CN104473956A - Yupingfeng powder active ingredient composition and preparation method and application thereof - Google Patents
Yupingfeng powder active ingredient composition and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of preparation of traditional Chinese medicines. The invention specifically relates to a Yupingfeng powder active ingredient composition and a preparation method and application thereof. The Yupingfeng powder active ingredient composition is produced by combining astragaloside, atractylenolide I and prim-o-glucosylcimifugin according to different proportions. According to the Yupingfeng powder active ingredient composition provided by the invention, traditional Chinese medicines are combined with the modern science and technology, the western medicine standard is used for research and development of the traditional Chinese medicine, the medicinal effects and the chemical components are presented in a standardized manner, and the modern traditional Chinese medicine with safety, stability and controllable quality is further researched and developed. The composition has three major factors, namely safety, effectiveness and controllable quality of the medicine. The composition is favorable for solving the problem of enabling the traditional Chinese medicine to be in line with the development trend of international medicines, and is favorable for realizing modernization and internationalization of the traditional Chinese medicine. The composition inherits and develops the traditional Chinese medicine, combines with the modern medicine technology, and is a key for enabling the traditional Chinese medicine to enter the international market.
Description
One, technical field
The invention belongs to Chinese drug preparation technique field.Be specifically related to a kind of YUPINGFENG SAN active ingredient composition and its preparation method and application.
Two, background technology
Normal immunne response is that body maintains Equilibrium, resists the main mechanism of foreign aggression, and superfluous immunne response then can cause various autoimmune disease, allergy and various inflammatory reactions etc.To suppress the existing immunosuppressant for the purpose of superfluous immunne response, because its effect lacks selectivity to biological cells and tissues, often cause comprehensive suppression of immunne response, thus produce serious toxic and side effects, as glucocorticoid, cyclophosphamide etc.Ciclosporin A finds in the metabolite of ring spore mycete, complex is formed by combining with ring Avidin, and then suppress the function of Calcineurin, further inflammation-inhibiting transcribing in early days by gene (as the IL-2) that activate, thus play immunosuppressive action.But ciclosporin A lacks selectivity, also has inhibitory action to the body normal immunological function that T cell is undertaken.And the patient of life-time service ciclosporin A, often can be observed medicine to kidney and neural toxicity, and the risk that infectious disease and tumor occur increases.In addition, molecular targeted para-immunity inhibitor be development in recent years get up based on the neotype immunosuppressant of antibody class preparation, but its suppression for immunne response is still nonselective often, can cause serious toxic and side effects.
Searching optionally can suppress immunocyte or the molecule of in disease development process, playing the part of pathologic role, and does not affect or less affect the molecule of other normal immunocytes, is realize selectivity immunosuppressant important step.From a series of heat clearing Chinese medicine, found Chinese medicine and the composition thereof that optionally can act on immunne response different phase, these Chinese medicines and composition are to the different phases selectively inhibitory action of delayed hypersensitivity (DTH).
YUPINGFENG SAN comes from " danxi's experiential therapy " of Yuan Dynasty's ZHU Dan-xi, and its effect is benefiting QI for strengthening the superficies hidroschesis, cures mainly exterior deficiency, aversion to wind spontaneous perspiration, the empty susceptible ailment said due to cold or exposure person of body.Medicine consists of the Radix Astragali, Radix Saposhnikoviae each one or two, the Rhizoma Atractylodis Macrocephalae two liang; Pill taken with boiled water can be ground into powder, or drink to be decocted in water for oral dose by same ratio.Experimentation shows, YUPINGFENG SAN has mediator's body immunity function; In recent years, be widely used in clinical, to the empty hyperhidrosis of children's's body, prevent the diseases such as weak repeated respiratory tract infections in children, urticaria, rheumatic arthritis, allergic dermatitis, nephritis, Meniere disease all to have good efficacy.
Chinese medicine is the principle composition prescription according to seven emotions and conjunction, monarch.The method of modern study Chinese medicinal formulae composition of prescription, the composition of prescription that mainly Study of Traditional Chinese Medicine effectively becomes (group) to divide on component level, the quantitative analysis of compatibility of drugs prescription can be realized, contribute to the action target spot that clear and definite Chinese medicine effectively becomes (group) to divide, inquire into the possible mechanism of action of Chinese medicinal formulae, for modernization of cmm lays the foundation, for Chinese medicine Opening International Market provides foundation.
