CN115645509A - Pharmaceutical composition for treating alopecia areata, preparation method and content measurement - Google Patents
Pharmaceutical composition for treating alopecia areata, preparation method and content measurement Download PDFInfo
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Abstract
The invention relates to a pharmaceutical composition for treating alopecia areata, a preparation method and content measurement, wherein the pharmaceutical composition comprises cacumen biotae, cowherb seed, caulis perllae, chinese mahonia, safflower, ginger, fleece-flower root and mulberry leaf. The result of the compound content detection method shows that the separation degree, the peak shape and the peak purity are good, the reproducibility is good, and the method is suitable for detecting the quercetin tincture.
Description
Technical Field
The invention relates to the field of medicine invention, in particular to a medicine composition for treating alopecia areata, a preparation method and content measurement.
Background
Alopecia areata is a T cell mediated autoimmune disease, is called as 'oily wind', 'ghost shaving head', 'pluck', 'plum coat alopecia' and the like in traditional Chinese medicine, is represented as circular or oval speckle-shaped non-scar alopecia of scalp, and is one of clinical common skin diseases. Epidemiological research shows that the prevalence rate of alopecia areata in China is 0.27%, and foreign research shows that the prevalence rate of people for the lifetime is about 2%.
The pathogenesis of alopecia areata is complex, and the exact etiology and pathogenesis of alopecia areata are not completely clear. Vascular endothelial growth factor, serum inflammatory factor and the like in the microcirculation of the hair follicle play a key role in the pathogenesis of alopecia areata. Clinically, according to baldness, the baldness can be divided into patch type, reticular type, creeping type, diffuse type and the like, and according to the progress of the disease condition, the progressive period (active period), the stationary period (resting period) and the convalescent period can be divided. Alopecia areata belongs to a loss-compatible disease, can cause negative emotions such as anxiety, inferior and the like of patients, and seriously influences the physiological and psychological health of the patients. The western medicine treatment method comprises local topical or skin lesion local injection of glucocorticoid, laser pulse, hair follicle growth stimulation, systemic use of immunosuppressant, glucocorticoid and the like, but the disease is easy to relapse after drug withdrawal due to toxic and side effects and adverse reactions, particularly, the systemic use of the glucocorticoid can achieve good effect in a short period, but the reduction is too fast or the relapse rate of drug withdrawal is higher, the clinical effect is not satisfactory, so the foreign guideline rating does not belong to the A-grade recommended treatment means, and at present, no FDA approved treatment is available.
Alopecia areata is located on scalp, and oral administration medicine is difficult to locally aggregate on scalp lesion to reach effective concentration. Research shows that the medicine concentration in the tissues of the local application part is obviously higher than that in blood, certain blood medicine concentration can be maintained, the traditional Chinese medicine externally treats the disease site directly, the operation is simple and safe, and the traditional Chinese medicine is accepted by most patients.
The invention discloses a Chinese medicinal composition for treating alopecia areata and a preparation method thereof, wherein the Chinese medicinal composition comprises the following Chinese medicinal raw materials in parts by weight: 3000-3500 g of fresh cacumen biotae, 1300-1500 g of polygonum multiflorum, 1200-1400 g of eclipta, 500-550 g of ginger, 300-350 g of dried pepper, 250-300 g of safflower, 250-300 g of ligusticum wallichii, 200-250 g of ginseng, 200-250 g of ailanthus bark and 8-10L of 95% ethanol. The traditional Chinese medicine is prepared from pure traditional Chinese medicines, has no toxic or side effect, can achieve the effects of nourishing, protecting and growing hair, and has the total effective rate of over 95 percent for treating alopecia areata proved by clinical use.
The application number CN201310458025.1 has the following defects: (1) the concentration of ethanol is high, and the skin is easily damaged; (2) The dried pepper in the raw material has large irritation to the skin and is difficult to accept by patients; and (3) the raw materials contain ginseng, so the cost is high.
