CN107865944A - A kind of clarification process and method of quality control with the pharmaceutical composition for repairing skin barrier effect - Google Patents
A kind of clarification process and method of quality control with the pharmaceutical composition for repairing skin barrier effect Download PDFInfo
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Abstract
The invention discloses a kind of clarification process and method of quality control of the pharmaceutical composition for having and repairing skin barrier effect,The medicine is by radix cynanchi atrati,Pilea fasciata,Ginkgo leaf,False-yellowflower milkwort root or herb,Stone precipice tea and the taste medicinal material of roxburgh anoectochilus terminal bud 6 composition,Specific clarification process is to clarify pharmaceutical composition extract solution with II type ZTC 1+1 natural clarifying agents,Specific method of quality control is to radix cynanchi atrati in pharmaceutical composition with thin-layered chromatography,Ginkgo leaf carries out thin layer discriminating,In combination with high effective liquid chromatography for measuring pharmaceutical composition Hernandulcin,Polygalic acid,The content of camellianin A and anoectochilus roxburghii glycosides,The clarification process and method of quality control that the present invention uses,It is few with equipment investment,Operating procedure is simple,It is big to overcome traditional alcohol deposition method consumption alcohol amount,Ethanol recovery is cumbersome,The defects of medicine multicomponent complicated assay method,Quickly,Effectively ensure that the inherent quality of composition,Curative effect and stability,Improve the controllability of pharmaceutical composition quality standard.
Description
Technical field
The present invention detects technical field of traditional Chinese medicine preparation using the clarification process and day analytical instrumentation of natural clarifying agent,
It is related to a kind of method of quality control for the pharmaceutical composition for repairing skin barrier.
Background technology
In recent years, widely using with glucocorticoid external preparation, steroid dependent dermatitis gradually increases, it has also become
The common disease of dept. of dermatology.The disease has polymorphic infringement, to hormone dependant, the features such as recurrent exerbation, drastically influence patient's
Appearance and mental health.In general, potent hormone ointment uses more than 1.5 months in face, weak to imitate or swash containing micro
The violation cosmetics of element use (1-2) for a long time, all can locally induce steroid dependent dermatitis.Long-term external application sugar cortical hormone
Element causes Skin barrier destruction, and atrophy occurs in skin, and cuticula is thinning, papilla regression, while inflammation inducing factor expression
It is relevant, make skin of face inflammation recurrent exerbation and gradually aggravate, so as to produce dependence to hormone.Patient skin barrier function destroys
Afterwards, skin increases to the sensitiveness of extraneous various physicochemical irritations, the symptom after Exposure to Sunlight, wind, heat and feed irritable food
Aggravate.Therefore, anti-anaphylaxis, the moisturizing medical science skin care item that can recover skin barrier function should be used cooperatively, to reduce cutaneous sensibility.
For this case, emphasis of many researchers using skin repair product as research and development.Such as patent 201510725856.X, this is special
Profit discloses a kind of skin repair liquid and its application, and the skin repair liquid includes glycerine, 1,2- pentanediols, dipotassium glycyrrhizinate, people
Oligopeptides, Sodium Hyaluronate, aloe barbadensis Miller juice, water.Such as patent 201510888998.8, a kind of skin which disclose
Liquid and preparation method thereof is repaired, the reparation liquid is by safflower, Herba portulacae, rheum officinale, folium artemisiae argyi, rhizoma Gastrodiae, rice vinegar, lemongrass, lemon, crocodile egg
Stoste forms.Such as patent 201510540856.2, the patent discloses a kind of peptide composition for skin repair, the polypeptide by
Hexapeptide -3, copper peptide, palmityl tripeptides -1, pentapeptide -31 etc. form.Such as patent 201510812038.3, one kind which disclose
For the formulation preparation method and its preparation of skin repair, said preparation is by rheum officinale, borneol, Asian puccoon, garden burnet, giant knotweed, kuh-seng, awns
Nitre, the root of Dahurain angelica, Verbena officinalis, desmodium, climbing groundsel, ginkgo leaf, the stem of noble dendrobium, pearl, catechu, rhizoma atractylodis, honeysuckle etc. form.But these are produced
Product are not all started with from the pathologic, physiologic feature of steroid dependent dermatitis to be solved the problems, such as, therefore usage time is grown, and effect is but paid no attention to
To think, this just brings puzzlement for manufacturer and user, and many chemicals can cause irreversible injury to skin,
And natural plants cosmetics to no skin irritation, and can help skin gradually to recover normal function without using chemicals,
Therefore natural plants cosmetics enjoy consumers.