At present, on overseas Market of Chinese Materia Medica, China gathers around patented Chinese medicine and is only 0.3%; Whole world Market of Chinese Materia Medica sales volume 1 year about 80,000,000,000 dollars, China only accounts for 10%.HANYAO enterprise of Japan borrows group Chinese medicine to capture 80% of international Chinese patent medicine market, earning juice.The standard of Western medicine is used for the research and development to Chinese medicine, drug effect and chemical composition is given standardization and presents, this make world community more easily accept Japan produce Chinese patent medicine, i.e. " HANYAO ", this be also Japan " HANYAO " go to the world basic.
Although there is abundant natural resources of Chinese medicinal materials advantage in China, the extremely complicated competitiveness limited in the world of its compound recipe composition, change the status quo, must walk Road to Modernization.And illustrate the chemical compositions of compound compatibility and multi-target effect mechanism, be solve Chinese medicine and the medical development trend in the world to integrate with the only way which must be passed of problem.Must use traditional Chinese medicine theory and research and develop modern Chinese medicine in conjunction with modern science and technology, being the only way of the current Development of Traditional Chinese Medicine of China, is also the effective way of new drug research.Therefore, inherit and development Chinese medicine, and combine with modern medicine technology, be the key point that Chinese medicine steps into international market.
Three, summary of the invention
The present invention need solve problem be: a kind of YUPINGFENG SAN active ingredient composition and its preparation method and application is provided.Realize the modernization of Chinese medicine.
Technical scheme of the present invention is summarized as follows:
1. the composition of YUPINGFENG SAN active ingredient composition
YUPINGFENG SAN active ingredient composition of the present invention is by astragaloside (i.e. Radix Astragali saponin IV, Astragaloside IV, C
41h
68o
14), atractylenolide Ⅰ (Atractylenolide I, C
15h
18o
2), cimicifugoside (Prim-o-glucosylcimifugin, C
22h
28o
11), three combines according to different ratios, produces YUPINGFENG SAN active ingredient composition.
The principle active component of the Radix Astragali is astragaloside (i.e. Radix Astragali saponin IV, Astragaloside IV, C
41h
68o
14), there is antiinflammatory blood pressure lowering, antalgic and sedative, improve myocardial contraction and the effect such as diastole, scavenging free radicals; And have inhibitory action to delayed hypersensitivity (DTH), may be relevant with suppression IFN-γ.The principle active component of the Rhizoma Atractylodis Macrocephalae is atractylenolide Ⅰ (Atractylenolide I, C
15h
18o
2), there is the function of antiinflammatory, antitumor, regulating intestinal canal function and promotion absorption of nutrient ingredients.The principle active component of Radix Saposhnikoviae is cimicifugoside (Prim-o-glucosylcimifugin, C
22h
28o
11), have significantly antipyretic, analgesia, antiinflammatory effect.
2. the preparation method of YUPINGFENG SAN active ingredient composition of the present invention:
Take astragaloside (3mg-21mg), atractylenolide Ⅰ (2mg-14mg) and cimicifugoside (6mg-21mg) respectively, add Tween 80 (Tween-80) and the 24.5ml distilled water of 0.5ml 2%, be mixed with the mixed solution of astragaloside, atractylenolide Ⅰ and cimicifugoside, in mixed solution, the final concentration of each active component is respectively astragaloside 0.12-1.05mg/ml, atractylenolide Ⅰ 0.08-0.7mg/ml, cimicifugoside 0.24-1.05mg/ml.
In composition I mixed solution, the final concentration of each active component is respectively 0.12mg/ml astragaloside, 0.08mg/ml atractylenolide Ⅰ and 0.24mg/ml cimicifugoside.
In composition I I mixed solution, the final concentration of each active component is respectively 1.05mg/ml astragaloside, 0.7mg/ml atractylenolide Ⅰ and 1.05mg/ml cimicifugoside.
3. pharmacological evaluation
Use dinitrofluorobenzene (DNFB) to carry out exogenous stimulation, and gavage normal saline, after 5 days, use low concentration DNFB to excite, build the contact dermatitis mouse model of delayed hypersensitivity continuously.
On this basis, respectively to the experiment mice of model group, dexamethasone positive drug group, YUPINGFENG SAN (YPF), YPF active ingredient composition administration group, after excite 6 ~ 8 hours, respectively the ear thickness of experiment mice and body weight change are measured; Get blood in exciting latter 12 hours and plucking eyeball to mice, get centrifugal after the upper serum that obtains carry out ELISA detection, detect IL-4 and IFN-γ content respectively and carry out statistical analysis.
After sacrifice, get its spleen and weigh, and calculate index and spleen index.