In order to solve the problems, the invention group selects Chinese arborvitae twig which can enter blood to expel wind and tuber fleeceflower root which can tonify liver and kidney, nourish essence and blood, blacken hair and grow hair as monarch drugs according to the basic theory of traditional Chinese medicine and by combining the causes of alopecia areata; safflower capable of nourishing blood and growing hair, breaking blood and promoting blood circulation and stimulating the menstrual flow, cowherb seed, chinese mahonia which has the effects of expelling wind and removing obstruction in channels and clearing heat and removing toxicity are taken as ministerial drugs; the ginger capable of pungent energy and dispelling wind and relieving exterior syndrome and the mulberry leaves capable of improving eyesight and growing hair are taken as adjuvant medicines, and the ginger and the mulberry leaves can effectively nourish hair and grow hair and blacken hair when used together. The traditional Chinese medicine composition has the advantages of simple preparation method, mild medicinal effect, small irritation, easy acceptance by patients and low cost, can be used for treating alopecia areata, alopecia with symptoms of spot-shaped alopecia, round or oval shape, long course of disease, and symptoms of dizziness, tinnitus, dysphoria with feverish sensation in chest, soreness and weakness in waist and lower extremities, insomnia, red tongue or ecchymosis, little fur, wiry and rapid pulse or weak pulse and the like, and has wide clinical application value.
Disclosure of Invention
The invention aims to provide a medicine composition for treating alopecia areata.
Another object of the present invention is to provide a method for preparing a pharmaceutical composition for alopecia areata.
The invention further aims to provide a content detection method of the pharmaceutical composition for treating alopecia areata.
The pharmaceutical composition is prepared from the following medicines by weight: 20-100g of cacumen biotae, 10-50g of cowherb seed, 10-50g of lysimachia christinae hance, 10-50g of mahonia fortunei, 5-15g of safflower, 10-30g of ginger, 10-30g of polygonum multiflorum and 5-15g of mulberry leaf.
In a preferred embodiment of the method of the invention,
the pharmaceutical composition is prepared from the following medicines by weight: 30-80g of cacumen biotae, 20-40g of cowherb seed, 20-40g of lysimachia christinae hance, 20-40g of mahonia fortunei, 8-12g of safflower, 15-25g of ginger, 15-25g of polygonum multiflorum and 8-12g of mulberry leaf.
It is further preferred that the first and second liquid compositions are,
the pharmaceutical composition is prepared from the following medicines by weight: 50g of cacumen biotae, 30g of cowherb seed, 30g of caulis et folium gaultheriae yunnanensis, 30g of Chinese mahonia, 10g of safflower, 20g of ginger, 20g of fleece-flower root and 10g of mulberry leaf.
The preparation method of the pharmaceutical composition comprises the following steps:
pulverizing folium Platycladi, caulis et folium Gaultheriae Yunnanensis, folium Mahoniae, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml of 60-80% ethanol, soaking for 5-9 days, filtering, adding 60-80% ethanol to adjust total amount to 1000ml, and making into tincture.
Preferably, the first and second liquid crystal materials are,
the preparation method of the pharmaceutical composition comprises the following steps:
pulverizing folium Platycladi, caulis et folium Gaultheriae Yunnanensis, folium Mahoniae, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml of 65-75% ethanol, soaking for 6-8 days, filtering, adding 65-75% ethanol to adjust total amount to 1000ml, and making into tincture.
It is further preferred that the first and second liquid crystal compositions,
the preparation method of the pharmaceutical composition comprises the following steps:
pulverizing folium Platycladi, caulis et folium Gaultheriae Yunnanensis, folium Mahoniae, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml 70% ethanol, soaking for 7 days, filtering, adding 70% ethanol to adjust total amount to 1000ml, and making into tincture.
The content detection method comprises the following steps:
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01% ammonium acetate solution (60-80; the detection wavelength was 265nm.
(2) Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
(3) Preparation of a test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 20mL of 60-80% methanol, reflux-extracting for 20-40 min, cooling, supplementing 20mL with 60-80% methanol, shaking, filtering, and collecting the filtrate.
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
In a preferred embodiment of the method of the invention,
the content detection method comprises the following steps of (1) performing chromatographic condition and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01% ammonium acetate solution (70-75); the detection wavelength was 265nm.
It is further preferred that the first and second liquid crystal compositions,
the content detection method comprises the following steps of (1) performing chromatographic condition and system applicability test:
octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01% ammonium acetate solution (73); the detection wavelength was 265nm.
Preferably, the first and second liquid crystal materials are,
the preparation of the test solution in the content detection step (3) of the invention comprises the following steps: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 20mL of 70% methanol, reflux-extracting for 30min, cooling, adding 20mL of 70% methanol, shaking, filtering, and collecting the filtrate.