The preparation of compound Chinese medicinal preparation often includes extraction process, and obtained Chinese medical extract is a complicated mixing
System, including active component and impurity, wherein (even containing materials such as substantial amounts of minuteness particle, starch, tannin, protein, polysaccharide
Have the impurity such as medicinal material residue, silt), it is colloidal dispersion system to be collectively forming 1-100Knm, belongs to dynamics and thermodynamics is unstable
Determine system, the trend with spontaneous sedimentation.For retentive activity composition, these impurity are removed, makes solid-liquid rapid and is completely separated from,
The stability of destruction mixed system will be tried.Compound Chinese medicinal preparation uses aqueous extraction-alcohol precipitation technology more, and the technique has clarification medicine
Liquid, reduce the advantages that dose.But the technique is in clarification decoction simultaneously by the macromolecular of some alcohol insoluble matters in Chinese medicine such as egg
White matter and par-tial polysaccharide remove as impurity, and these compositions have extensive physiologically active and medical value.Clarificant method
It is to add a kind of fining agent in the Chinese medicine extract of suspension to remove corase particles in solution in a manner of adsorption bridging and charge neutrality,
To reach a kind of high efficiency technical for isolating and purifying purpose, ZTC1+1 natural clarifying agents are that one kind is extracted from food, with natural more
Sugar etc. is natural high molecular substance made of raw material, is made up of A, B2 kind component, and it is using " 1+1 " clarification skill that it, which clarifies principle,
Art, wherein 1 component plays main flocculation, another 1 component plays auxiliary flocculation, has preferable clarifying effect.
With the reach of science, the popularization and application of analytical instrument, Chinese medicine compound prescription detection and analysis and quality control is set constantly to carry
It is high and perfect.Thin layerChromatogramMethod (TLC), being will be suitableStationary phaseIt is coated on glass plate, plastics or aluminium substrate, it is uniform into one
Thin layer.After point sample, expansion, according toRf value(Rf) with the Rf value (Rf) of the suitable tester chromatogram of gained by the same method
Compare, be quick separating and a small amount of thing of qualitative analysis to carry out the discriminating of medicine, determination of foreign matter or the method for assay
A kind of critically important experimental technique of matter, it is also used for tracking reaction process.High performance liquid chromatography detection analytical technology is in Chinese medicine matter
Played an increasingly important role in amount control.It is that Chinese medicine or compound are carried out into go-on-go by analysis means such as spectral chromatographies
Survey, utilize the distinctive chemical profile of heterogeneity, the species and content of the intrinsic chemical composition contained by reflection Chinese medicine or compound.
Pharmaceutical composition is by radix cynanchi atrati, Pilea fasciata, ginkgo leaf, false-yellowflower milkwort root or herb, stone precipice tea and the taste medicinal material of roxburgh anoectochilus terminal bud 6
Composition, its active component is more, according to 2015 editions Chinese Pharmacopoeias, chromatographic condition and the system suitability side of each component medicinal material
Method is different, determines the target component content of each medicinal material in compound one by one, is unfavorable for evaluating and controls the quality of herbal mixture,
So as to ensure the stability of its effective component and quantity.
The present invention from substantial amounts of natural drug, has selected radix cynanchi atrati, Pilea fasciata, ginkgo leaf, Huang according to tcm theory
Flower omei meadowrue herb, stone precipice tea and roxburgh anoectochilus terminal bud are raw material, prepare and clarify the composition for obtaining described reparation skin barrier, its
In, described radix cynanchi atrati selects the dry root of Asclepiadaceae plant radix cynanchi atrati；Pilea fasciata selects Urticaceae Pilea；Bastard cold water
Colored drying herb；Ginkgo leaf selects the dried leaf of Ginkgoaceae plant Ginkgo biloba；False-yellowflower milkwort root or herb selects Polygalaceae polygala
The dry root of false-yellowflower milkwort root or herb；Roxburgh anoectochilus terminal bud selects the drying herb of orchidaceae is a perennial herb roxburgh anoectochilus terminal bud；Stone precipice tea
It is fragrant through Guangdong Provincial TCM Hospital Pharmaceutical Department Chen Yan from the yellow auspicious wooden dried leaf of the bright leaf of Theaceae Yang Tong platymisciums, the above-mentioned prepared slices of Chinese crude drugs
Director pharmacist of traditional Chinese medicine identification meets national standard, and it is for future reference that sample voucher is deposited in Guangdong Provincial TCM Hospital Chinese medicine preparation laboratory.
According to《Chinese Pharmacopoeia》Recorded with document：
Radix cynanchi atrati is radix cynanchi atrati from Asclepiadaceae plant radix cynanchi atrati or the dry root or rhizome of Cynanchum versicolor Bunge.Radix cynanchi atrati is bitter, salty, cold, returns
Stomach, liver and kidney channel, function clearing heat and cooling blood, inducing diuresis for treating strangurtia, detoxification sore treatment.For the heating of warm evil wound battalion, fever due to yin deficiency, hectic fever due to yin labor it is hot,
Postpartum fever caused by blood deficiency, heat gonorrhea, blood strangury, swollen ulcer drug.
Pilea fasciata also known as fasciate clearweed herb, blue or green medicine, common clearweed herb, amsonia elliptica, anise, palace fiber crops, are Urticaceae Pilea
The herb of Pilea fasciata, the province such as China Guangxi, Guizhou, Sichuan and Yunnan is distributed in, there is clearing heat and detoxicating, promoting blood circulation by removing wind, reason
The effect of gas relieves pain, cures mainly high fever, tonsillitis swelling and pain, thrush, traumatic injury, fracture, arthralgia pain due to rheumatism, Guangxi it is among the people with it in being good for
Spleen, lactagogue, cough-relieving, anti-inflammatory, preventing and treating adnexitis and gynecopathy such as pelvic inflammatory disease.
