CN110193046A - A kind of response phase method optimization acne washout side extracting method - Google Patents
A kind of response phase method optimization acne washout side extracting method Download PDFInfo
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Abstract
The invention belongs to treat acne technical field, a kind of response phase method optimization acne washout side extracting method is disclosed；It is that index carries out comprehensive score according to content and paste-forming rate using the content of high effective liquid chromatography for measuring Fructus Forsythiae ester A, forsythin, epiberberine, coptisine, palmatine, jamaicin；By experiment of single factor, extracting method is primarily determined；Response surface experiments are designed by Box-Behnken Design method and determine optimum optimization extracting method, select concentration to do Extraction solvent for 80% ethyl alcohol, solid-liquid ratio 1:11 is extracted 2 times, 100min minutes each.The optimum extraction process of acne washout side provided by the invention be be condensed back extract, select concentration for 80% ethyl alcohol, solid-liquid ratio 1:11, extraction twice, each 100min.Extracting method of the invention for acne washout side extraction stablize it is feasible, and for its shaped preparation it is further develop effective foundation is provided.
Description
Technical field
The invention belongs to treat acne technical field more particularly to a kind of response phase method optimization extraction side, acne washout side
Method.
Background technique
Currently, the prior art commonly used in the trade is such that
Acne is a kind of chronic inflammatory skin of pilosebaceous follicle, is common in puberty men and women.The morbidity machine of acne
System is not fully understood, and majority thinks to increase with androgen levels, the high level of propionibacterium acnes, follicular orifice epithelium angling mistake
Degree, sebum secretion increases and blood microcirculation is unsmooth related.And in traditional Chinese medicine angle, the lesion of skin be often the five internal organs to
The also useful information of outer reception and registration.Acne is often caused by heat evil, and it is excessively serious that heat evil invades body, changes the raw poison of fire, and calcination body fluid leads to blood
It coagulates and becomes silted up.Leading acnegenic heat evil illness often has wind and heat in the lung meridian card, syndrome of dampness-heat of spleen and stomach, heat toxin to stop up Sheng card, syndrome of hyperactivity of fire due to yin deficiency etc..It seeks
The perseverance pathogenesis of acne cause of disease is complicated, for its main pathogenesis using inhibit sebaceous glands function, adjust endocrine, it is anti-infective,
The drugs such as antiandrogen and anti-inflammatory, anti-hair follicle sebum angling are treated, in recent years in medicament selection, treatment method and clinical efficacy
Equal everyways have biggish development.Acne voluntarily can fully recover or mitigate after puberty, but take correctly control not in time
Treatment measure, more after then will appear pachylosis even uneven phenomenon.The psychological burden of people is caused, while also affecting society
It hands over.Therefore acne has become a kind of generally existing very important disease.Cuo caused by treatment propionibacterium acnes at present
The external drug of the skin diseases such as sore, mostly antibiotics chemicals easily produce though this kind of drug has certain curative effect
Raw antibody-resistant bacterium, and side effect is larger.And Chinese medicine, complicated component, mechanism of action multiplicity, it is not easy drug resistance, Chinese medicine is about Cuo
The treatment means of sore are mostly based on for oral administration, and the method that dual-purpose is applied with, effect is preferable, and uneasy to recur.
In conclusion existing problem is: the skin diseases such as acne caused by existing treatment propionibacterium acnes
External drug, mostly antibiotics chemicals are also easy to produce antibody-resistant bacterium though this kind of drug has certain curative effect.Such as:
S. aureus L-forms, streptococcus pneumonia, escherichia coli are to tetracycline resistant；The R-plasmid that S. aureus L-forms carry generates, and makes in 50S subunit
23SrRNA on adenine methyl, generate to the drug resistance of erythromycin；S. aureus L-forms most strains are to minocycline drug resistance.
Erythromycin and clindamycin are main topical antibiotic types, but not can be used as acne trouble using topical antibiotic
The monotherapy of person, often with topical Retinoids class Drug combination, local biostearin local treatment would generally cause to stimulate
And drying, it may cause pigmentation after scar and inflammation
It is generated in view of sebum and the exception of the various procedures such as sebocyte cell differentiation, proliferation and inflammation may cause acne
Occur, therefore Most patients are needed using combined treatment.The therapeutic scheme of this complexity can damage the compliance for the treatment of,
And aggravate acne, this has subsequent affect to quality of life, is in early days vital with positive treatment therefore.
Although current modern medicine has certain in-depth study for the pathogenesis of this disease, there are many treatments at present
Medicinal application is in the clinical treatment of acne, including tretinoin medicines, antibiotic and hormone etc., but the mesh of said medicine treatment
Be directed primarily to the regulatory pathway of acne Pathological Physiology, rather than inhibit the influence of the final product of these approach, and
Due to the recurrent exerbation feature of this disease, drug need to be used for a long time, therefore since the adverse reaction of drug bring also increases, such as different
Tretinoin, maximum side effect are serious teratogenesis, and common tetracycline antibiotics may cause nausea, are vomitted, diarrhea, food
The side effects such as Guan Yan, candidiasis, photaesthesia and benign intracranial pressure increase.
It solves the difficulty and meaning of above-mentioned technical problem: causing the mechanism multiplicity of acne, treat method, the drug of acne
Development in pluralism.But due to pathogenic mechanism complexity, various treatment methods are difficult to perfection.The symptom of many patients is sent out repeatedly
Make, prolonged administration of drugs does not improve, and, also in that Pathogenesis of Acne is complicated, medication cannot suit the medicine to the illness for this, and treatment is difficult.Together
When, many drugs are easy to produce side effect in application process, keep treatment more difficult.The present invention is derived from old docter of TCM's clinical experience
Side has significant curative effect, and external application can be to avoid oral drugs bring adverse reaction.Plurality of active ingredients is investigated, to acne
Treatment have more specific aim, improve treatment curative effect.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of response phase methods to optimize extraction side, acne washout side
Method.