Advantageous effects
1. The medicine composition has the effects of nourishing blood, promoting blood circulation and growing hair. Can be used for treating alopecia areata with symptoms of alopecia, circular or elliptical alopecia, and long course of disease, manifested by dizziness, tinnitus, dysphoria with feverish sensation in chest, legs, soreness of waist and legs, and insomnia. The traditional Chinese medicine composition is simple in preparation method, low in cost, mild in drug effect, small in irritation, easy to accept by patients, free of adverse reactions such as local erythema, scales and alopecia aggravation, and has a clinical popularization value.
2. The clinical curative effect test result of the tincture of the invention shows that the total effective rate of the treatment is 93.3%, the difference is significantly higher than 83.3% of the control group, the tincture of the invention has statistical significance (P is less than 0.05), and the tincture of the invention can improve the treatment effect of alopecia areata.
3. According to the clinical efficacy test of the invention, through comparison of IL-12, IL-18 and IFN-gamma levels before and after treatment, after 3 months of treatment, the IL-12, IL-18 and IFN-gamma levels of two groups of serum are obviously lower than those before treatment, and the IL-12, IL-18 and IFN-gamma levels of the group of application are lower than those of a control group, and the differences have statistical significance (P is less than 0.05), so that the tincture provided by the invention can improve the IL-12, IL-18 and IFN-gamma levels of the serum of a patient in combination with the compound glycyrrhizin tablet, and the treatment effect is improved.
4. According to the preparation method, through the investigation of the concentration of the ethanol extracted from the medicinal materials, the extraction rate of the effective component quercetin of the cacumen biotae and the effective component vaccaria segetalis flavonoid glycoside of the vaccaria segetalis in the formula is taken as an evaluation index, and the ethanol concentration in the preparation process is optimized. When the medicine is soaked by 70 percent ethanol and 90 percent ethanol, the extraction rates of the quercitrin and the cowherb seed flavonoid glycoside are obviously higher than 50 percent ethanol, and the 70 percent ethanol concentration is equivalent to the extraction rate of the 90 percent ethanol concentration, so that the 70 percent ethanol concentration is selected as the preparation process parameter of the invention to save the production cost.
5. According to the preparation method, through the investigation of the medicinal material soaking time, the result shows that after the medicinal material is soaked in 70% ethanol for 7 days, the extraction rates of the quercetin and the cowherb seed flavonoid glycoside are changed slowly, the extraction rates are equivalent after the medicinal material is soaked for 7 days, and in order to save the production cost, the preparation process parameters are selected to be soaking for 7 days.
6. According to the invention, through the investigation of the method for measuring the content of the quercetin, the problems of poor separation degree, poor peak shape, poor accuracy, poor stability and the like appear in the result of detection according to the pharmacopoeia standard, and aiming at the problems, the mobile phase, the proportion, the wavelength, the extraction solvent, the extraction method and the like in the method for measuring the content of the quercetin are investigated. The mobile phase examination result shows that: the detection result by taking acetonitrile-0.01 percent ammonium acetate solution (73) as a mobile phase shows that the separation degree, the peak shape and the peak purity are good, the reproducibility is good, and the method is suitable for the inspection of the quercetin tincture; the wavelength investigation result shows that: at about 265nm, the quercetin in the tincture has maximum absorption, high peak response and stable baseline, so that 265nm is selected as the preferable detection wavelength; the investigation result of the extraction solvent shows that: the sample is treated by methanol and 70 percent methanol, the peak areas are not greatly different, but are larger than the peak areas of other solvent treatment samples, so that the '70 percent methanol' is taken as a preferred extraction solvent in consideration of cost; the investigation result of the extraction method shows that: reflux extraction is adopted for 30min and 60min, the peak area of a sample is large, and the reflux extraction for 30min is selected as a preferred extraction mode for cost consideration.
7. The method for measuring the content of the quercetin is subjected to method verification and investigation, and a linear investigation result shows that the injection concentration of the quercetin is in a good linear relation within the range of 50.013 mu g/ml-2500.65 mu g/ml; the precision test result shows that the sample introduction is repeated for 6 times, the precision of the relative standard deviation verification method is calculated, the RSD% value is calculated to be 1.40%, and the instrument precision is good; the result of the repeatability test shows that 6 test sample solutions are prepared, the peak area of the quercitrin is measured, and the RSD value is 1.39 percent, which shows that the measuring method of the quercitrin has good repeatability; the stability test result shows that the quercetin is relatively stable within 24 hours.