Ginkgo leaf is the dried leaf of Ginkgoaceae plant Ginkgo biloba.Ginkgo leaf is a kind of rare Chinese medicine, sweet, bitter, puckery, flat, is returned
The heart, lung channel.Function is promoting blood circulation and removing blood stasis, removes obstruction in channels to relieve pain, astringes the lung and relieving asthma, change turbid lipid-loweringing, for treat obstruction of collaterals by blood stasis, chest impediment and cardialgia, in
Windage yaw paralysis, deficiency syndrome of the lung cough and asthma, hyperlipidemia.Research thinks that the main active in ginkgo leaf has flavonoids, terpene lactones at present
Class, organic acid, additionally containing polypenthylene alcohols ester, polysaccharide, alkyl phenolic acid compound and some other nutritional ingredients
Deng.Ginkgo biloba p.e has the function that to mitigate neural cell injury and cerebral ischemic.
False-yellowflower milkwort root or herb also known as chrysanthemum ginseng, chrysanthemum hang water lotus, and it is the drying of Polygalaceae polygala false-yellowflower milkwort root or herb
Root, it is a kind of long folk medicine of applicating history.It has invigorating the spleen, dissipate blood stasis and smoothing collaterals and other effects, is conventionally used to treat overstrain
Under the weak, prolapse of uterus, infantile malnutrition, edema due to deficiency of the spleen, band, the disease such as irregular menstruation, traumatic injury, acute, chronic hepatitis.Newest
Research finds that it has good antioxidant radical activity, and its saponin component has very strong effect for reducing blood fat.
Stone precipice tea is the yellow auspicious wooden dried leaf of the bright leaf of Theaceae Yang Tong platymisciums, also known as clint tea, Shi Yancha, tor tea,
Red light, the bright yellow auspicious wood of leaf of Adinandra nitida, bright leaf, is distributed mainly on the ground such as China Guangxi, Guangdong and Guizhou, aboundresources is among the people
Using the with a long history of the plant.Stone precipice tea gives off a strong fragrance, the then sweet cool sweetness of taste elder generation slight bitter, have it is clearing heat and detoxicating, protect liver is bright
Mesh, anti-inflammatory moistening lung, beauty treatment decompression, Adjust-blood lipid, stomach strengthening and digestion promoting etc. act on, and are all very high wild plants of a kind of health care and medical value
Thing.The compositions such as flavonoids, triterpene saponin, Tea Polyphenols, polysaccharide are mainly contained in the tea of stone precipice.
Roxburgh anoectochilus terminal bud is orchidaceae is a perennial herb roxburgh anoectochilus terminal bud, is very rare Chinese herbal medicine, is known as " god
The laudatory titles such as grass " " black ginseng ".Whole body is medicinal, and taste is put down, and property is sweet, has clearing heat and cooling blood, removing dampness and detoxicating, the function such as prolong life, can
Treat cough with lung heat, treating rheumatic ostealgia, diabetes, cystitis etc..Rich in polysaccharide, flavonoids, trace element, amino acid, phenols, three
The many kinds of substance such as terpene.
At present, both at home and abroad there is not yet radix cynanchi atrati, Pilea fasciata, ginkgo leaf, false-yellowflower milkwort root or herb, stone precipice tea and roxburgh anoectochilus terminal bud are carried
The composition of thing is taken to be used for the relevant report for repairing skin barrier.
Compared with prior art, the beneficial effects of the invention are as follows：Pharmaceutical composition provided by the invention, for the knot of skin
Structure and the mechanism of steroid dependent dermatitis skin damage, have the work for improving epidermal cell microcirculation and promoting skin metabolism
With, can accelerate regeneration with repair etc. effect；The clinical trial of scientific and precise further proves that it has good reparation skin
Skin barrier action, and without skin irritation, adverse reaction is low, fool proof, and social required quantity is big, is especially suitable for being added to cream
In the cosmetics such as emulsion, there is good social and economic benefit.
The content of the invention
Present invention aims at according to modern technologies, there is provided a kind of to have the Chinese medicine combination for repairing skin barrier effect
The High-speed clarification of thing, efficient method of quality control, drug regimen amount of substance is evaluated and controlled on the whole, ensure to repair skin screen
The effect of hindering Chinese medicine is more stable, reliable.
The present invention is to be achieved through the following technical solutions：
A kind of clarification process with the pharmaceutical composition for repairing skin barrier effect, take with reparation skin barrier effect
Chinese medicine preparation conventional ethanol soak extraction, soak is concentrated under reduced pressure, concentrate macroporous resin adsorption, 5%-95% ethanol
Elution, eluent concentration, as described has the pharmaceutical composition for repairing skin barrier effect, is placed in 60 DEG C of water-baths, first adds
Enter the II type ZTC1+1 natural clarifying agent B components of 4% effluent volume, the II types ZTC1+1 that 2% volume is added after 1h is naturally clear
Clear agent component A, during which stirred 1 minute per half an hour and make impurity component coagulative precipitation, then with 4000r/min in centrifuge
10min is centrifuged, takes supernatant；Described II type ZTC1+1 natural clarifying agents：It is one kind using natural polysaccharide as made of raw material
Natural high molecular substance, it is made up of two components of A, B, component A is yellowish-brown soluble solids powder or light brown liquid, mainly
Amylase-containing, it is made into 1% aqueous solution use；B component is light yellow soluble solids powder or light brown liquid, mainly containing shallow lake
The complex enzyme of powder enzyme, the solution that 1% concentration is made into 1% acetic acid use.
