CN115645509B - Pharmaceutical composition for alopecia areata, preparation method and content measurement - Google Patents
Pharmaceutical composition for alopecia areata, preparation method and content measurement Download PDFInfo
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Abstract
The application relates to a pharmaceutical composition for alopecia areata, a preparation method and content measurement, wherein the pharmaceutical composition comprises cacumen biotae, semen vaccariae, garden balsam, chinese mahonia, safflower, ginger, polygonum multiflorum and mulberry leaf, the preparation method is simple, the cost is low, the drug effect is mild, the irritation is small, the patient is easy to accept, and adverse reactions such as local erythema, scales and aggravation of alopecia are avoided, so that the pharmaceutical composition has clinical popularization value for the treatment of alopecia areata. The result of the method for detecting the content of the compound shows that the separation degree, peak shape and peak purity are good, the reproducibility is good, and the method is suitable for detecting the quercetin tincture.
Description
Technical Field
The application relates to the field of medicine application, in particular to a pharmaceutical composition for alopecia areata, a preparation method and content measurement.
Background
Alopecia areata is a T cell mediated autoimmune disease, and is called "oily wind", "ghost head", "hair drawing", "Mei Yitu" and the like in traditional Chinese medicine, and is expressed as circular or oval plaque-like non-scarring alopecia of scalp, which is one of the common skin diseases in clinic. Epidemiological studies have shown that the prevalence of alopecia areata in China is 0.27%, and foreign studies have shown that the lifetime prevalence of the population is about 2%.
The pathogenesis of alopecia areata is complex, and the exact etiology and pathogenesis are not completely clear. Vascular endothelial growth factor, serum inflammatory factor and the like in hair follicle microcirculation play a key role in the onset process of alopecia areata. Clinically, it can be classified into patch type, net type, creeping type, diffuse type, etc. according to the form of alopecia, and can be classified into an development period (active period), a stabilization period (stationary period) and a recovery period according to the progress of the disease. Alopecia areata belongs to a damage-tolerance disease, and can cause negative emotions such as anxiety, spelt and the like of patients, and seriously affect the physiological and psychological health of the patients. Western medicine treatment methods comprise local external application or local injection of glucocorticoid, laser pulse, hair follicle growth stimulation, systemic use of immunosuppressant, glucocorticoid and the like, but toxic and side effects and adverse reactions are caused, and the system is easy to relapse after drug withdrawal, and particularly the systemic use of glucocorticoid can achieve good effects in a short period, but the decrement is too fast or the drug withdrawal relapse rate is higher, and the clinical effect is not satisfactory, so that foreign guideline rating does not belong to class A recommended treatment means, and no FDA approved treatment method exists at present.
Alopecia areata is located on the scalp, and the oral medicine is difficult to gather on the local lesions of the scalp to reach effective concentration. The research shows that the medicine concentration in the tissue of the local medicine application place is obviously higher than the concentration in blood, can maintain a certain blood medicine concentration, and the traditional Chinese medicine is directly treated by external treatment to the affected part, so that the operation is simple and safe, and is accepted by most patients.
The application number CN201310458025.1 is named as a traditional Chinese medicine composition for treating alopecia areata and a preparation method thereof, and discloses that the medicine is composed of the following traditional Chinese medicine raw materials in parts by weight: 3000-3500 g of fresh biota orientalis, 1300-1500 g of polygonum multiflorum, 1200-1400 g of eclipta, 500-550 g of ginger, 300-350 g of dried chilli, 250-300 g of safflower, 250-300 g of ligusticum wallichii, 200-250 g of ginseng, 200-250 g of cortex ailanthi and 8-10L of 95% ethanol. The application is prepared from pure traditional Chinese medicines, has no toxic or side effect, can achieve the effects of nourishing hair, protecting hair and growing hair, and has the total effective rate of more than 95% for treating alopecia areata proved by clinical use.