The invention is realized in this way with forsythiaside A, forsythin, epiberberine, coptisine, palmatine, jamaicin
Content, dry cream rate are inspection target, using single-factor design experiment, to Extraction solvent, extraction time, extraction time, solid-liquid ratio
It is investigated, obtains being extracted 2 times, each 1.5h with 10 times 70% of ethyl alcohol, effect is more excellent.On the basis of single factor experiment,
Using forsythiaside A, forsythin, epiberberine, coptisine, palmatine, the content of jamaicin, dry cream rate as inspection target, to mention
Time, concentration of alcohol, solid-liquid ratio is taken as a result to select concentration for 80% with response phase method Optimized Extraction Process for investigation factor
Ethyl alcohol does Extraction solvent, and solid-liquid ratio 1:11 is extracted 2 times, and each 100min minutes effect is optimal.Specifically includes the following steps:
Step 1: choosing the good coptis, Fructus Forsythiae, dandelion, purslane, the root bark of Chinese wolf-berry, and fresh loguat leaf is spare；
Step 2: using high effective liquid chromatography for measuring forsythiaside A, forsythin, epiberberine, coptisine, palmatine,
The content of jamaicin is that index carries out comprehensive score according to the content of upper six kinds of ingredients and paste-forming rate；
Step 3: by experiment of single factor, primarily determining extracting method, extracts medical fluid through being condensed back, filters to obtain the final product；
Step 4: response surface experiments are designed by Box-Behnken Design method and determine optimum optimization extracting method, are selected
It selects the ethyl alcohol that concentration is 80% and does Extraction solvent, solid-liquid ratio 1:11 is extracted 2 times, 100min minutes each.
Further, in step 1, loguat leaf is fresh loguat leaf, that is, adopts and use, due to loguat leaf larger, after weighing,
Before use, brushing away the villus of loquat leaf back with brush, it is then cut into uniform fritter, is put into round-bottomed flask and is mentioned
It takes.
Further, in step 3, extracting method is primarily determined specifically:
By prescription dosage, coptis 1.0g, Fructus Forsythiae 6.0g, dandelion 3.0g, purslane 10.0g, ground are weighed with electronic balance
Fresh loguat leaf is cut into small pieces by bone skin 3.0g, fresh loguat leaf 5.0g, is put into ethyl alcohol, 10 times material of the 500ml round-bottomed flask with 70%
Liquor ratio, extract 2 times, the condition of each 90min extracts.
Further, in step 4, Box-Behnken Design method designs response surface experiments specifically:
On the basis of single factor experiment, using extraction time, concentration of alcohol, solid-liquid ratio as experimental factor, each factor is set
3 levels are set, are indicated with code 1,0, -1, with forsythiaside A, hydrochloric acid coptisine, forsythin, Berberine hydrochloride, hydrochloric acid bar
Ma Ting, hydrochloric acid epiberberine, dry cream rate comprehensive score (Y) be index analyzed.
Another object of the present invention is to provide a kind of response phase method optimization acne washout sides by coptis 1.0g, Fructus Forsythiae
6.0g, purslane 10.0g, dandelion 3.0g, loguat leaf 5.0g and root bark of Chinese wolf-berry 3.0g composition.
In conclusion advantages of the present invention are as follows: the present invention in acne washout side from old docter of TCM's many years practise medicine through
It tests, and obtains significant curative effect, this side compares with other drugs, is not likely to produce antibody-resistant bacterium, no obvious adverse reaction, treatment effect
Fruit is significant.The present invention is under guidance of traditional Chinese medicine theory, basic using Chinese medicine compound prescription scientific meaning as development theories, to meet Chinese medicine
The technique preferred method of nonlinear interaction characteristic has investigated the plurality of active ingredients in side, has been effective as development technique basis
Treatment acne lays the foundation, and also scientific and reasonable, safely and effectively Chinese medicine external acne preparation lays the foundation to develop, and enriches
The diversification of traditional Chinese medicine research is conducive to the development of adolescent psychology physiological health and local economy.The present invention uses response surface
Optimization, it is contemplated that test random error, while response surface optimization method uses complicated unknown functional relation in zonule
Simple primary or quadratic polynomial simulation is the effective means of solving practical problems to be fitted.Prediction model obtained is
Continuously, compared with orthogonal experiment, advantage is: can be continuously to each level of experiment in experiment condition searching process
It is analyzed, and orthogonal experiment can only analyze an isolated experimental point.Acne washout side provided by the invention is most
Good extraction process be be condensed back extract, select concentration for 80% ethyl alcohol, solid-liquid ratio 1:11, extraction twice, every time
100min.The extracting method for acne washout side extraction stablize it is feasible, and for its shaped preparation it is further develop provide
Effective foundation.
Detailed description of the invention
Fig. 1 is response phase method optimization acne washout side extracting method flow chart provided in an embodiment of the present invention.
Fig. 2 is acne washout quadrat sampling product Fructus Forsythiae chromatographic condition chromatogram provided in an embodiment of the present invention.
Fig. 3 is acne washout quadrat sampling product coptis chromatographic condition chromatogram provided in an embodiment of the present invention.
Fig. 4 is forsythiaside A standard items provided in an embodiment of the present invention and forsythin standard chromatogram.
Fig. 5 is epiberberine provided in an embodiment of the present invention, hydrochloric acid coptisine, palmatin hydrochloride, Berberine hydrochloride standard
Product chromatogram.
Fig. 6 is forsythiaside A standard curve and forsythin standard curve schematic diagram provided in an embodiment of the present invention.
Fig. 7 is epiberberine standard curve and hydrochloric acid coptisine standard curve schematic diagram provided in an embodiment of the present invention.
Fig. 8 is palmatine standard curve and Berberine hydrochloride standard curve schematic diagram provided in an embodiment of the present invention.