Component A, precision measure 1.0g II type ZTC1+1 natural clarifying agents and added on a small quantity in distilled water, stir into pasty state, then
The viscose that distilled water is formulated into 100mL is added, 24h is swelled, is filtered with double gauze；
B component, it is molten that precision measures the acetic acid that a small amount of volume fraction of 1.0g II type ZTC1+1 natural clarifying agents addition is 1%
Liquid, pasty state is stirred into, then the acetum for adding volume fraction to be 1% is configured to 100mL viscose, 24h is swelled, with double-deck yarn
Cloth filters..
A kind of clarification process and method of quality control with the pharmaceutical composition for repairing skin barrier effect, its feature exist
In this method comprises the following steps：
(1) the medicine Chinese medicine radix cynanchi atrati is differentiated with thin-layered chromatography, including：
A. the preparation of test sample：This product pharmaceutical composition 2mL is taken, adds methanol 30mL, ultrasonic extraction 20 minutes, filters,
Filtrate is evaporated, and residue adds methanol 1mL to make dissolving, as need testing solution；
B. the preparation of control medicinal material solution：Radix cynanchi atrati control medicinal material powder 1g is taken, adds methanol 30mL, ultrasonic extraction 20 minutes,
Filtration, filtrate are evaporated, and residue adds methanol 1mL to make dissolving, as radix cynanchi atrati medicinal material solution；
C. radix cynanchi atrati indentification by TLC：Tested according to thin-layered chromatography, draw control medicinal material solution and sample solution each 2 respectively
μ L, put respectively on same silica gel g thin-layer plate, using n-butanol-ethyl acetate-water (4: 1: 5) as solvent, deploy, take out,
Dry, spray ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, is inspected under white light；
(2) the medicine Chinese medicine ginkgo leaf is differentiated with thin-layered chromatography, including：
A. the preparation of test sample：This product pharmaceutical composition 2mL is taken, adds ethanol 20mL, ultrasonic extraction 30 minutes, filters,
Water bath method, residue add methanol 1mL to make dissolving, take supernatant as need testing solution；
B. the preparation of control medicinal material solution：Ginkgo leaf control medicinal material 1g is taken, adds ethanol 20mL, heating and refluxing extraction 10min,
Filtration, water bath method, residue adds methanol 1mL to make dissolving, as control medicinal material solution；
C. ginkgo leaf indentification by TLC：According to thin-layered chromatography test, respectively draw ginkgo leaf control medicinal material solution and
Each 5 μ L of test sample, put respectively on same silica gel g thin-layer plate, using ethyl acetate-butanone-formic acid-water (5: 3: 1: 1) as
Solvent, deploy, take out, dry, spray：3% alchlor ethanol solution, hot blast drying, put and examined under ultraviolet lamp (365nm)
Depending on；
(3) content of anoectochilus roxburghii glycosides in high effective liquid chromatography for measuring medicine is used, including：
A. high-efficient liquid phase chromatogram condition：Inertsil ODS-3 chromatographic columns, octadecylsilane chemically bonded silica are filler,
Mobile phase is methanol A- formic acid waters, gradient elution is 0~10min 30%A → 35%A, 10~45min 35%A → 45%A,
45~60min 45%A → 75%A, equilibration time 15min, flow velocity 0.8mL/min, the μ L of sample size 10,24 DEG C of column temperature, PDA inspections
Survey device acquisition range：190~400nm；Detection wavelength 215nm；
B. the preparation of need testing solution：Take and repair the Chinese medicine preparation that skin barrier acts on, conventional ethanol soak extraction 24 is small
When, with filtered through gauze, it is concentrated under reduced pressure as the 2%-5% of original weight amount, macroporous resin adsorption on concentrate, 5%-95% ethanol is washed
It is de-, concentrate eluant, add methanol constant volume to i.e. every milliliter of decoction active compound containing 1g, with methanol dilution 5,10,15 times it is standby；
C. the preparation of reference substance solution：Precision weighs Hernandulcin, polygalic acid, camellianin A and anoectochilus roxburghii glycosides pair
According to each 2mg of product, respectively plus methanol prepares the reference substance solution of different multiples mass concentration；
D. linear relationship is investigated：The accurate reference substance solution for drawing above-mentioned various concentrations, high performance liquid chromatograph enter respectively
The μ L of sample 10, linear regression is carried out with the response signal and concentration that measure, obtains the regression equation of standard items；
E. determination method：It is accurate respectively to draw reference substance and each 10 μ L of need testing solution, inject hplc determination.