The application number CN201310458025.1 has the following disadvantages: (1) high ethanol concentration, which is vulnerable to skin damage; (2) The dry chilli in the raw materials has great skin irritation and is unacceptable to patients; the raw material (3) contains ginseng, and the cost is high.
In order to solve the problems, the application team selects arborvitae twig capable of entering blood to expel wind and polygonum multiflorum capable of nourishing liver and kidney, nourishing essence and blood, blackening hair and growing hair as principal drugs according to the basic theory of traditional Chinese medicine and combining with the cause of alopecia areata; safflower and seed of cowherb which can nourish blood, promote hair growth, break blood, activate blood and promote menstruation, the bone penetrating incense which can dispel wind and dredge collaterals and Chinese mahonia which can clear heat and detoxify are combined as ministerial drugs; the ginger capable of dispelling wind and relieving exterior syndrome and the mulberry leaf capable of improving eyesight and growing hair are taken as adjuvant drugs, and the combined use of the ginger can effectively nourish hair, promote hair growth and blacken hair. The preparation method is simple, the drug effect is mild, the irritation is small, the patient is easy to accept, the cost is low, the alopecia is marked by plaque-like falling, the alopecia is circular or oval, the course of the disease is long, and symptoms such as dizziness, tinnitus, dysphoria with feverish sensation in the chest and the back, soreness and weakness of the waist and the legs, insomnia, red tongue or ecchymosis, little fur, wiry pulse, weakness and the like can be caused, so that the alopecia areata-treating drug has a wide clinical application value.
Disclosure of Invention
The application aims to provide a pharmaceutical composition for alopecia areata.
Another object of the present application is to provide a method for preparing a pharmaceutical composition for alopecia areata.
It is still another object of the present application to provide a method for detecting the content of a pharmaceutical composition for alopecia areata.
The medicine composition disclosed by the application is prepared from the following medicines in parts by weight: 20-100g of biota orientalis, 10-50g of semen vaccariae, 10-50g of garden balsam, 10-50g of Chinese mahonia, 5-15g of safflower, 10-30g of ginger, 10-30g of polygonum multiflorum and 5-15g of mulberry leaf.
Preferably, the method comprises the steps of,
the medicine composition disclosed by the application is prepared from the following medicines in parts by weight: 30-80g of cacumen biotae, 20-40g of semen vaccariae, 20-40g of garden balsam, 20-40g of Chinese mahonia, 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 composition of the present application,
the medicine composition disclosed by the application is prepared from the following medicines in parts by weight: 50g of biota orientalis, 30g of semen vaccariae, 30g of garden balsam, 30g of Chinese mahonia, 10g of safflower, 20g of ginger, 20g of polygonum multiflorum and 10g of mulberry leaf.
The preparation method of the pharmaceutical composition comprises the following steps:
pulverizing folium Platycladi, herba speranskiae tuberculatae, 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, and adding 60-80% ethanol to adjust total volume to 1000ml to obtain tincture.
Preferably, the method comprises the steps of,
the preparation method of the pharmaceutical composition comprises the following steps:
pulverizing folium Platycladi, herba speranskiae tuberculatae, 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, and adding 65-75% ethanol to adjust total volume to 1000ml to obtain tincture.
It is further preferred that the composition of the present application,
the preparation method of the pharmaceutical composition comprises the following steps:
pulverizing folium Platycladi, herba Speranskiae Tuberculatae, mahonia, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml of 70% ethanol, soaking for 7 days, filtering, and adding 70% ethanol to adjust total volume to 1000ml to obtain tincture.
The content detection method provided by the application comprises the following steps:
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.01% ammonium acetate solution (60-80:20-40) is taken as a mobile phase; the detection wavelength was 265nm.
(2) Preparation of a control solution: taking appropriate amount of quercetin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg per 1 ml.
(3) Preparation of test solution: precisely measuring 1mL of tincture, placing in conical flask with plug, precisely adding 60-80% methanol 20mL, reflux extracting for 20-40 min, cooling, adding 60-80% methanol to 20mL, shaking, and filtering to obtain filtrate.