Fig. 9 is contour map provided in an embodiment of the present invention and three-dismensional effect surface chart.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is further elaborated with reference to the accompanying drawing；
As shown in Figure 1, response phase method provided in an embodiment of the present invention optimizes acne washout side extracting method, specifically include with
Lower step:
S101: choosing the good coptis, Fructus Forsythiae, dandelion, purslane, the root bark of Chinese wolf-berry, and fresh loguat leaf is spare；
S102: using high effective liquid chromatography for measuring forsythiaside A, forsythin, epiberberine, coptisine, palmatine, small
The content of bark of a cork tree alkali is that index carries out comprehensive score according to the content of upper six kinds of ingredients and paste-forming rate；
S103: by experiment of single factor, primarily determining extracting method, extracts medical fluid through being condensed back, filters to obtain the final product；
S104: response surface experiments are designed by Box-Behnken Design method and determine optimum optimization extracting method, are selected
The ethyl alcohol that concentration is 80% does Extraction solvent, and solid-liquid ratio 1:11 is extracted 2 times, 100min minutes each.
In step S101, loguat leaf provided in an embodiment of the present invention be fresh loguat leaf, it is current now to adopt, due to loguat leaf compared with
Big, after weighing, before use, brushing away the villus of loquat leaf back with brush, it is then cut into uniform fritter, is put into round bottom
It is extracted in flask.
In step S103, extracting method is primarily determined specifically:
By prescription dosage, coptis 1.0g, Fructus Forsythiae 6.0g, dandelion 3.0g, purslane 10.0g, ground are weighed with electronic balance
Fresh loguat leaf is cut into small pieces by bone skin 3.0g, fresh loguat leaf 5.0g, is put into ethyl alcohol, 10 times material of the 500ml round-bottomed flask with 70%
Liquor ratio, extract 2 times, the condition of each 90min extracts.
In step S104, Box-Behnken Design method design response surface experiments provided in an embodiment of the present invention are specific
Are as follows:
On the basis of single factor experiment, using extraction time, concentration of alcohol, solid-liquid ratio as experimental factor, each factor is set
3 levels are set, are indicated with code 1,0, -1, with forsythiaside A, hydrochloric acid coptisine, forsythin, Berberine hydrochloride, hydrochloric acid bar
Ma Ting, hydrochloric acid epiberberine, dry cream rate comprehensive score (Y) be index analyzed.
Response phase method optimization provided in an embodiment of the present invention acne washout side is by coptis 1.0g, Fructus Forsythiae 6.0g, purslane
10.0g, dandelion 3.0g, loguat leaf 5.0g and root bark of Chinese wolf-berry 3.0g composition.
Application principle of the invention is further elaborated below with reference to specific herbal medicine efficacy；
Coptis heat-clearing and damp-drying drug, purging fire for removing toxin, main component jamaicin has and its extensive antimicrobial spectrum, to golden yellow
Staphylococcus and propionibacterium acnes have stronger antibacterial action.Berberine to total type influenza virus, gram-positive bacteria,
Gramnegative bacterium, fungi etc. have inhibiting effect, can reach the effect that low concentration is antibacterial and high concentration is sterilized.Coptis conduct
Traditional antibacterial anti-inflammatory Chinese medicine, jamaicin have an inhibiting effect to Acute and chronic inflammation, jamaicin can by the influence to protease,
To play antibacterial action, jamaicin anti-inflammatory mechanism is more conducive to the treatment of acne and all kinds of inflammation.The coptis it is main at
Berberine hydrochloride is divided to have inhibiting effect to propionibacterium acnes.
Fructus Forsythiae is clearing heat and detoxicating, dispersing swelling and dissipating binds.Its main component has forsythiaside A, volatile oil etc..To Active Ingredients of Forsythia Suspensa into
In-depth study is gone, discovery Forsythoside (forsythiaside) has significant antibacterial action, wherein forsythiaside A
The content highest of (forsythiaside A).The internal and external bacteriostatic activity of Forsythoside, obtains Forsythoside in body
It is inside and outside, there is the conclusion of significant inhibitory activity to staphylococcus aureus.Forsythoside has good bacteriostasis, and
And content is higher, is not likely to produce drug resistance, therefore Fructus Forsythiae can be used as a good selection of antibacterials.In the dry fruit of Fructus Forsythiae
Volatile oil rich in is the main component in Fructus Forsythiae steam-volatile oil, has antibacterial, anti-oxidant, anti-inflammatory effect.
In Vitro Bacteriostasis of the nopinene to ten kinds of bacterial strains (streptococcus pneumonia, staphylococcus aureus, Escherichia coli the second grade) in existing analysis
Test, it was therefore concluded that, nopinene has significant bacteriostatic activity to various bacteria.
Purslane clearing heat and promoting diuresis, removing toxicity for detumescence.Purslane is a kind of medicinal material of dual-purpose of drug and food, the chemical component containing there are many,
Such as flavonoids, organic acid, volatile oil and polysaccharide have nutrition, medicinal dual value.There is purslane the antibacterial of wide spectrum to make
With.Purslane ethanol extract or water extract have significant inhibition killing effect to shigella dysenteriae.The antibacterial machine of purslane flavones
Reason is that the cell membrane of destruction thallus makes its content leak outside and reach antibacterial purpose.Purslane has superoxide radical significant clear
Except effect, up to 90% or more.
Dandelion is clearing heat and detoxicating, resolving carbuncle and expulsing boil.Effective component in dandelion is mainly caffeic acid, chlorogenic acid, wherein green
Ortho acid is the principle active component of dandelion antibacterial.Chlorogenic acid extracting solution makees the inhibition of Escherichia coli and gold-coloured staphylococci
With chlorogenic acid is significant to the antibacterial effect living of staphylococcus aureus and Escherichia coli.In addition to this, the polysaccharide in dandelion
It is a kind of widely used ingredient.Song little Yong et al. is measured the antibacterial action of the polysaccharide extracted in dandelion, obtains, Pu
The polysaccharide of public English-Chinese extraction has preferable bacteriostatic activity, and with the raising of polysaccharide concentration, bacteriostatic activity is all the more obvious.
