
490nmvalue, the variable concentrations Uropoly acid-peptide surveyed respectively is at the cell proliferation inhibition rate (cell proliferation inhibition rate=(1-experimental group is on average inhaled brightness value/matched group and on average inhaled brightness value) × 100%) of different time, take drug level as abscissa, cell proliferation inhibition rate is vertical coordinate, draw Drug dose response. curve, result is as Fig. 1.When after this selecting cell proliferation rate the highest, corresponding drug level repeats to do three batches of cell experiments, and the average inhibition of statistical analysis three batches experiment, adopt T-test statistical method to analyze the significance of difference experimental group and matched group, result as shown in Figure 2.
The concentration table of table 1-1 different pharmaceutical preparation
Asiaticoside reaches maximum 75.69% when its concentration is 64mg/ml to people's cicatrical fibrosis cell proliferation inhibition rate as seen from Figure 1, curcumin cell proliferation inhibition rate when concentration is 0.016mg/ml reaches maximum 52.99%, retinoic acid cell proliferation suppression ratio when concentration is 0.32mg/ml reaches maximum 67.93%, and allantoin cell proliferation inhibition rate when concentration is 0.16mg/ml reaches maximum 77.46%, and Uropoly acid-peptide concentration is maximum to the suppression ratio of people's fibroblasts from hypertrophic scars when being 6.4mg/ml, maximal percentage inhibition is 88.45%, other four kinds of medicines (* * P < 0.01) are significantly higher than by the suppression ratio of T check analysis Uropoly acid-peptide on cell proliferation, the highest to the suppression usefulness of people's fibroblasts from hypertrophic scars.
Table 1-2MTT method detects different pharmaceutical to the effect of fibroblasts from hypertrophic scars
Test example 2, Uropoly acid-peptide are to the Inhibition test of rabbit ear hypertrophic cicatrix
1, the foundation of cicatrix animal model
Get the Japanese screech owl white rabbit of 3-4 month size, male and female, body weight 2.2-2.6kg, ketamine (15mg/kg) auricular vein injecting anesthetic, be fixed on operating board by prostrate for rabbit, often surveying rabbit facies ventralis equal excision diameter is the full thickness skin of 10mm, strikes off perichondrium, and two pick up the ears 5 places totally, each wound surface interval more than 15mm, wound surface exposes, and after wound surface epithelization 20d, scar hyperplasia block is formed.
2, Uropoly acid-peptide ointment preparation method
2.1 ointment base preparations:
Ointment base composition Borneolum Syntheticum, Mentholum, Camphora, thymol all grind to form segmentation, and cross 120 mesh sieves, this zoopery needs the substrate total amount used to be as required: mass percent 52% (0.5% Borneolum Syntheticum+48% vaseline+2% Mentholum+1% Camphora+0.5% thymol) * 180 each cicatrixs of wound surface * of substrate smear ointment amount 50mg=4680mg, prepares the above-mentioned substrate of 5.2g altogether.Take vaseline 4.8g, be heated to 50 ~ 60 DEG C, make it melt into thick paste, constant temperature is placed for subsequent use.Take respectively and ground and the fine powder sieved: Borneolum Syntheticum 50mg, Mentholum 200mg, Camphora 100mg, thymol 50mg, by above-mentioned fine powder mix homogeneously, add in the liquid thick paste of vaseline while stirring, until evenly, constant temperature keeps thick paste state for subsequent use.
The preparation of the Uropoly acid-peptide ointment of 2.2 variable concentrations:
According to table 2-2 different pharmaceutical ointment material composition mass percent table, known Uropoly acid-peptide ointment test group establishes 7 variable concentrations altogether, the Uropoly acid-peptide ointment of each concentration is parallel smears 5 wound surface, each wound surface need smear ointment base 50mg, therefore the Uropoly acid-peptide ointment that can be calculated often kind of concentration needs preparation: 5 each cicatrixs of parallel wound surface * smear ointment amount 50mg=250mg, prevent the loss in ointment preparation and use procedure, the Uropoly acid-peptide ointment of often kind of concentration prepares 300mg altogether.