Meanwhile, cell pyrolysis liquid and protease inhibitor are added to mouse spleen tissue, use Syrup-homogenizing instrument to carry out homogenate; Centrifugal, get supernatant, measure histone stock protein concentration by BCA method; Then, westernblotting method is adopted to detect IL-6 expression.
4. experimental result
Result of study shows, after the administration of YUPINGFENG SAN active ingredient composition, Mouse Weight obviously declines (P<0.05); Compare with YUPINGFENG SAN with dexamethasone, the difference that the Mouse Weight of composition I declines is little; But compare with model group mice, the fall of composition I obviously reduces (table 1).
YUPINGFENG SAN active ingredient composition significantly can reduce and excites rear mice ear degree (P<0.01), and the inhibitory action of composition I and composition I I is all higher than dexamethasone and YUPINGFENG SAN (table 1).
YUPINGFENG SAN active ingredient composition significantly can reduce index and spleen index (p<0.01), has certain immunosuppressive action (table 1).
YUPINGFENG SAN active ingredient composition significantly can both raise the content (p<0.001) of IL-4 in mice serum, and the activity of composition I and composition I I and dexamethasone are quite (table 1).
YUPINGFENG SAN active ingredient composition significantly can reduce the content (p<0.05) of IFN-γ in mice serum, the activity more closely Sai meter Song (table 1) of composition I.
From the ratio of IFN-γ/IL-4, the ratio of model group mice is significantly higher than normal mouse, and after administration, this ratio significantly declines again.YUPINGFENG SAN active ingredient composition significantly can both reduce IFN-γ/IL-4 ratio, wherein the ratio (table 1) of the ratio of composition I more closely Sai meter Song.This shows, they have the function regulating Th1/Th2 unbalance.
Table 1, YUPINGFENG SAN compound recipe and effective ingredient complex thereof are on the impact of IL-6 expression in IFN-γ in DTH Mouse Weight, ear swelling degree, index and spleen index, serum and IL-4 level and spleen
Note: Mouse Weight before Mouse Weight-sensitization after body weight change value=sensitization; Ear swelling degree is the thickness difference of left and right ear; Index and spleen index=spleen weight/Mouse Weight × 100; The gray value of mouse spleen IL-6 represents the destination protein relative amount of certain sample divided by the gray value of internal reference mGAPDH.*P<0.05,**P<0.01，***P<0.001
Western Blotting testing result shows, compared with Normal group, model group mouse spleen IL-6 expression significantly raises (p<0.001).Compared with model group, YUPINGFENG SAN active ingredient composition I significantly can reduce IL-6 expression (p<0.05), and, active and dexamethasone suitable (table 1).
Feature of the present invention is: the Chinese medicine compound based on Clinical practice is transformed, and its effective ingredient is carried out reasonable combination, produces brand-new composition of medicine, i.e. YUPINGFENG SAN active ingredient composition.
Said composition had both remained the effective ingredient of YUPINGFENG SAN, specify that again the composition of medicine, guaranteed the controllability of quality.Because YUPINGFENG SAN is Clinical practice medicine, therefore, said composition has again reliable safety.Experiment shows, activity and the YUPINGFENG SAN of said composition are close, and thus, said composition has the effectiveness similar with YUPINGFENG SAN.Said composition has possessed the feature that original new drug has, treatment respiratory tract infection, bronchial asthma, allergic rhinitis, chronic urticaria, skin pruritus, allergic dermatitis, rheumatic arthritis, nephritis and immunoregulatory medicine can be prepared into, also can apply in preparation treatment malignant tumor, chronic hepatitis, chronic bronchitis ancillary drug.
The invention has the beneficial effects as follows: Chinese medicine combines with modern science and technology by the present invention, the standard of Western medicine is used for the research and development of Chinese medicine, drug effect and chemical composition are given standardization present, and research and develop safety, stable, quality controllable modern Chinese medicine.Namely said composition has possessed the large key element of the safety of medicine, effectiveness and quality controllability three.This is conducive to solving the medical development trend in Chinese medicine and the world and integrates with problem, contributes to the modernization and the internationalization that realize Chinese medicine.Inherit and development Chinese medicine, and combine with modern medicine technology, be the key point that Chinese medicine steps into international market.
Four, detailed description of the invention
1. experimental technique
BALB/c mouse (normal group)
BALB/c mouse inbred lines (purchased from Nanjing University's model animal center), cleaning grade, body weight 18-22g, male and female half and half.Adaptability is fed.Experiment first day used 0.2ml normal saline (0.9%NaCl) gavage twice continuously by the 5th day, every minor tick 6 hours.