As preferable, repair the pharmaceutical composition of skin barrier clarification process and method of quality control test sample it is molten
Liquid is Aqueous extracts, and specific method is：The Chinese medicine preparation for repairing skin barrier effect is taken, conventional ethanol soak extraction 24 hours, is used
Filtered through gauze, it is concentrated under reduced pressure as the 2%-5% of original weight amount, macroporous resin adsorption on concentrate, 5%-95% ethanol elutions, concentration
Eluent, add methanol constant volume to i.e. every milliliter of decoction active compound containing 1g, with methanol dilution 5,10,15 times it is standby.
Preferably, the clarification process of the pharmaceutical composition of described reparation skin barrier and method of quality control are used to extract
Method identical medicinal composition tablets, granule or medicine composition pour containing radix cynanchi atrati, Pilea fasciata, ginkgo leaf, chrysanthemum
The compound of lotus, stone precipice tea and roxburgh anoectochilus terminal bud medicinal material.
Preferably, the clarification process of pharmaceutical composition of skin barrier and the assay side of method of quality control are being repaired
In method：Test sample dilutes 5 times, crosses 0.22 μm of filter membrane, 10 μ L enter liquid chromatograph, the content of anoectochilus roxburghii glycosides is：10.51μg/ml.
Preferably, described thin-layered chromatography differentiates in radix cynanchi atrati, ginkgo leaf, radix cynanchi atrati with n-butanol-ethyl acetate-water (4:
1: 5) be used as solvent, the colour developing of spray ethanol solution of sulfuric acid, ginkgo leaf using ethyl acetate-butanone-formic acid-water (5: 3: 1: 1) as
Solvent, spray：3% alchlor ethanol solution develops the color.
The present invention is with the identification composition Hernandulcin of Pilea fasciata medicinal material, the identification composition of false-yellowflower milkwort root or herb medicinal material
Polygalic acid, the identification composition camellianin A of stone precipice tea medicinal material, the composition anoectochilus roxburghii glycosides of identifying of roxburgh anoectochilus terminal bud medicinal material are index, the party
Method avoids medicine group and the overlapping of (peak) is responded under same chromatographic condition into medicinal material Chinese medicine thing composition, with containing for active component
Measurement is set to control standard, more rationally, truly reflects a kind of Traditional Chinese medicine composition for having and repairing skin barrier effect
Quality.
Figure of description
Fig. 1 is radix cynanchi atrati control medicinal material figure.
Fig. 2 is ginkgo leaf control medicinal material figure.
Fig. 3 is Hernandulcin under 215nm wavelength, polygalic acid, the chromatogram of camellianin A and anoectochilus roxburghii glycosides reference substance
Figure.
Fig. 4 is HPLC chromatogram under the pharmaceutical composition 215nm wavelength for repairing skin barrier.
Fig. 5 is placebo solution HPLC chromatogram under 215nm wavelength.
Embodiment
A kind of clarification process and method of quality control with the pharmaceutical composition for repairing skin barrier effect of the present invention, should
Clarification process is carried out as follows：
Clarification process and each component content
(1) preparation of fining agent：II type ZTC1+1 natural clarifying agents are by A, the components of B two composition.Component A：Precision measures
Added in 1.0g component As on a small quantity in distilled water, stir into pasty state, add the viscose that distilled water is formulated into 100mL, be swelled
24h, filtered and produced with double gauze.B component：Precision measures the acetic acid for adding that a small amount of volume fraction is 1% in 1.0g B components
Solution, pasty state is stirred into, then the acetum for adding volume fraction to be 1% is configured to 100mL viscose, 24h is swelled, with bilayer
Filtered through gauze, produce.
(2) fining agent A, B addition sequences：According to specification, ZTC natural clarifying agents mainly select according to the property of solution
Different orders of addition, general Chinese medicine compound prescription are sour environment relative to isoelectric points of proteins (pH=4.8), and order of addition is first
Component A after B, B component dosage are 2 times of component A.
(3) evaluation index：Hernandulcin contents retention rate (X1), polygalic acid content retention rate are chosen respectively
(X2), camellianin A content retention rate (X3), anoectochilus roxburghii glycosides content retention rate (X4), solid content clearance (X5) refer to as evaluation
Mark carries out comprehensive grading, compound retention rate weight is set into 0.7, solid content clearance X5 weights are set to 0.3, i.e. OD%=
(0.7X1+0.7X2+0.7X3+0.7X4)/4+0.3X5.Calculation formula is as follows：
Content × 100% of compound in content/extract solution of compound is extracted after compound retention rate=clarification
Solid content before solid content clearance=(solid content after solid content-clarification before clarification)/clarification ×
100%
(4) technique is investigated：It is investigation factor with fining agent addition, solid-liquid ratio, temperature, time, it is each to choose 3 optimal water
It is flat, with L9(34) orthogonal arrage tested, comprehensive grading is calculated with compound retention rate and solid content clearance, carries out variance point
Analysis, the results are shown in Table 2.Fining agent addition and extract solution solid-liquid ratio are commented synthesis it can be seen from the variance analysis of orthogonal test
Divide and have a significant impact, temperature and time is not statistically significant to the clarifying effect of extract solution.Found in experiment, fining agent B component
For 4% when the clear effect of pharmaceutical composition Aqueous extracts it is good, determine II type ZTC 1+1 natural clarifying agents clarify pharmaceutical composition extract solution
Optimum process condition be fining agent addition B component 4%, B component is that 2% solid-liquid ratio is 1: 5, and temperature is 60 DEG C, during reaction
Between 1h.