Take 3mg, 6mg, 12mg, 24mg, 48mg, 96mg, 144mg Uropoly acid-peptide fine powder, add to respectively in 141mg, 138mg, 132mg, 120mg, 96mg, 48mg, 0mg propylene glycol, stir evenly to obtain Uropoly acid-peptide propylene glycol solution.By above-mentioned solution, add in 2.1 substrate of the 156mg that 7 parts have prepared respectively, fully stir evenly condensation and obtain Uropoly acid-peptide ointment, by the Uropoly acid-peptide ointment formulation of above variable concentrations, with packaging of aluminium foil bag sealing, then irradiation sterilization, stand-by in 4 DEG C of preservations.
The preparation of 2.3 variable concentrations positive control drug ointment:
Such as show shown in 2-2 containing percent mass contained by Asiaticoside, curcumin, retinoic acid and allantoin four kinds of drug ointments medicines for other four kinds, preparation method is with 2.2.
The preparation of 2.4 blank ointment:
The ointment of blank group: take 144mg propylene glycol and 2.1 substrate that 156mg prepares and fully mix homogeneously condensation.
Table 2-1 different pharmaceutical ointment material composition mass percent table
Table 2-2 different pharmaceutical ointment formulation prescription table
3, Uropoly acid-peptide research that the cicatrix of rabbit ear scar model animal is affected
This experiment is divided into 6 groups: (1) blank group; (2) positive controls (2-1: curcumin, 2-2: retinoic acid, 2-3: Asiaticoside, 2-4: allantoin; (3) Uropoly acid-peptide group.
36 rabbits, every rabbit is provided with 5 wound surface, amounts to 180 wound surface.Often kind of ointment positive control medicine group, all has 7 different ointment formulation prescriptions, and often kind of ointment formulation prescription is parallel smears 5 wound surface, and namely often kind of ointment positive control medicine group needs 7*5=35 wound surface.Uropoly acid-peptide group also needs 35 wound surface by that analogy.Blank ointment base matched group (does not add any raw material, only add substrate, each substrate mass percent is as follows, propylene glycol: Borneolum Syntheticum: vaseline: Mentholum: Camphora: Moschus grass meal=48: 0.5: 48: 2: 1: 0.5) only have 1 preparation prescription, parallelly smears 5 wound surface.So whole rabbit experiment needs: 4 kinds of positive control ointment *, 7 preparation prescription *, 5 parallel wound surface+1 Uropoly acid-peptide testing drug ointment * 7 preparation prescription * 5 parallel wound surface+1 blank ointment * 1 preparation prescription *, 5 wound surface=180 wound surface.Corresponding drug ointment is coated with outside the synulotic position of the rabbit ear, and soft until medicaments uniformity; Once a day, often locate cicatrix and be about 50mg, continuously process 28 days, after the 28th day, detect following index:
Scar hyperplasia is observed: cut hypertrophy block in 24hr after drug withdrawal in 4 weeks, take hypertrophy block weight, and calculates often group hypertrophy block growth inhibition ratio, and computing formula is as follows:
Suppression ratio=(the average cicatrix weight of 1-medication group average cicatrix weight/blank group) × 100%
Result as shown in Figure 3, result reaches maximum to the suppression ratio of the rabbit ear scar hyperplasia block when showing that in positive controls, 2-1 to 2-4 four kinds of raw materials quality percentage ratios are followed successively by 32%, 0.16%, 3.2%, 1.6%, and maximum is respectively 76.68%, 50.46,56.78,68.93%; When Uropoly acid-peptide raw materials quality percentage ratio is 16%, reach maximum to the scar hyperplasia block suppression ratio of the rabbit ear, suppression ratio is 89.28%.By T check analysis, as shown in Figure 4, show that the Emax (maximum efficiency) of Uropoly acid-peptide to scar hyperplasia block suppression ratio is significantly higher than the Emax (* * P < 0.01) of other four kinds of positive control medicines.
2. collagen fiber surface density in cicatrix: the section of the often kind of medicine cut being made after fixing, dehydration, transparent, waxdip, embedding about 4 μm to the maximum cicatrix of rabbit ear scar hyperplasia block suppression ratio, adopt the dyeing of VG staining, namely iron haematoxylin mixed liquor dyeing 5min is used after conventional dewaxing, hydrochloride alcohol breaks up, ammonia is anti-blue, VG liquid 1% acid fuchsin 2, saturated picric acid 8 dyeing 5min, below often distillation washing 1min is all used between step, 95% ethanol breaks up and dewaters afterwards, dimethylbenzene is transparent, neutral gum mounting.Take pictures under the microscope in cicatrix section central authorities and each random selecting of sidepiece 5 visuals field, collagen fiber are red after VG dyeing, utilize Computer digital image analysis to calculate the averaged areal density of collagen fiber.