Drawings
FIG. 1 is a line graph of investigation results of different ethanol concentrations;
figure 2 is a line graph of the results of different dip times.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
The formula is as follows: 50g of cacumen biotae, 30g of cowherb seed, 30g of caulis et folium gaultheriae yunnanensis, 30g of Chinese mahonia, 10g of safflower, 20g of ginger, 20g of fleece-flower root and 10g of mulberry leaf.
Example 2
The formula is as follows: 20g of cacumen biotae, 10g of cowherb seed, 10g of caulis et folium gaultheriae yunnanensis, 10g of Chinese mahonia, 5g of safflower, 10g of ginger, 10g of polygonum multiflorum and 5g of mulberry leaf.
Example 3
The formula is as follows: 100g of cacumen biotae, 50g of cowherb seed, 50g of caulis et folium gaultheriae yunnanensis, 50g of Chinese mahonia, 15g of safflower, 30g of ginger, 30g of polygonum multiflorum and 15g of mulberry leaf.
Example 4
The formula is as follows: 30g of cacumen biotae, 20g of cowherb seed, 20g of caulis et folium gaultheriae yunnanensis, 20g of Chinese mahonia, 8g of safflower, 15g of ginger, 15g of polygonum multiflorum and 8g of mulberry leaf.
Example 5
40g of cacumen biotae, 25g of cowherb seed, 25g of caulis et folium gaultheriae yunnanensis, 25g of Chinese mahonia, 12g of safflower, 25g of ginger, 25g of polygonum multiflorum and 12g of mulberry leaf.
Example 6
60g of cacumen biotae, 35g of cowherb seed, 35g of caulis et folium gaultheriae yunnanensis, 35g of Chinese mahonia, 10g of safflower, 28g of ginger, 28g of polygonum multiflorum and 10g of mulberry leaf.
Example 7
80g of cacumen biotae, 20g of cowherb seed, 30g of caulis et folium gaultheriae yunnanensis, 30g of Chinese mahonia, 15g of safflower, 20g of ginger, 20g of polygonum multiflorum and 10g of mulberry leaf.
Example 8
90g of cacumen biotae, 40g of cowherb seed, 30g of caulis et folium gaultheriae yunnanensis, 40g of Chinese mahonia, 5g of safflower, 20g of ginger, 10g of polygonum multiflorum and 15g of mulberry leaf.
The formulations of examples 1-8 were prepared according to any of the following methods.
Example 9
The preparation method comprises the following steps: pulverizing folium Platycladi, caulis et folium Gaultheriae Yunnanensis, folium Mahoniae, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml 70% ethanol, soaking for 7 days, filtering, adding 70% ethanol to adjust total amount to 1000ml, and making into tincture.
Example 10
The preparation method comprises the following steps: pulverizing folium Platycladi, caulis et folium Gaultheriae Yunnanensis, folium Mahoniae, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml of 60% ethanol, soaking for 5 days, filtering, adding 60% ethanol to adjust total amount to 1000ml, and making into tincture.
Example 11
The preparation method comprises the following steps: pulverizing folium Platycladi, caulis et folium Gaultheriae Yunnanensis, folium Mahoniae, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 80% ethanol 1000ml, soaking for 9 days, filtering, adding 80% ethanol to adjust total amount to 1000ml, and making into tincture.
Example 12
The preparation method comprises the following steps: pulverizing folium Platycladi, caulis et folium Gaultheriae Yunnanensis, folium Mahoniae, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml 75% ethanol, soaking for 6 days, filtering, adding 75% ethanol to adjust total amount to 1000ml, and making into tincture.
The tinctures prepared in examples 9-12 were tested according to any of the following content testing methods.
Example 13 Quercetin content assay
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01% ammonium acetate solution (73); the detection wavelength was 265nm.
(2) Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
(3) Preparation of a test solution: precisely measuring 1mL of tincture, placing into a conical flask with a plug, precisely adding 20mL of 70% methanol, reflux-extracting for 30min, cooling, adding 20mL of 70% methanol, shaking, filtering, and collecting the filtrate.
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 14 Quercetin content assay
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01% ammonium acetate solution (60; the detection wavelength was 265nm.
(2) Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
(3) Preparation of a test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 20mL of 60% methanol, reflux-extracting for 20 min, cooling, supplementing 20mL with 60% methanol, shaking, filtering, and collecting the filtrate.
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 15 Quercetin content assay
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01% ammonium acetate solution (80); the detection wavelength was 265nm.