The L of table 29(34) orthogonal experiments
(5) each component content：With the optimised process bar of II type ZTC 1+1 natural clarifying agents clarification pharmaceutical composition extract solution
Part：Fining agent addition B component 4%, B component are that 2% solid-liquid ratio is 1: 5, and temperature is 60 DEG C, reaction time 1h, prepares sample.
High-efficient liquid phase chromatogram condition：Inertsil ODS-3 chromatographic columns, eluent gradient are shown in Table 1, equilibration time 15min, flow velocity
0.8mL/min, 24 DEG C of column temperature, PDA detector acquisition ranges：190~400nm；Detection wavelength 215nm, the μ L of sample introduction 10.Selection
215nm wavelength determines Hernandulcin, polygalic acid, camellianin A and anoectochilus roxburghii glycosides content respectively.Sample continuous acupuncture 3 times,
Average.Every gram of crude drug is measured by method of quality control of the present invention, the content of anoectochilus roxburghii glycosides is：10.51μg/ml.
As Figure 1-5, a kind of clarification process and matter with the pharmaceutical composition for repairing skin barrier effect of the present invention
Amount control method, the method for quality control are carried out as follows：
(1) preparation of need testing solution：Take and repair the Chinese medicine preparation that skin barrier acts on, conventional ethanol soak extraction 24 is small
When, with filtered through gauze, it is concentrated under reduced pressure as the 2%-5% of original weight amount, macroporous resin adsorption on concentrate, 5%-95% ethanol is washed
It is de-, concentrate eluant, add methanol constant volume to i.e. every milliliter of decoction active compound containing 1g, with methanol dilution 5,10,15 times it is standby；
(2) the preparation precision of reference substance solution weighs Hernandulcin, polygalic acid, camellianin A and anoectochilus roxburghii glycosides
2mg, respectively plus methanol prepares different multiples mass concentration reference substance solution.
(3) linear relationship is investigated：The accurate reference substance solution for drawing above-mentioned various concentrations, high performance liquid chromatograph enter respectively
The μ L of sample 10, linear regression is carried out with the response signal (peak area) and concentration that measure, obtains the regression equation of 5 kinds of standard items.
Hernandulcin Y=253.53X+42.37 (R2=0.9996), concentration is linearly good in 3.245~30.35g/mL scopes
It is good., polygalic acid Y=288.53X+34.05 (R2=0.9997), concentration is linearly good in 2.245~22.34g/mL scopes.,
Camellianin A Y=228.53X+38.48 (R2=0.9999), concentration is linearly good in 3.545~35.35g/mL scopes., gold
Line lotus glycosides Y=267.15 X+51.31 (R2=0.9995), concentration is linearly good in 4.245~42.35g/mL scopes.
(4) assay method：Determined according to high-efficient liquid phase chromatogram condition, chromatographic condition and system suitability and Inertsil
ODS-3 chromatographic columns, mobile phase are shown in Table 1, equilibration time 15min, flow velocity 0.8mL/min, the μ L of sample introduction 10,24 DEG C of column temperature, PDA detections
Device acquisition range：190~400nm；Detection wavelength 215nm.
The eluent gradient of table 1 elutes table
(5) efficiency evaluation is carried out to the assay method of above-mentioned determination, including precision, repeatability, stability etc..
2. radix cynanchi atrati, ginkgo leaf are differentiated with thin-layered chromatography in silver medal bits miraculous cure compositions：
(1) radix cynanchi atrati thin-layered chromatography differentiates
The preparation of test sample：This product pharmaceutical composition 2mL is taken, adds methanol 30mL, ultrasonic extraction 20 minutes, filters, filter
Liquid is evaporated, and residue adds methanol 1mL to make dissolving, as need testing solution；
The preparation of control medicinal material solution：Radix cynanchi atrati control medicinal material powder 1g is taken, adds methanol 30mL, ultrasonic extraction 20 minutes, filter
Cross, filtrate is evaporated, and residue adds methanol 1mL to make dissolving, as radix cynanchi atrati medicinal material solution；
Radix cynanchi atrati indentification by TLC：Tested according to thin-layered chromatography, draw control medicinal material solution and each 2 μ of sample solution respectively
L, put respectively on same silica gel g thin-layer plate, using n-butanol-ethyl acetate-water (4: 1: 5) as solvent, deploy, take out,
Dry, spray ethanol solution of sulfuric acid, it is clear to be heated to spot development at 105 DEG C, is inspected under white light；
(2) ginkgo leaf thin-layered chromatography differentiates
The preparation of test sample：This product pharmaceutical composition 2mL is taken, adds ethanol 20mL, ultrasonic extraction 30 minutes, filtration, water
Bath is evaporated, and residue adds methanol 1mL to make dissolving, takes supernatant as need testing solution；