Loguat leaf stopping nausea and vomiting by lowering the adverse flow of QI, clearing lung and relieving cough.In loguat leaf, triterpenoids have good anti-infectious function
Energy.The bacteriostasis alcohol extracting of loquat-leaf extract is mentioned greater than water.To the inhibitory effect of bacterium by by force to weak are as follows: Staphylococcus aureus
Bacterium, Escherichia coli, bacillus subtilis.Ursolic acid and its derivative have antiviral and antitumor and anti-inflammatory antibacterial in loguat leaf
Equal pharmacological activity.
Root bark of Chinese wolf-berry clearing heat and cooling blood moves back hectic fever due to yin labor heat.The decocting liquid and alcohol extract of the root bark of Chinese wolf-berry have preferable antibacterial activity, ground
Bone severe edema due to hypofunction of the spleen decocting liquid has stronger bacteriostasis to dermatophyte.Isolated lignin glycosides compound in the root bark of Chinese wolf-berry, such
Compound has apparent inhibiting effect to human body cause illness's property fungi of methicillin-resistant and staphylococcus aureus.
Application principle of the invention is further elaborated below with reference to specific experiment；
Experiment 1；
1. instrument and reagent
(1) instrument
SHIMADZ Μ LC-2030C 3D high performance liquid chromatograph；SHIMADZΜ Shim-pack GIST C18 5μm
HPLC chromatogram column (250 × 4.6mmI.D)；0.22 μm of 6 aperture of nylon organic system miillpore filter (the limited public affairs of Tianjin navigation equipment
Department)；ZNHW type electric jacket (Yuhua Instrument Co., Ltd., Gongyi City)；JOYN-10A type ultrasonic cleaner (Shanghai Qiao Yue
Electronics Co., Ltd.)；PHS-3E acidometer (Shanghai You Ke instrument and meter Co., Ltd)；SQP type analysis balance (Sai Duolisike
Instrument (Beijing) Co., Ltd is learned, I grades of accuracy, is numbered: 35090245)；JOYN-10A type thermostat water bath (Shanghai Qiao Yue electricity
Sub- Co., Ltd).
(2) reagent
This experiment agents useful for same mainly has standard items, and source see the table below 1.
Other analytical reagents have: Chromatographic Pure Methanol, trifluoroacetic acid aqueous solution, pure 95 ethyl alcohol of analysis, phosphoric acid, potassium dihydrogen phosphate, ten
Sodium dialkyl sulfate etc..
1 reagent source of table
(3) medicinal material
2 crude drug source of table
2. test method
(1) HPLC measures content
1) chromatographic condition
Chromatographic column is 5 μm of HPLC chromatogram columns (250 × 4.6mml) of SHIMADZ Μ Shim-pack GIST C18.
The chromatographic condition of Fructus Forsythiae: -0.2% phosphate aqueous solution (B) of acetonitrile (A), gradient elution, 0~30min, 15%~
30%A；Flow velocity is 1ml/min；Detection wavelength is 277nm；Column temperature is 35 DEG C.
The chromatographic condition of the coptis: acetonitrile -0.05%mol/L potassium dihydrogen phosphate (50:50) (is added ten in every 100ml
Sodium dialkyl sulfate 0.4g, then with phosphorus acid for adjusting pH value for 4.0)；Flow velocity is 1ml/min；Detection wavelength is 345nm；Column temperature is
35℃。
As shown in Fig. 2, acne washout quadrat sampling product Fructus Forsythiae chromatographic condition chromatogram provided in an embodiment of the present invention.
In figure, from left to right chromatographic peak is respectively forsythiaside A, forsythin.
As shown in figure 3, acne washout quadrat sampling product coptis chromatographic condition chromatogram provided in an embodiment of the present invention.
In figure, from left to right chromatographic peak is respectively epiberberine, hydrochloric acid coptisine, palmatin hydrochloride, Berberine hydrochloride.
2) preparation of reference substance solution
The process for preparing forsythiaside A standard items is as follows, weighs forsythiaside A with the assay balance precision of ten a ten thousandths
Standard items 8.13mg, is added 25ml volumetric flask, and constant volume is configured to the forsythiaside A standard solution of 0.3252mg/ml.
The standard solution preparation process of forsythin is as follows, critically weighs forsythin standard items with same method
1.16mg is added 25ml volumetric flask with dropper constant volume and is configured to the forsythin standard solution of 0.0464mg/ml.
Preparing for hydrochloric acid epiberberine standard solution is as follows, and epiberberine standard items precision weighs 0.88mg, is added
25ml volumetric flask, constant volume obtain the forsythin standard solution that concentration is 0.0352mg/ml.
The process for preparing hydrochloric acid coptisine standard solution is as follows, critically weighs coptisine standard items 0.82mg, is added
25ml volumetric flask is configured to the forsythin standard solution of 0.0328mg/ml by constant volume.
The preparation process of hydrochloric acid horse spit of fland standard solution is as follows, and precision weighs palmatine standard items 0.62mg, and 25ml is added
Volumetric flask, constant volume are configured to the forsythin standard solution of 0.0248mg/ml.
The process of Berberine hydrochloride standard items preparation is as follows, and precision weighs Berberine hydrochloride standard items 0.96mg, is added
25ml volumetric flask, constant volume are configured to the standard solution that concentration is 0.0384mg/ml Berberine hydrochloride.
As shown in figure 4, forsythiaside A standard items provided in an embodiment of the present invention and forsythin standard chromatogram.
As shown in figure 5, epiberberine provided in an embodiment of the present invention, hydrochloric acid coptisine, palmatin hydrochloride, Berberine hydrochloride
Standard chromatogram.