(2) Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
(3) Preparing a test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 20mL of 80% methanol, reflux-extracting for 40 min, cooling, adding 80% methanol to make up for 20mL, shaking, filtering, and collecting the filtrate.
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 16 Quercetin content assay
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01% ammonium acetate solution (65); the detection wavelength was 265nm.
(2) Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
(3) Preparation of a test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 20mL of 60% methanol, reflux-extracting for 25 min, cooling, supplementing 20mL with 60% methanol, shaking, filtering, and collecting the filtrate.
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 17 Quercetin content assay
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01% ammonium acetate solution (75; the detection wavelength was 265nm.
(2) Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
(3) Preparation of a test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 20mL of 75% methanol, reflux-extracting for 30min, cooling, supplementing 20mL with 75% methanol, shaking, filtering, and collecting the filtrate.
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
Example 18 Quercetin content assay
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01% ammonium acetate solution (78); the detection wavelength was 265nm.
(2) Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
(3) Preparation of a test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 20mL of 70% methanol, reflux-extracting for 25 min, cooling, supplementing 20mL with 70% methanol, shaking, filtering, and collecting the filtrate.
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
In order to further verify the effectiveness of the invention, a series of verification tests are carried out, specifically as follows:
1 prescription composition, theoretical basis
1.1 prescription composition
50g of cacumen biotae, 30g of cowherb seed, 30g of caulis et folium gaultheriae yunnanensis, 30g of Chinese mahonia, 10g of safflower, 20g of ginger, 20g of fleece-flower root and 10g of mulberry leaf.
1.2 theoretical basis
In the medicine formula, the Chinese arborvitae twig, which belongs to lung, liver and spleen channels, the tuber fleeceflower root, which tonifies liver and kidney, nourishes essence and blood, blackens hair and grows hair, the safflower and the raw cowherb seed, which break blood and activate blood circulation to dredge meridians, nourish blood and grow hair, the bone-penetrating essence, the wind-dispelling and collateral-dredging, the swelling-eliminating and pain-relieving, the Chinese mahonia, which clears away heat and toxic material and the swelling, can grow hair by external washing, are ministerial with the cowherb seed, the raw Jiang Weixin, which can dispel the wind and relieve the exterior syndrome by pungent energy, the mulberry leaf, which is bitter and sweet in taste and cold in nature, belongs to lung and liver channels, has the efficacies of improving eyesight and growing hair, dispelling wind and clearing heat, and the efficacies of nourishing blood and activating blood, nourishing hair and growing hair by the combination of various medicines.
2 study of the preparation Process
2.1 examination of ethanol concentration
The extraction rate of effective components quercetin of Chinese arborvitae twig and cowherb seed and flavonoid glycoside of cowherb seed in the prescription is used as evaluation indexes, and the ethanol concentration in the preparation process is optimized.
Weighing 3 parts of medicinal material decoction pieces in 1/2 prescription amount according to the formula proportion of the embodiment 1, adding 500mL of ethanol with different concentrations according to the requirements of the table 3, soaking for 7 days, filtering, respectively measuring the content of quercetin and the content of cowherb seed flavonoid glycoside in the filtrate, and calculating the extraction rate, wherein the result is shown in the table 1 and the figure 1.
TABLE 1 test results of ethanol concentration investigation
As can be seen from the test results in Table 1 and FIG. 1, the extraction rates of quercetin and vaccaria flavonoid glycoside are both significantly higher than 50% ethanol when the extract is soaked in 70% and 90% ethanol concentrations, and the 70% ethanol concentration is selected as the preparation process parameter of the invention in order to save production cost because the extraction rates of 70% ethanol concentration and 90% ethanol concentration are equivalent.
2.2 investigation of immersion time
Weighing 4 parts of medicinal material decoction pieces in 1/2 prescription amount according to the formula proportion in the embodiment 1, and respectively adding 70% ethanol
500mL, soaking and filtering according to the time requirements in the table 4, respectively measuring the contents of quercetin and cowherb seed flavonoid glycoside in the filtrate, and calculating the extraction rate, wherein the results are shown in the table 2 and the figure 2.
Table 2 table of test results of investigation of immersion time
As can be seen from the test results in Table 2 and FIG. 2, after the soaking in 70% ethanol for 7 days, the extraction rates of quercetin and cowherb seed flavonoid glycoside change slowly, which indicates that the extraction rates are equivalent after the soaking for 7 days, and the soaking for 7 days is selected as the preparation process parameter of the invention in order to save the production cost.