The structure (DTH mice, pathologic group/model group) of DNFB sensitized mice model
BALB/c mouse inbred lines (purchased from Nanjing University's model animal center), cleaning grade, body weight 18-22g, male and female half and half.Adaptability feeds a few days.Test the previous day, remove the hair of mouse web portion, smear 1%2,4-dinitrofluorobenzene (DNFB) (acetone: olive oil=4:1) 20ul sensitization morning in first day.Experiment first day used 0.2ml normal saline (0.9%NaCl) gavage twice continuously by the 5th day, every minor tick 6 hours.In 6th day morning, auris dextra is smeared 0.5% dinitrochlorobenzene 20ul and is excited, and simultaneously left ear is smeared equivalent solvent (olive oil) and contrasted.
Dexamethasone positive drug experiment (positive drug group)
Get DTH mice 10, experiment first day was normally raised to the 4th day, tested the 5th day intraperitoneal administration once.Diluted by the dexamethasone acetum of original content 5mg/ml for 1mg/ml, every only intraperitoneal administration 0.2ml, actual administration concentration is 10mg/kg.
The preparation of YUPINGFENG SAN and administration (YUPINGFENG SAN administration group)
YUPINGFENG SAN (YPFS) prescription rolls up 150 " studying carefully former side " from " Yifangleiju ", medicine composition Radix Saposhnikoviae 1 liang (30g), Radix Astragali (processed with honey) 2 liang (60g), the Rhizoma Atractylodis Macrocephalae 2 liang (60g).
Take the Radix Astragali, the Rhizoma Atractylodis Macrocephalae and Radix Saposhnikoviae prepared slices of Chinese crude drugs 150g altogether respectively in the ratio of 2:2:1, pulverize grinding, add 300ml water, be made into the suspension that final concentration is 0.5g crude drug/ml; The actual dosage of YUPINGFENG SAN powder is 5g crude drug/kg.
BALB/c mouse inbred lines (purchased from Nanjing University's model animal center), cleaning grade, body weight 18-22g, male and female half and half.Test the previous day, remove the hair of mouse web portion, smear 1%2,4-dinitrofluorobenzene (acetone: olive oil=4:1) 20ul sensitization in first day.From second day, continuous five days gastric infusions, every mice was administered once every day, each 0.2ml.In 6th day morning, auris dextra is smeared 0.5% dinitrofluorobenzene 20ul and is excited, and simultaneously left ear is smeared equivalent solvent and contrasted.Start after 6 ~ 8h to carry out follow-up index measurement and analysis.
From experiment first day by the 5th day, each gastric infusion 0.2ml of YPFS powder group every mice, every every day is administered twice, 6 hours, interval.
Each group of mice is normally raised under equivalent environment, feeding SPF maintenance type Mus grain and distilled water.
The preparation of YUPINGFENG SAN active ingredient composition and administration
Take astragaloside (3mg-21mg), atractylenolide Ⅰ (2mg-14mg) and cimicifugoside (6mg-21mg) respectively, add Tween 80 (Tween-80) and the 24.5ml distilled water of 0.5ml 2%, be mixed with the mixed solution of astragaloside, atractylenolide Ⅰ and cimicifugoside.
In composition I mixed solution, the final concentration of each active component is respectively 0.12mg/ml astragaloside, 0.08mg/ml atractylenolide Ⅰ and 0.24mg/ml cimicifugoside.
Test first day by the 5th day, every experiment mice each gastric infusion 0.2ml medicinal liquid, daily once.Actual dosage astragaloside is 1.2mg/kg, and atractylenolide Ⅰ is 0.8mg/kg, and cimicifugoside is 2.4mg/kg.Each group of mice is normally raised under equivalent environment, feeding SPF maintenance type Mus grain and distilled water.
In composition I I mixed solution, the final concentration of each active component is respectively 1.05mg/ml astragaloside, 0.7mg/ml atractylenolide Ⅰ and 1.05mg/ml cimicifugoside.
Test first day by the 5th day, every only each gastric infusion 0.2ml medicinal liquid, daily once.Actual dosage astragaloside is 10.5mg/kg, and atractylenolide Ⅰ is 7mg/kg, and cimicifugoside is 10.5mg/kg.Each group of mice is normally raised under equivalent environment, feeding SPF maintenance type Mus grain and distilled water.
2. experiment detects
The body weight change of mice
Respectively at after experiment stimulation the previous day and after within the 6th day, exciting rear 6 ~ 8h, body weight record is carried out to each group every mice.The absolute value (g) of Mouse Weight change before and after experiment with computing and relative value's (percentage ratio).
The ear swelling degree of mice
After within 6th day, exciting after 6 ~ 8h, first observe difference in mice left and right ear form, color.Redr and more swollen, thicken situation.And the difference of left and right ear thickness is measured with calibrator.