3) preparation of test solution
By prescription dosage, coptis 1.0g, Fructus Forsythiae 6.0g, dandelion 3.0g, purslane 10.0g, ground are weighed with electronic balance
Fresh loguat leaf is cut into small pieces by bone skin 3.0g, fresh loguat leaf 5.0g, is put into ethyl alcohol, 10 times material of the 500ml round-bottomed flask with 70%
Liquor ratio, the condition for extracting 2 times, each 90min are extracted medical fluid through being condensed back, are filtered to obtain the final product.
4) linear relationship is investigated
Precision draws 2,6,10,14,18,22 μ l of forsythiaside A reference substance solution, is surveyed according to above-mentioned chromatographic condition
It is fixed, peak area is recorded, with reference substance sample volume X (μ l) for abscissa, integrating peak areas value (Y) is ordinate, and it is bent to draw standard
Line, the equation of linear regression of forsythiaside A are y=759,748.13302x+113,354.05238, R2=0.9997, Fructus Forsythiae ester
Glycosides A linear relationship in 0.6504 μ of μ g~7.1544 g of sample volume is good.
Precision draws 2,4,8,12,16,20 μ l of forsythin reference substance solution, is measured according to above-mentioned chromatographic condition, remembers
Peak area is recorded, with reference substance sample volume X (μ l) for abscissa, integrating peak areas value (Y) is ordinate, draws standard curve, even
The equation of linear regression for sticking up glycosides is y=863,938.53330x+1,278.27123, R2=0.99995, forsythin is in sample volume
Linear relationship is good in 0.0928 μ of μ g~0.9280 g.
Precision draws 2,6,10,14,18,22 μ l of hydrochloric acid epiberberine reference substance solution, carries out according to above-mentioned chromatographic condition
Measurement records peak area, and with reference substance sample volume X (μ l) for abscissa, integrating peak areas value (Y) is ordinate, draws standard
Curve, the equation of linear regression of hydrochloric acid epiberberine are y=2753299x-21619, R2=0.9992, hydrochloric acid epiberberine exists
Linear relationship is good in 0.0704 μ of μ g~0.7744 g of sample volume.
Precision draws 2,6,10,14,18,22 μ l of hydrochloric acid coptisine reference substance solution, is surveyed according to above-mentioned chromatographic condition
It is fixed, peak area is recorded, with reference substance sample volume X (μ l) for abscissa, integrating peak areas value (Y) is ordinate, and it is bent to draw standard
Line, the equation of linear regression of hydrochloric acid coptisine are y=2303888x+4367.2, R2=0.9998, hydrochloric acid coptisine is in sample volume
Linear relationship is good in 0.0464 μ of μ g~0.5104 g.
Precision draws 2,6,10,14,18,22 μ l of palmatin hydrochloride reference substance solution, is surveyed according to above-mentioned chromatographic condition
It is fixed, peak area is recorded, with reference substance sample volume X (μ l) for abscissa, integrating peak areas value (Y) is ordinate, and it is bent to draw standard
Line, the equation of linear regression of palmatin hydrochloride are y=2959073x+2752.1, R2=0.99998, palmatin hydrochloride is in sample introduction
It is good to measure linear relationship in 0.0496 μ of μ g~0.5456 g.
Precision draws 2,6,10,14,18,22 μ l of Berberine hydrochloride reference substance solution, and above-mentioned chromatographic condition is measured, and remembers
Peak area is recorded, with reference substance sample volume X (μ l) for abscissa, integrating peak areas value (Y) is ordinate, draws standard curve, salt
The equation of linear regression of sour jamaicin be y=5,853,362x+4,757, R2=0.99997, Berberine hydrochloride is in sample volume
Linear relationship is good in 0.0768 μ of μ g~0.8448 g.
As shown in fig. 6, forsythiaside A standard curve provided in an embodiment of the present invention and forsythin standard curve schematic diagram.
As shown in fig. 7, epiberberine standard curve provided in an embodiment of the present invention and the signal of hydrochloric acid coptisine standard curve
Figure.
As shown in figure 8, palmatine standard curve provided in an embodiment of the present invention and Berberine hydrochloride standard curve schematic diagram.
5) study on the stability
Take same acne washout side test solution, respectively at 0h, 2h, 4h, 8h, 10h, for 24 hours when, respectively 10 μ l of sample introduction,
Thus come measure forsythiaside A, hydrochloric acid coptisine, forsythin, hydrochloric acid epiberberine, Berberine hydrochloride, palmatin hydrochloride six
The peak area of a effective component, and calculate the RSD of its peak area.
As a result forsythiaside A, hydrochloric acid coptisine, forsythin, hydrochloric acid epiberberine, Berberine hydrochloride, palmatin hydrochloride peak
The RSD of area, which is computed, to be obtained, respectively 1.76%, 1.94%, 2.12%, 2.04%, 2.04%, 2.09%, this result
It is good in internal stability for 24 hours to show test sample.
6) precision is investigated
The 10 μ l of reference substance solution configured before taking, selected chromatographic condition carry out carrying out assay to it, with
Product continuous sample introduction 6 times, the peak area of six effective component is recorded respectively.Forsythiaside A, hydrochloric acid coptisine, forsythin, hydrochloric acid
Epiberberine, Berberine hydrochloride, palmatin hydrochloride peak area RSD, be computed and obtain, respectively 0.62%, 0.99%,
0.82%, 0.33%, 0.27%, 0.29%, it should be statistics indicate that the precision of institute's measuring method be good.
7) repeatability is investigated
Test solution is prepared according to the above method, 6 parts altogether, measures its forsythiaside A, hydrochloric acid coptisine, forsythin, salt
Sour epiberberine, Berberine hydrochloride, palmatin hydrochloride peak area, and calculate its content, show that their RSD is respectively
0.94%, 2.31%, 1.14%, 0.41%, 1.37%, 1.68%, the results show that method repeatability is good.
(2) single factor experiment
The present invention carries out methodological study by single factor experiment, primarily determines extraction process.