3 clinical efficacy evaluation test
3.1 sources of cases
60 alopecia areata patients who are treated in traditional Chinese medicine hospitals in Guizhou province, guizhou province from 3 months in 2018 to 2 months in 2020 are collected. Referring to the diagnosis standard of alopecia areata in the Chinese and western medicine integrated dermatosis: the hair is flaked and shed in a short time or suddenly and shows single or multiple times; the skin of the alopecia part has no atrophy or scar; the color of the skin of the alopecia part is normal, and no obvious inflammatory reaction exists.
3.2 methods of treatment
The patients are divided into a control group and an observation group by adopting a random digital table method, and the comparison difference of the sex, age and disease course clinical data of the two groups of patients has no statistical significance (P is more than 0.05) and has comparability.
The control group was given western routine treatment: 50mg of compound glycyrrhizin tablet for oral administration, 3 times per day. Observation group: the tincture prepared in the embodiment 1 of the invention is added on the basis of the treatment of a control group, the tincture is applied to the affected part once a day, the affected part is uniformly smeared with the tincture once a day, one month is a treatment course, the treatment effect is comprehensively compared after 3 treatment courses of continuous use, SPSS23.0 statistical software is adopted to analyze experimental data, the metering data is expressed as +/-s, t test is adopted, the counting data is expressed as cases (%), and x 2 test is adopted. P < 0.05 is statistically significant.
The evaluation standard of curative effect is as follows: refer to the Chinese and Western medicine integrated diagnosis and treatment efficacy determination Standard (draft) for 5 skin diseases, which is set by the dermatological society of Chinese medical and Western medicine. And (3) healing: the hair at the alopecia areata is grown completely, the color, the thickness and the distribution density of the hair are close to normal, and the hair tension test is negative; the effect is shown: the growth of new hair at the alopecia areata is more than 70 percent, the color glossiness and the distribution density of the new hair are close to those of hair in normal areas, more vellus hairs become terminal hairs, and the result of a light-drawing test is negative; the method has the following advantages: the growth rate of new hairs at the alopecia areata is 10% -70%, vellus hairs grow, the growth rate of the new hairs is slow, and the results of the light-drawing test can be negative or positive under the condition of thick hairs or white hairs; and (4) invalidation: no new hair growth, or less than 10% new hair growth, or continuing to lose hair, and the result of the light-pull test is positive. Total effective rate = (number of cure cases + number of significant cases + number of effective cases)/total number of cases × 100%.
3.3 results
3.3.1 comparison of therapeutic effects, the results are shown in Table 3.
TABLE 3 comparison of clinical efficacy of patients
Note: p < 0.05 compared to control group
The results in table 3 show that the total effective rate of the patients in the observation group is 93.3%, which is significantly higher than 83.3% of the patients in the control group, the difference is significant, and the statistical significance is achieved (P is less than 0.05). The tincture prepared by the invention can improve the treatment effect of alopecia areata.
3.3.2 comparison of IL-12, IL-18, IFN- γ levels before and after treatment
Before and 3 months after treatment, 3mL of fasting peripheral venous blood is drawn from morning of two groups of patients respectively, serum samples are obtained after routine centrifugation, and the levels of serum interleukin-12 (IL-12), interleukin-18 (IL-18) and gamma interferon (IFN-gamma) are measured by an enzyme-linked immunosorbent assay. The results are shown in Table 4.
TABLE 4 comparison of cytokine levels in serum before and after treatment
P < 0.05 compared to before treatment; p < 0.05 compared to the control group.
After 3 months of treatment, the levels of IL-12, IL-18 and IFN-gamma in the serum of the two groups are obviously lower than those before treatment, the levels of IL-12, IL-18 and IFN-gamma in the observed group are lower than those of the control group, and the differences are statistically significant (P is less than 0.05). The tincture of the invention is combined with the compound glycyrrhizin tablet to treat the diseases, and the tincture of the invention can improve the serum IL-12, IL-18 and IFN-gamma levels of patients and improve the treatment effect.
4 examination of Quercetin content measurement method
4.1 method sources, refer to detection standard of cacumen Platycladi in the 2020 pharmacopoeia.
Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol-0.01 mol/L potassium dihydrogen phosphate solution-glacial acetic acid (40; the detection wavelength was 254nm. The number of theoretical plates is not less than 1500 calculated according to the peak of quercetin.
Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
Preparation of a test solution: precisely weighing 1mL of the tincture, placing the tincture in a conical flask with a plug, precisely adding 20mL of methanol, sealing the plug, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the weight loss with methanol, shaking up, filtering, and taking a subsequent filtrate to obtain the tincture.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
As a result: the detection result is found to have the problems of poor separation degree, poor peak shape, poor accuracy, poor stability and the like, and the mobile phase, the proportion, the wavelength, the extraction solvent, the extraction method and the like in the quercetin content method are examined aiming at the problems, and the method comprises the following specific steps:
4.2 mobile phase investigation
Precisely measuring 1mL of tincture, placing the tincture in a conical flask with a plug, precisely adding 20mL of methanol, sealing the plug, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, complementing the weight loss with methanol, shaking up, filtering, and taking a subsequent filtrate, wherein the test is carried out according to the mobile phase in the table 5, and the test result is as follows according to the chromatographic condition under the item of '4.1':
TABLE 5 examination of mobile phases
As can be seen from table 5, the results of the detection using "acetonitrile-0.01% ammonium acetate solution (73).
4.3 detection wavelength investigation
Precisely measuring 1mL of tincture, placing the tincture in a conical flask with a plug, precisely adding 20mL of methanol, sealing the plug, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, complementing the weight loss with methanol, shaking up, filtering, taking the subsequent filtrate, and recording the absorption spectrum within the range of 190-400 nm.
As a result, the quercetin of the tincture has the maximum absorption at about 265nm, the peak response is high, and the baseline is stable, so that 265nm is selected as the preferable detection wavelength.
4.4 examination of extraction solvent
The extraction solvent was screened with a mobile phase of "acetonitrile-0.01% ammonium acetate solution (60)" and a wavelength of "265 nm":
precisely measuring 1mL of tincture, placing in a conical flask with a plug, treating a sample with 20mL of methanol, 70% methanol, acetonitrile, ethyl acetate and glacial acetic acid as solvents respectively, sealing the plug, weighing, ultrasonically treating for 30 minutes, cooling, weighing again, supplementing the lost weight with the corresponding solvents respectively, shaking up, filtering, taking the subsequent filtrate, and detecting according to the method under the item '4.1', wherein the result is shown in Table 6.
TABLE 6 solvent investigation table for quercetin content method
As a result: the peak areas were comparable but larger for the samples treated with methanol and 70% methanol than for the other solvent treated samples. Therefore, "70% methanol" is the preferred extraction solvent for cost reasons.
4.5 examination of solvent extraction method and extraction time
Taking the mobile phase as acetonitrile-0.01% ammonium acetate solution (73) "and the wavelength as" 265nm ", taking the extraction solvent as" 70% methanol ", precisely measuring 1mL of tincture, extracting by the extraction method of Table 7, and detecting according to the chromatographic conditions under the item" 4.1", the results are shown in Table 7.
TABLE 7 examination table of the extraction method of quercetin content
As a result: reflux extraction is adopted for 30min and 60min, the peak area of a sample is large, and the reflux extraction for 30min is selected as a preferred extraction mode for cost consideration.
4.6 conclusion: through the investigation experiment, the preferable method for measuring the content of the quercetin is obtained as follows:
chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.01% ammonium acetate solution (73); the detection wavelength was 265nm.
Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
Preparation of a test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 20mL of 70% methanol, reflux-extracting for 30min, cooling, adding 20mL of 70% methanol, shaking, filtering, and collecting the filtrate.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
4.7 methodological validation study
4.7.1 Linear test
Precision fetchingThe concentration of the quercitrin reference substance solution is 50.013 mug/ml, 100.026 mug/ml, 500.130 mug/ml, 1000.26 mug/ml and 2500.65 mug/ml, the corresponding concentration is subjected to regression analysis by the chromatographic peak area of the standard working solution, the linear equation is y =147.74x 2025.5, R is obtained 2 =0.999, indicating that the concentration of quercetin in the sample is in good linear relationship in the range of 50.013 μ g/ml-2500.65 μ g/ml, and the result is shown in table 8.
TABLE 8 Quercetin Linear test results
4.7.2 instrumental precision test
Taking a quercetin control solution with the concentration of 50.013 mug/ml, repeatedly injecting for 5 times under the chromatographic condition of the item of 4.6, measuring the peak area of the quercetin, calculating the RSD% value to be 1.40%, indicating that the precision of the instrument is good, and the measurement result is shown in Table 8.