The preparation of control medicinal material solution：Ginkgo leaf control medicinal material 1g is taken, adds ethanol 20mL, heating and refluxing extraction 10min, is filtered
Cross, water bath method, residue adds methanol 1mL to make dissolving, as control medicinal material solution；
Ginkgo leaf indentification by TLC：Tested according to thin-layered chromatography, draw ginkgo leaf control medicinal material solution and confession respectively
Each 5 μ L of test agent, put respectively on same silica gel g thin-layer plate, exhibition is used as using ethyl acetate-butanone-formic acid-water (5: 3: 1: 1)
Agent is opened, is deployed, takes out, dries, is sprayed：3% alchlor ethanol solution, hot blast drying, put and inspected under ultraviolet lamp (365nm)；
A kind of clarification process and method of quality control with the pharmaceutical composition for repairing skin barrier effect of the present invention, it is excellent
The preparation of the need testing solution of choosing：The preparation of this method publicity product solution：The Chinese medicine preparation for repairing skin barrier effect is taken, it is conventional
Ethanol soak extraction 24 hours, with filtered through gauze, it is concentrated under reduced pressure as the 2%-5% of original weight amount, macroreticular resin is inhaled on concentrate
It is attached, 5%-95% ethanol elutions, concentrate eluant, add methanol constant volume to i.e. every milliliter of decoction active compound containing 1g, with methanol dilution 5,
10th, 15 times it is standby；
The present invention is a kind of to be had in the clarification process and method of quality control for repairing the pharmaceutical composition that skin barrier acts on,
Preferable assay method：High-efficient liquid phase chromatogram condition determines, chromatographic condition and system suitability： Inertsil ODS-3
Chromatographic column, eluent gradient are shown in Table 1, equilibration time 15min, flow velocity 0.8mL/min, 24 DEG C of column temperature, PDA detectors collection model
Enclose：190~400nm；Detection wavelength 215nm, the μ L of sample introduction 10.
Method of quality control of the present invention, due to a length of 240nm of Hernandulcin maximum absorption wave, polygala soap
The a length of 202nm of maximum absorption wave of glycosides, a length of 215 nm of maximum absorption wave of camellianin A and anoectochilus roxburghii glycosides, therefore select 215nm ripples
It is long to determine anoectochilus roxburghii glycosides content respectively.
Present inventor is known through investigating, when using acetonitrile-water, acetonitrile-formic acid water, methanol-water solution is as flowing
Phase gradient elute when, drug ingedient separation it is undesirable, with methyl alcohol-formic acid aqueous systems elute, each composition of medicine do not have it is overlapping, and
Peak shape is excellent.Medicine group is evaluated and controlled on the whole to this experimental program by the quality control to active constituents of medicine content
Compound quality, more rationally, truly reflect a kind of quality for the Traditional Chinese medicine composition for repairing skin barrier.
Claims (6)
- A kind of 1. clarification process with the pharmaceutical composition for repairing skin barrier effect, it is characterised in that：Take and repair skin screen The Chinese medicine preparation conventional ethanol soak extraction of barrier effect, soak are concentrated under reduced pressure, and concentrate macroporous resin adsorption, use 5%- 95% ethanol elution, eluent concentration, it is the described pharmaceutical composition for repairing skin barrier effect, is placed in 60 DEG C of water Bath, the II type ZTC1+1 natural clarifying agent B components of 4% effluent volume are first added, the II types ZTC1+1 of 2% volume is added after 1h Natural clarifying agent component A, during which stirred per half an hour and make impurity component coagulative precipitation in 1 minute, then in centrifuge with 4000r/min centrifuges 10min, takes supernatant；Described II type ZTC1+1 natural clarifying agents：It is a kind of using natural polysaccharide as original Natural high molecular substance made of material, it is made up of two components of A, B, component A is yellowish-brown soluble solids powder or light brown Liquid, main amylase-containing, be made into 1% the aqueous solution use；B component is light yellow soluble solids powder or light brown liquid Body, the complex enzyme of main amylase-containing, the solution that 1% concentration is made into 1% acetic acid use.Component A, precision measure 1.0g II type ZTC1+1 natural clarifying agents and added on a small quantity in distilled water, stir into pasty state, add Distilled water is formulated into 100mL viscose, is swelled 24h, is filtered with double gauze；B component, precision measure 1.0g II type ZTC1+1 natural clarifying agents and add the acetum that a small amount of volume fraction is 1%, stir Pasty state is mixed, then the acetum for adding volume fraction to be 1% is configured to 100 mL viscose, 24h is swelled, with double gauze mistake Filter.