1) Extraction solvent is investigated
Prescription dosage is pressed by prescription, weighs coptis 1.0g, Fructus Forsythiae 6.0g, dandelion 3.0g, purslane with electronic balance
Fresh loguat leaf is cut into small pieces, is put into 500ml round-bottomed flask by 10.0g, root bark of Chinese wolf-berry 3.0g, fresh loguat leaf 5.0g, respectively with water and
70% ethyl alcohol as solvent extraction medical fluid, by high performance liquid chromatography survey forsythiaside A, forsythin, hydrochloric acid epiberberine,
The content of hydrochloric acid coptisine, palmatin hydrochloride, Berberine hydrochloride, while its dry cream rate is measured, after comprehensive score, discovery is extracted
1.5h effect is more excellent.
2) extraction time is investigated
Prescription dosage is pressed by prescription, weighs coptis 1.0g, Fructus Forsythiae 6.0g, dandelion 3.0g, purslane with electronic balance
Fresh loguat leaf is cut into small pieces, is put into 500ml round-bottomed flask, extracts respectively by 10.0g, root bark of Chinese wolf-berry 3.0g, fresh loguat leaf 5.0g
1h,1.5h,2h.Forsythiaside A, forsythin, hydrochloric acid epiberberine, hydrochloric acid coptisine, hydrochloric acid are surveyed by high performance liquid chromatography
The content of palmatine, Berberine hydrochloride, while its dry cream rate is measured, after comprehensive score, it is found that 70% ethyl alcohol proposes effect than pure water
Fruit.
3) solid-liquid ratio is investigated
Prescription dosage is pressed by prescription, weighs coptis 1.0g, Fructus Forsythiae 6.0g, dandelion 3.0g, purslane with electronic balance
Fresh loguat leaf is cut into small pieces, is put into 500ml round-bottomed flask, uses medicinal material respectively by 10.0g, root bark of Chinese wolf-berry 3.0g, fresh loguat leaf 5.0g
8 times, 10 times, 12 times of amount.Forsythiaside A in each extracting solution, forsythin, hydrochloric acid table barberry are surveyed by high performance liquid chromatography
The content of alkali, hydrochloric acid coptisine, palmatin hydrochloride, Berberine hydrochloride, while measuring its dry cream rate, after comprehensive score, discovery with
10 times of medicinal material amount of solvent extraction effect is preferable.
4) extraction time is investigated
Prescription dosage is pressed by prescription, weighs coptis 1.0g, Fructus Forsythiae 6.0g, dandelion 3.0g, purslane with electronic balance
Fresh loguat leaf is cut into small pieces, is put into 500ml round-bottomed flask by 10.0g, root bark of Chinese wolf-berry 3.0g, fresh loguat leaf 5.0g, extract 1 respectively,
2,3 times.By high performance liquid chromatography survey forsythiaside A in each extracting solution, forsythin, hydrochloric acid epiberberine, hydrochloric acid coptisine,
The content of palmatin hydrochloride, Berberine hydrochloride, while its dry cream rate is measured, after comprehensive score, it is preferable that 2 effects are extracted in discovery.
(3) response phase method Optimized Extraction Process
1) experimental design
On the basis of single factor experiment, determine that the investigation factor of acne washout side extraction process is extraction time, ethyl alcohol
Concentration, solid-liquid ratio, according to the principle that Box-Behnken Design is designed, 3 levels are arranged in each factor, with code 1,0, -1
It indicates, with forsythiaside A, hydrochloric acid coptisine, forsythin, Berberine hydrochloride, palmatin hydrochloride, hydrochloric acid epiberberine, dry cream
The comprehensive score (Y) of rate is that index is investigated, and practical operation physics amount represented by code is shown in Table, and experiment arranges and result is shown in
Table 3, table 4.
+ 10% (palmatin hydrochloride extracted amount/maximum of comprehensive score=20% (forsythiaside A extracted amount/maximum extracted amount)
Extracted amount)+20% (forsythin extracted amount/maximum extracted amount)+10% (hydrochloric acid epiberberine extracted amount/maximum extracted amount)+
20% (Berberine hydrochloride extracted amount/maximum extracted amount)+10% (hydrochloric acid coptisine extracted amount/maximum paste-forming rate)+10% (dry cream
Rate/maximum paste-forming rate).
3 response surface design factor of table and level
4 response surface contrived experiment of table and result
2) simulation fitting
Polynary quadratic regression is carried out to the data in table 2 using expert data analysis software Design Expert 8.0.6
Fitting, obtain concentration of alcohol A, solid-liquid ratio B, extraction time C and forsythiaside A, forsythin, Berberine hydrochloride, dry cream rate it is comprehensive
Close the secondary multinomial regression equation between scoring, regression equation are as follows:
Comprehensive score (Y)=+ 0.81-0.030A-0.035B-8.264E-003C+0.025AB+0.061AC+0.060BC+
0.041A2-0.013B2-0.076C2 (R2=0.8544, P < 0.05, F=4.57；Lose=0.0496 < 0.05, F=of quasi- degree
6.86).By the fitting result it is found that model F value is 4.57, larger, P < 0.05 shows that model has conspicuousness.Lose quasi- item F
It is worth smaller, P=0.0569 > 0.05, loses quasi- item and do not have conspicuousness, i.e., the model is good in regression region fitting.Precision letter
It makes an uproar than measuring, signal-to-noise ratio=8.658 are greater than 4, carry out data statistic analysis to experimental result with the model, it is found that it meets reality
Requirement is tested, that is, the model can be used to be analyzed and predicted the extraction process of acne washout side.
3) prediction and optimization of technological parameter
According to the fitting of quadratic polynomial regression equation as a result, fixed one of independent variable factor level, draws synthesis
The contour map and three-dismensional effect surface chart of scoring (Y) and other two independents variable, predict acne washout side Extraction technique.
As shown in figure 9, contour map provided in an embodiment of the present invention and three-dismensional effect surface chart.