3. cicatrix I, type III collagen ratio: after medication 28d, with in 2., get often kind of medicine and section is made to the cicatrix that rabbit ear scar hyperplasia block suppression ratio is maximum, dye through picrosirius red staining, namely celestine blue liquid dye 5min is entered after conventional dewaxing, distillation washing three times, use sirius red saturated picric acid dye liquor 30min afterwards, dehydrated alcohol differentiation and dehydration, dimethylbenzene is transparent, neutral gum mounting, preserve image in polarized light microscopy Microscopic observation afterwards, shown in red or the yellow fibers of type i collagen fiber, the shown in green reticular fiber of type III collagen, Computer digital image analysis is utilized to calculate I, type III collagen area occupied percent.
Below 2. ~ collagen fiber surface density that 3. each scar tissue block records in Testing index and I, type III collagen ratiometric result are as shown in following table 2-3, result shows that Uropoly acid-peptide makes to become fibre density and type i collagen and type III collagen ratio significantly to decline compared with other several drugses in rabbit ear cicatrix, and curative effect is the most outstanding.
Table 2-3 collagen fiber surface density and I, type III collagen ratio test result
In sum, result of the present invention shows that Uropoly acid-peptide significantly can suppress the fibroblastic propagation of cicatrix of skin, reduces the index of cicatrix, thus may be used for the medicine preparing treatment cicatrix.
Claims (10)
1. a Uropoly acid-peptide prepares the application in the medicine for the treatment of cicatrix.
2. application according to claim 1, is characterized in that, described Uropoly acid-peptide is the preparation containing Uropoly acid-peptide.
3. application according to claim 2, is characterized in that, described preparation is external preparation, and described external preparation is gel, ointment, spray or transdermal patch.
4. application according to claim 3, is characterized in that, the amount containing Uropoly acid-peptide in described external preparation is 1%-48% in mass.
5. application according to claim 1, it is characterized in that, described Uropoly acid-peptide comprises hippuric acid, phenylacetylglutamine, 4-hydroxyl phenylacetic acid and 5-HIAA, and the percentage ratio that the amount of wherein said hippuric acid, phenylacetylglutamine, 4-hydroxyl phenylacetic acid and 5-HIAA accounts for gross mass is 28.5% ~ 37.75%.
6. application according to claim 5, it is characterized in that, in described Uropoly acid-peptide, every 1mg Uropoly acid-peptide is containing 0.10mg ~ 0.15mg hippuric acid, 0.15mg ~ 0.20mg phenylacetylglutamine, 10 μ g ~ 20 μ g4-hydroxyl phenylacetic acids, 5-HIAA 2.5 μ g ~ 7.5 μ g.
7. application according to claim 6, is characterized in that, described Uropoly acid-peptide is adopted and prepared with the following method:
1) get freshly voided urine, add HCl adjust ph, carry out ultrafiltration, collect the filtrate of molecular weight lower than 10kD; Then by filtrate with XAD-8 silicagel column on 1.5l/min flow velocity, first use 20L distilled water with 3l/min adsorption column afterwards, then add in XAD-8 post with 15L ethanol with the flow velocity of 1.0l/min, collect the eluent of coloured moiety; Vacuum drying treatment, is slowly warming up to 50 DEG C, until solvent all evaporates; After this carry out lyophilization, obtain the dry product of Uropoly acid-peptide composition;
2) dissolving dry product to concentration with distilled water is 40mg/ml, uses NaOH adjust ph, obtains Uropoly acid-peptide crude product solution; Crude product solution is put 4 DEG C of cryogenic conditions 120hr, get supernatant and carry out filtering with microporous membrane, filtrate uses ultrafilter membrane ultrafiltration, removing thermal source and virus.
8. application according to claim 7, is characterized in that, described step 1) in pH value be 1.5-3.0.
9. application according to claim 7, is characterized in that, described step 2) in pH value be 6.5-7.5.