TABLE 8 results of instrumental precision tests
4.7.3 repeatability test
Precisely measuring 1mL of tincture, preparing 6 parts of test solution according to the preparation method of the test solution under the item of '4.6', precisely absorbing 10 mu L of each test solution, injecting the test solution into a liquid chromatograph, analyzing according to chromatographic conditions, measuring the peak area of quercetin, and measuring the RSD value to be 1.39%, which shows that the measuring method of quercetin has good repeatability; the results are shown in Table 9.
TABLE 9 results of the repeatability tests
4.7.4 stability test
Taking one part of the tincture sample solution, standing at room temperature, injecting samples at 0,4,8, 12, 12, 16 and 24h under the chromatographic condition of item 4.6, respectively, and measuring peak area, wherein RSD% is 1.40%, which shows that quercetin is relatively stable in 24 h. The stability test results are shown in Table 10.
TABLE 10 stability test results
Although the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto without departing from the spirit of the invention.
Claims (10)
1. The pharmaceutical composition for treating alopecia areata is characterized by being prepared from the following medicines in parts by weight: 20-100g of cacumen biotae, 10-50g of cowherb seed, 10-50g of lysimachia christinae hance, 10-50g of mahonia fortunei, 5-15g of safflower, 10-30g of ginger, 10-30g of polygonum multiflorum and 5-15g of mulberry leaf.
2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is prepared from the following drugs by weight: 30-80g of cacumen biotae, 20-40g of cowherb seed, 20-40g of lysimachia christinae hance, 20-40g of mahonia fortunei, 8-12g of safflower, 15-25g of ginger, 15-25g of polygonum multiflorum and 8-12g of mulberry leaf.
3. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is prepared from the following drugs by weight: 50g of cacumen biotae, 30g of cowherb seed, 30g of caulis et folium gaultheriae yunnanensis, 30g of Chinese mahonia, 10g of safflower, 20g of ginger, 20g of fleece-flower root and 10g of mulberry leaf.
4. A process for preparing a pharmaceutical composition according to claims 1-3, wherein the process comprises: pulverizing folium Platycladi, caulis et folium Gaultheriae Yunnanensis, chinese Mahonia, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml of 60-80% ethanol, soaking for 5-9 days, filtering, adding 60-80% ethanol to adjust total amount to 1000ml, and making into tincture.
5. The method of preparing the pharmaceutical composition of claim 4, wherein the method comprises: pulverizing folium Platycladi, caulis et folium Gaultheriae Yunnanensis, chinese Mahonia, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml of 65-75% ethanol, soaking for 6-8 days, filtering, adding 65-75% ethanol to adjust total amount to 1000ml, and making into tincture.
6. The method of preparing the pharmaceutical composition of claim 5, wherein the method comprises: pulverizing folium Platycladi, caulis et folium Gaultheriae Yunnanensis, folium Mahoniae, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml 70% ethanol, soaking for 7 days, filtering, adding 70% ethanol to adjust total amount to 1000ml, and making into tincture.
7. A content detection method for the tincture prepared in claim 6, which comprises the following steps:
(1) Chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; the proportion of the components is 60-80:20-40 acetonitrile-0.01% ammonium acetate solution as mobile phase; the detection wavelength was 265nm.
(2) Preparation of control solutions: taking a proper amount of quercetin control, precisely weighing, and adding methanol to obtain solution containing 50 μ g per 1 ml.
(3) Preparation of a test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 20mL of 60-80% methanol, reflux-extracting for 20-40 min, cooling, supplementing 20mL with 60-80% methanol, shaking, filtering, and collecting the filtrate.
(4) The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
8. The assay method according to claim 7, wherein the chromatographic conditions and system suitability test of step (1) is: octadecylsilane chemically bonded silica is used as a filling agent; the proportion is 70-75:25-30 acetonitrile-0.01% ammonium acetate solution as mobile phase; the detection wavelength was 265nm.
9. The assay method according to claim 8, wherein the chromatographic conditions and system suitability test of step (1) is: octadecylsilane chemically bonded silica is used as a filling agent; in a ratio of 73:27 acetonitrile-0.01% ammonium acetate solution as mobile phase; the detection wavelength was 265nm.
10. The assay method according to claim 7, wherein the sample solution in step (3) is prepared by: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 20mL of 70% methanol, reflux-extracting for 30min, cooling, adding 20mL of 70% methanol, shaking, filtering, and collecting the filtrate.
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