It is obtained by upper figure, comprehensive score (Y) is influenced not to be simple linear between significant each factor and response
Relationship, AC, BC, C2 influence comprehensive score significant.Influence B > A > C of the extraction process influence factor to comprehensive score.
It keeps one of independent variable constant, analyzes the quantity relative ratio relationship of other two variable.
Fixed concentration of alcohol, the interaction figure for two factor of solid-liquid ratio and extraction time drawn by Data Analysis Software can be seen
It arrives, response is a curved surface.Between at the extraction it is shorter in the case of, comprehensive score is reduced with the raising of solid-liquid ratio；It is mentioning
In the case where taking the time longer, comprehensive score is increased with the raising of solid-liquid ratio.In the biggish situation of solid-liquid ratio, with mentioning
It takes the increase of time and is reduced after increasing；In the lesser situation of solid-liquid ratio, comprehensive score declines with increasing for extraction time.
The interaction figure of fixed extraction time, two factor of solid-liquid ratio and extraction time drawn by Data Analysis Software can be seen
It arrives, response is a curved surface.Comprehensive score first reduces and increases afterwards with the increase of concentration of alcohol.It is comprehensive when concentration of alcohol is lower
Scoring increases with the reduction of solid-liquid ratio；Under the higher situation of concentration of alcohol, the increase of solid-liquid ratio influences not comprehensive score
Greatly.
Fixed solid-liquid ratio is constant, and the interaction figure of two factor of solid-liquid ratio and extraction time drawn by Data Analysis Software can be with
See, response is a curved surface.Comprehensive score first first increases with the increase of extraction time to be reduced afterwards.Shorter feelings between at the extraction
Under shape, comprehensive score is reduced with the increase of concentration of alcohol；With the extension of extraction time, comprehensive score with concentration of alcohol increasing
Add first to reduce and increase afterwards, with the extension of extraction time, influence of the variation of concentration of alcohol to comprehensive score is gradually reduced.
In conclusion all factors are integrated, and consider practical operability, the extraction scheme of response surface design optimization is
Concentration of alcohol 80%, solid-liquid ratio are 10 times, extraction time 100min.
(4) verification test
With said extracted scheme: the ethyl alcohol of 80% concentration, 10 times of solid-liquid ratios, extraction 2 times, each 100min are extracted
Verifying, discovery comprehensive score result is good, determines that it is optimum extraction process.
3. analysis
(1) initial stage inquires data
At experiment initial stage to this Six-element medicine of the coptis, Fructus Forsythiae, the root bark of Chinese wolf-berry, dandelion, purslane, loguat leaf in acne washout side
The effect of and the clinical application in terms for the treatment of acne done preliminary understanding.It was found that this Six-element medicine treatment acne be mostly due to
Its antibacterial action, in particular, the party belongs to exterior-applied prescription, the power of antibacterial action determines its curative effect to a certain extent.Pass through
To the access of document, find the Berberine hydrochloride in the coptis, the forsythiaside A, forsythin in Fructus Forsythiae be antibacterial action mainly at
Point, antibacterial effect is the most significant, and representative.General flavone antibacterial action is also more significant in purslane, but is not single
Ingredient it is improper to survey its content with high-efficient liquid phase technique, if with ultraviolet method, due to limited time, it is contemplated that actual conditions and benefit
Just it not measures.The ursolic acid in chlorogenic acid, loguat leaf in dandelion has antibacterial effect, but its ingredient is in a variety of medicinal materials
All exist, does not have representativeness.Thus the present invention is substantially determined in acne washout side, with Berberine hydrochloride, forsythiaside A,
The extraction process that the content of forsythin is index to optimize acne washout side.
Then, pharmacopeia is browsed, learns coptis pharmacopeia to three epiberberine, coptisine, palmatine ingredients when consulting the coptis
The survey assays commented have been done more.By structure to this three ingredients and effect inquiry, discovery epiberberine, coptisine,
This four constituent structure of palmatine, jamaicin are similar, function is close, all have good bacteriostasis, are just included in optimization together
In the testing index of acne washout side extraction process.Meanwhile dry cream rate is to judge in medicinal material Contents of Main Components is how many and medicinal material
One important indicator of quality, therefore using this as an index for measuring extraction process.
To sum up, present invention determine that the index of optimization acne washout side extraction process: the content of forsythiaside A, forsythin
Content, the content of hydrochloric acid epiberberine, the content of hydrochloric acid coptisine, the content of palmatin hydrochloride, the content of Berberine hydrochloride,
Dry cream rate (presence mostly in the form of hydrochloride of epiberberine, coptisine, palmatine, jamaicin).
(2) high effective liquid chromatography for measuring content
It is easy to operate when for later period optimization technique since the ingredient for needing to measure is more, in the debugging of mobile phase
The present invention has descended many time.
It is the proportion of the mobile phase of the high performance liquid chromatography referring to the pharmacopeia coptis, Fructus Forsythiae tie element first.Through testing
It was found that assay can not be carried out under same proportion of mobile phase, while to this 6 ingredients.Then pass through centering National IP Network
On the document that is recorded consulted, it is intended to find can measure simultaneously the coptis, in Fructus Forsythiae effective component proportion of mobile phase, to text
Offer institute's support method to be tested, choose more feasible scheme to water phase, the ratio of organic phase, pH value, the model of chromatographic column into
Preferably flowing phase method is selected in row adjustment.Through a long time test, epiberberine in the coptis, coptisine chromatographic peak always without
Method is kept completely separate, and separating degree is unable to reach requirement, therefore the present invention has finally selected a preferably chromatographic condition, can measure simultaneously
Forsythiaside A, forsythin in Fructus Forsythiae.And in the coptis four effective components due to the chromatographic condition with coptis pharmacopeia it is available very
Good isolated chromatographic peak, therefore select to survey its content with the method for coptis pharmacopeia efficient liquid phase measurement content.