10. application according to claim 7, is characterized in that, described Uropoly acid-peptide is adopted and prepared with the following method:
1) get freshly voided urine, add 1mol/lHCl and regulate its pH to 2.0, by nylon cloth collecting by filtration urine, ultrafiltration apparatus carries out ultrafiltration, in ultrafiltration apparatus, add deionized water, washes bubble ultrafilter membrane, the urine of collection is poured in charging bucket, opens nanofiltration, collect the filtrate of molecular weight lower than 10kD; Get XAD-8 silica gel, by soak with ethanol, load in bag to be positioned over and mould in bucket, make adsorption column, with 95% alcohol flushing pillar, use the deionized water wash adsorption column of same volume afterwards; Then the urine after filtering is added in post with the flow velocity of 1.5l/min, add and first add adsorption column with distilled water with the flow velocity of 3l/min afterwards, then add in XAD-8 post with 95% ethanol with the flow velocity of 1.0l/min, collection coloured moiety eluent; Through vacuum drying treatment, slowly heating up and controlling feed temperature is 50 DEG C and all evaporates to solvent; Lyophilization, obtains the dry product of Uropoly acid-peptide composition;
2) dried residue is dissolved with distilled water, its concentration is made to be 40mg/ml, after regulating the pH value of Uropoly acid-peptide crude product to 7.4 with 2mol/lNaOH, 120hr is placed in 4 DEG C of cryogenic conditions, get supernatant, use the filtering with microporous membrane of 1 μm and 0.45 μm successively, then use the ultrafilter membrane ultrafiltration of 10000 molecular weight, removing thermal source, then through UltiporVF
tMdV50 filter, except virus, to obtain final product.
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| CN107753516A (en) * | 2016-08-19 | 2018-03-06 | 泰凌(中国)投资有限公司 | A kind of Uropoly acid-peptide of low moisture absorption freezes compound powder and preparation method thereof |
| CN108478585A (en) * | 2018-03-30 | 2018-09-04 | 上海璞萃生物科技有限公司 | A kind of anti-inflammatory composition and its preparation method and application |
| CN111135205A (en) * | 2020-01-08 | 2020-05-12 | 长沙医学院 | Compound centella gel preparation, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107753516A (en) * | 2016-08-19 | 2018-03-06 | 泰凌(中国)投资有限公司 | A kind of Uropoly acid-peptide of low moisture absorption freezes compound powder and preparation method thereof |
| CN107753516B (en) * | 2016-08-19 | 2020-11-20 | 泰凌(中国)投资有限公司 | Low-moisture-absorption uroacitide freeze-dried powder composition and preparation method thereof |
| CN107661355A (en) * | 2017-09-29 | 2018-02-06 | 泰凌生物制药江苏有限公司 | A kind of method for the inactivation of virus being used in Uropoly acid-peptide semi-finished product preparation process |
| CN108478585A (en) * | 2018-03-30 | 2018-09-04 | 上海璞萃生物科技有限公司 | A kind of anti-inflammatory composition and its preparation method and application |
| CN111135205A (en) * | 2020-01-08 | 2020-05-12 | 长沙医学院 | Compound centella gel preparation, preparation method and application thereof |
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Effective date of registration: 20160405 Address after: 200020, 5 floor, city building, No. 45, Nanchang Road, Shanghai, Luwan District Applicant after: NT PHARMA (CHINA) INVESTMENT Co.,Ltd. Applicant after: Zhou Mingtao Applicant after: NT BIOPHARMACEUTICALS JIANGSU CO.,LTD. Applicant after: Tai Ling Pharmaceutical (Changsha) Co.,Ltd. Applicant after: Jiangsu No. 1 Pharmaceutical Co.,Ltd. Address before: 200020, 5 floor, city building, No. 45, Nanchang Road, Shanghai, Luwan District Applicant before: NT PHARMA (CHINA) INVESTMENT Co.,Ltd. Applicant before: Zhou Mingtao |
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Denomination of invention: Application of uroacitides to preparation of cicatrix treatment medicines Effective date of registration: 20200408 Granted publication date: 20190412 Pledgee: Haier financial factoring (Chongqing) Co.,Ltd. Pledgor: Suzhou First Pharmaceutical Co.,Ltd.|Tai Ling Pharmaceutical (Changsha) Co.,Ltd.|NT BIOPHARMACEUTICALS JIANGSU CO.,LTD.|NT PHARMA (CHINA) INVESTMENT Co.,Ltd.|Zhou Mingtao Registration number: Y2020310000007 |
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