- A kind of 2. method of quality control with the pharmaceutical composition for repairing skin barrier effect, it is characterised in that this method bag Include following steps：(1) the medicine Chinese medicine radix cynanchi atrati is differentiated with thin-layered chromatography, including：A. the preparation of test sample：This product pharmaceutical composition 2mL is taken, adds methanol 30mL, ultrasonic extraction 20 minutes, filtration, filtrate It is evaporated, residue adds methanol 1mL to make dissolving, as need testing solution；B. the preparation of control medicinal material solution：Radix cynanchi atrati control medicinal material powder 1g is taken, adds methanol 30mL, ultrasonic extraction 20 minutes, filters, Filtrate is evaporated, and residue adds methanol 1mL to make dissolving, as radix cynanchi atrati medicinal material solution；C. radix cynanchi atrati indentification by TLC：Tested according to thin-layered chromatography, draw control medicinal material solution and each 2 μ L of sample solution respectively, Put respectively on same silica gel g thin-layer plate, using n-butanol-ethyl acetate-water (4: 1: 5) as solvent, deploy, take out, dry in the air It is dry, ethanol solution of sulfuric acid is sprayed, it is clear to be heated to spot development at 105 DEG C, is inspected under white light；(2) the medicine Chinese medicine ginkgo leaf is differentiated with thin-layered chromatography, including：A. the preparation of test sample：This product pharmaceutical composition 2mL is taken, adds ethanol 20mL, ultrasonic extraction 30 minutes, filtration, water-bath It is evaporated, residue adds methanol 1mL to make dissolving, takes supernatant as need testing solution；B. the preparation of control medicinal material solution：Ginkgo leaf control medicinal material 1g is taken, adds ethanol 20mL, heating and refluxing extraction 10min, is filtered Cross, water bath method, residue adds methanol 1mL to make dissolving, as control medicinal material solution；C. ginkgo leaf indentification by TLC：Tested according to thin-layered chromatography, draw ginkgo leaf control medicinal material solution respectively and for examination Each 5 μ L of sample, put respectively on same silica gel g thin-layer plate, expansion is used as using ethyl acetate-butanone-formic acid-water (5: 3: 1: 1) Agent, deploy, take out, dry, spray：3% alchlor ethanol solution, hot blast drying, put and inspected under ultraviolet lamp (365nm)；(3) Hernandulcin in high effective liquid chromatography for measuring medicine, polygalic acid are used, camellianin A and anoectochilus roxburghii glycosides contain Amount, including：A. high-efficient liquid phase chromatogram condition：Inertsil ODS-3 chromatographic columns, octadecylsilane chemically bonded silica are filler, flowing Be mutually methanol A- formic acid waters, gradient elution is 0~10min 30%A → 35%A, 10~45min 35%A → 45%A, 45~ 60min 45%A → 75%A, equilibration time 15min, flow velocity 0.8mL/min, the μ L of sample size 10,24 DEG C of column temperature, PDA detectors Acquisition range：190~400nm；Detection wavelength 215nm；B. the preparation of need testing solution：The Chinese medicine preparation for repairing skin barrier is taken, conventional ethanol soak extraction 24 hours, uses gauze Filtering, it is concentrated under reduced pressure as the 2%-5% of original weight amount, macroporous resin adsorption on concentrate, 5%-95% ethanol elutions, concentration elution Liquid, add methanol constant volume to i.e. every milliliter of decoction active compound containing 1g, with methanol dilution 5,10,15 times it is standby；C. the preparation of reference substance solution：Precision weighs Hernandulcin, polygalic acid, camellianin A and anoectochilus roxburghii glycosides reference substance Each 2mg, respectively plus methanol prepares the reference substance solution of different multiples mass concentration；D. linear relationship is investigated：The accurate reference substance solution for drawing above-mentioned various concentrations respectively, the μ of high performance liquid chromatograph sample introduction 10 L, linear regression is carried out with the response signal and concentration that measure, obtains the regression equation of standard items；E. determination method：It is accurate respectively to draw reference substance and each 10 μ L of need testing solution, inject hplc determination.
- 3. according to claim 1, a kind of clarification process and matter with the pharmaceutical composition for repairing skin barrier effect described in 2 Amount control method, this method need testing solution are alcohol extract, and specific method is：The Chinese medicine preparation for repairing skin barrier is taken, it is conventional Ethanol soak extraction 24 hours, with filtered through gauze, it is concentrated under reduced pressure as the 2%-5% of original weight amount, macroporous resin adsorption on concentrate, 5%-95% ethanol elutions, concentrate eluant, add methanol constant volume to i.e. every milliliter of decoction active compound containing 1g, with methanol dilution 5,10, 15 times standby.
- 4. according to claim 1, a kind of clarification process and matter with the pharmaceutical composition for repairing skin barrier effect described in 2 Amount control method, it is characterised in that：This method is used for extracting method identical medicinal composition tablets, granule or medicine composition Compound containing radix cynanchi atrati, Pilea fasciata, ginkgo leaf, false-yellowflower milkwort root or herb, stone precipice tea and roxburgh anoectochilus terminal bud medicinal material.
- 5. according to claim 1, a kind of clarification process and matter with the pharmaceutical composition for repairing skin barrier effect described in 2 Amount control method, it is characterised in that in content assaying method：Test sample dilutes 5 times, crosses 0.22 μm of filter membrane, and 10 μ L enter liquid phase color Spectrometer, the content of anoectochilus roxburghii glycosides are：10.50μg/ml.
- 6. according to claim 1, a kind of clarification process and matter with the pharmaceutical composition for repairing skin barrier effect described in 2 Amount control method, it is characterised in that：Described thin-layered chromatography differentiates in radix cynanchi atrati, ginkgo leaf that radix cynanchi atrati is with n-butanol-acetic acid second Ester-water (4: 1: 5) is used as solvent, and the colour developing of spray ethanol solution of sulfuric acid, ginkgo leaf are with ethyl acetate-butanone-formic acid-water (5: 3: 1 : solvent 1) is used as, is sprayed：3% alchlor ethanol solution develops the color.
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