To sum up, present invention determine that in the coptis, Fructus Forsythiae effective component efficient liquid phase measurement content chromatographic condition.
(3) extraction of sample
The coptis, Fructus Forsythiae, dandelion, purslane, the root bark of Chinese wolf-berry medicinal material be to be processed because medicinal material volume itself compared with
Small, mostly coarse granule shape can be put into round-bottomed flask.Loguat leaf is fresh loguat leaf, current now to adopt, since loguat leaf is larger
, after weighing, before use, brushing away the villus of loquat leaf back with brush, it is then cut into uniform fritter, is put into round bottom burning
It is extracted in bottle.
The extracting solution of acne washout side, i.e. sample are untreated.Chromatographic condition is tested in efficient liquid phase early period
When, once root it is documented that, sample solvent is volatilized, with 75% methanol be redissolved, peak shape, resolution etc. with it is untreated
Sample there is no notable difference.Sample is also without concentration, this is because in preliminary experiment early period, the sample without concentration
Appoint to study carefully and can run out of preferable peak in efficient liquid phase assay.
(4) optimization of extraction process
First the present invention by single factor experiment to the concentration of extraction time of acne washout side, Extraction solvent ethyl alcohol, mention
The ratio of the number, extraction liquor capacity and the medicinal material that take has carried out having carried out preliminary screening to the extraction process of acne washout side
Optimization, obtained with the ethyl alcohol of 70% concentration, extract 2 times, each 90min, solid-to-liquid ratio be 10:1 extraction process, basis herein
On, I to extraction scope carried out it is certain reduce the scope, and devise sound with the range with Box-Behnken Design method
It should interview and test, further optimization has been carried out to extracting method, more accurately extraction scheme is obtained with this.
4. result
The optimum extraction process of acne washout side be be condensed back extract, select concentration for 80% ethyl alcohol, solid-liquid ratio
For 1:11, extract twice, each 100min.The extraction scheme is stablized feasible for the extraction of acne washout side, and forms for it
The further development of preparation provides experimental basis.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (6)
1. a kind of response phase method optimizes acne washout side extracting method, which is characterized in that outside the response phase method optimization acne
The side's of washing extracting method is extracted 2 times with 10 times 70% of ethyl alcohol, each 1.5h；Concentration is selected to do Extraction solvent for 80% ethyl alcohol,
Solid-liquid ratio is 1:11, is extracted 2 times, 100min minutes each.
2. response phase method as described in claim 1 optimizes acne washout side extracting method, which is characterized in that the response surface
Method optimize acne washout side extracting method the following steps are included:
Step 1: choosing the good coptis, Fructus Forsythiae, dandelion, purslane, the root bark of Chinese wolf-berry, and fresh loguat leaf is spare；
Step 2: high effective liquid chromatography for measuring forsythiaside A, forsythin, epiberberine, coptisine, palmatine, barberry are used
The content of alkali is that index carries out comprehensive score according to the content of upper six kinds of ingredients and paste-forming rate；
Step 3: by experiment of single factor, primarily determining extracting method, extracts medical fluid through being condensed back, filters to obtain the final product；
Step 4: response surface experiments are designed by Box-Behnken Design method and determine optimum optimization extracting method, are selected dense
Degree does Extraction solvent for 80% ethyl alcohol, and solid-liquid ratio 1:11 is extracted 2 times, 100min minutes each.
3. response phase method as claimed in claim 2 optimizes acne washout side extracting method, which is characterized in that the step 1
In, loguat leaf is fresh loguat leaf, it is current now to adopt, due to loguat leaf larger, after weighing, before use, brushing away loquat with brush
Then the villus of leaf back is cut into uniform fritter, is put into round-bottomed flask and extracts.
4. response phase method as claimed in claim 2 optimizes acne washout side extracting method, which is characterized in that the step 3
In, primarily determine extracting method specifically:
By prescription dosage, coptis 1.0g, Fructus Forsythiae 6.0g, dandelion 3.0g, purslane 10.0g, the root bark of Chinese wolf-berry are weighed with electronic balance
3.0g, fresh loguat leaf 5.0g, fresh loguat leaf is cut into small pieces, and is put into ethyl alcohol of the 500ml round-bottomed flask with 70%, 10 times of feed liquids
Than, extract 2 times, the condition of each 90min extracts.
5. response phase method as claimed in claim 2 optimizes acne washout side extracting method, which is characterized in that the step 4
In, Box-Behnken Design method designs response surface experiments specifically:
On the basis of single factor experiment, using extraction time, concentration of alcohol, solid-liquid ratio as experimental factor, each factor is arranged 3
Level indicates with code 1,0, -1, with forsythiaside A, hydrochloric acid coptisine, forsythin, Berberine hydrochloride, palmatin hydrochloride,
Hydrochloric acid epiberberine, dry cream rate comprehensive score (Y) be index analyzed.
6. a kind of response that the optimization of the response phase method as described in Claims 1 to 5 any one acne washout side extracting method obtains
Face method optimizes acne washout side, which is characterized in that the response phase method optimization acne washout side is by coptis 1.0g, Fructus Forsythiae
6.0g, purslane 10.0g, dandelion 3.0g, loguat leaf 5.0g and root bark of Chinese wolf-berry 3.0g composition.
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| CN101259172A (en) * | 2007-03-06 | 2008-09-10 | 上海家化联合股份有限公司 | Chinese medicinal composition for treating acne, preparation and application thereof |
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| CN101259172A (en) * | 2007-03-06 | 2008-09-10 | 上海家化联合股份有限公司 | Chinese medicinal composition for treating acne, preparation and application thereof |
| CN104491522A (en) * | 2014-12-06 | 2015-04-08 | 周连才 | Pharmaceutical composition for treating seborrheic dermatitis |
| CN104490728A (en) * | 2014-12-08 | 2015-04-08 | 罗昕 | Acne-removing facial cleanser |
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Application publication date: 20190903 |