ELISA detects
After ether or sodium phenobarbital anesthetized mice, pluck eyeball and get blood, getting supernatant after the centrifugal 10min of 2500rmp uses ELISA to detect box (Mouse IL-4ELISA Kit 96T, Mouse IFN-gamma ELISAKit, Lian Ke Bioisystech Co., Ltd), measure IL-4 and IFN-γ content.
The index and spleen index of mice
Aseptic taking-up mouse spleen in super-clean bench, takes sky EP pipe quality in advance, subtracts each other and obtains spleen weight again divided by the corresponding body weight of mice, obtain every 100g body weight spleen weight (mg) after measuring EP pipe and spleen gross weight.
The extraction of mouse spleen protein
The spleen tissue getting mice is respectively about 50mg, puts into 2ml centrifuge tube, respectively adds 500ul western and IP cell pyrolysis liquid and 20ul Roche Protease Inhibitor, Syrup-homogenizing instrument homogenate (60Hz, 70s).Leave standstill 1h, 10000g, 4 DEG C of centrifugal 15min on ice.Get supernatant, be tissue protein stock solution.Measure histone stock protein concentration by BCA method, with ddH2O, histone stock solution is adjusted to equal-volume isoconcentration (4.201ug/ul), adds 5 × SDS sample-loading buffer of 1/4th volumes.Boiling water bath 5min ,-20 DEG C of preservations.
Western blotting detects
SDS-PAGE electrophoresis, goes to pvdf membrane by semidry method, and 5% defatted milk powder is closed, and incubate primary antibodie, 4 DEG C are slowly shaken overnight incubation, and TBST washes film 4 times, each 15min.Add two to resist, incubated at room 1h, TBST wash film, and ECL method detects.With Image J software analysis albumen gray value.
Statistical procedures
BM SPSS Statistics 19.0 software is utilized to carry out statistical analysis.Experimental data x ± s represents, comparing between two groups of means with t inspection, is that difference has significance with P<0.05.
Concrete grammar is: first use " exploration " normality to each group of DATA DISTRIBUTION in " descriptive statistics " to test, reject the exceptional value outside normal distribution.To " comparing average " under the data separate " analysis " retained, with DTH model group for reference, independent sample t-test is carried out to each group of sample, judge that whether the treatment of medicine is effective by significant difference size, if effectively according to significance concrete numerical value judge significant difference degree (P<0.001 as extremely significantly, labelling " * * * "; P<0.05 is very remarkable, labelling " * * "; P<0.01 is significantly, labelling " * ").
Claims (5)
1. a YUPINGFENG SAN active ingredient composition, is characterized in that, by astragaloside, atractylenolide Ⅰ and cimicifugoside, combining according to different ratios, produces YUPINGFENG SAN active ingredient composition.
2. the preparation method of YUPINGFENG SAN active ingredient composition according to claim 1, is characterized in that being made up of following steps:
Take astragaloside 3mg-21mg, atractylenolide Ⅰ 2mg-14mg and cimicifugoside 6mg-21m respectively;
Add Tween 80 and the 24.5ml distilled water of 0.5ml 2%, be mixed with the mixed solution of astragaloside, atractylenolide Ⅰ and cimicifugoside;
In mixed solution, the final concentration of each active component is respectively astragaloside 0.12-1.05mg/ml, atractylenolide Ⅰ 0.08-0.7mg/ml, cimicifugoside 0.24-1.05mg/ml.
3. YUPINGFENG SAN active ingredient composition described in claim 1 is preparing the application in immunomodulator.
4. the application of YUPINGFENG SAN active ingredient composition described in claim 1 in preparation treatment respiratory tract infection or bronchial asthma or allergic rhinitis or chronic urticaria or skin pruritus or allergic dermatitis or rheumatic arthritis or nephritis medicine.
5. the application of YUPINGFENG SAN active ingredient composition described in claim 1 in preparation treatment malignant tumor, chronic hepatitis, chronic bronchitis ancillary drug.
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| CN105878234A (en) * | 2016-06-02 | 2016-08-24 | 河南大学 | Application of atractylenolide I in preparation of medicine for enhancing immunity and healthcare products |
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| CN1836686A (en) * | 2005-03-23 | 2006-09-27 | 刘凯 | Chinese traditional effective part compound preparation for promoting organic body immune function, and its preparation method |
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| CN1836686A (en) * | 2005-03-23 | 2006-09-27 | 刘凯 | Chinese traditional effective part compound preparation for promoting organic body immune function, and its preparation method |
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