(4) Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
Preferably, the method comprises the steps of,
the chromatographic condition and system applicability test in the step (1) of the content detection method of the application is as follows: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.01% ammonium acetate solution (70-75:25-30) is taken as a mobile phase; the detection wavelength was 265nm.
It is further preferred that the composition of the present application,
the chromatographic condition and system applicability test in the step (1) of the content detection method of the application is as follows:
octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.01% ammonium acetate solution (73:27) was used as mobile phase; the detection wavelength was 265nm.
Preferably, the method comprises the steps of,
the preparation of the sample solution in the content detection step (3) comprises the following steps: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 70% methanol 20mL, reflux-extracting for 30min, cooling, supplementing 20mL with 70% methanol, shaking, and filtering to obtain subsequent filtrate.
Advantageous effects
1. The medicine composition has the effects of nourishing blood, activating blood and growing hair. Can be used for treating alopecia areata, with symptoms of alopecia, such as plaque-like drop, circular or oval shape, long course of disease, dizziness, tinnitus, dysphoria with feverish sensation in chest, waist and legs, and insomnia. The preparation method is simple, 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 aggravation of alopecia, and has clinical popularization value.
2. The clinical curative effect test result shows that the total effective rate of treatment is 93.3 percent, which is obviously higher than 83.3 percent of that of a control group, the difference is obvious, and the tincture has statistical significance (P is less than 0.05), which proves that the tincture prepared by the application can improve the treatment effect of alopecia areata.
3. The clinical curative effect test of the application passes the comparison of IL-12, IL-18 and IFN-gamma levels before and after treatment, the serum IL-12, IL-18 and IFN-gamma levels in two groups are obviously lower than those before treatment after 3 months, and the IL-12, IL-18 and IFN-gamma levels in the groups are lower than those in a control group, and the difference has statistical significance (P is less than 0.05), so that the tincture combined compound glycyrrhizin tablet can improve the serum IL-12, IL-18 and IFN-gamma levels of patients and improve the treatment effect.
4. The preparation method of the application passes the investigation of the concentration of ethanol extracted from medicinal materials, the extraction rate of the active ingredients of the monarch drug biota orientalis, namely quercetin and the active ingredient of the vaccaria segetalis, namely vaccaria segetalis flavonoid glycoside in the formula is used as an evaluation index, and the concentration of ethanol in the preparation process is optimized. When the extract is immersed by 70% and 90% ethanol, the extraction rate of quercetin and vaccaria segetalis flavonoid glycoside is obviously higher than 50% ethanol, and the 70% ethanol concentration is selected as the preparation process parameter for saving the production cost because the extraction rate of 70% ethanol concentration and 90% ethanol concentration are equivalent.
5. According to the preparation method, the soaking time of the medicinal materials is examined, and the result shows that after the medicinal materials are soaked in 70% ethanol for 7 days, the extraction rate of quercetin and vaccaria segetalis flavonoid glycoside is slowly changed, so that the extraction rate is equivalent after soaking for 7 days, and the preparation process parameters are selected after soaking for 7 days in order to save the production cost.
6. The application examines the mobile phase and the proportion, wavelength, extraction solvent and extraction method in the quercetin content method by examining the quercetin content determination method, and solving the problems of poor separation degree, poor peak shape, poor accuracy, poor stability and the like of the results in reference to pharmacopoeia standard detection. The mobile phase investigation result shows that: the detection result of acetonitrile-0.01% ammonium acetate solution (73:27) is used as a mobile phase, so that the separation degree, peak shape and peak purity are good, the reproducibility is good, and the method is suitable for the inspection of tincture quercetin; the wavelength investigation result shows that: about 265nm, the quercetin of the tincture has maximum absorption, the peak response is high, and the base line is stable, so that 265nm is selected as the optimal detection wavelength; the extraction solvent investigation result shows that: the peak areas of the samples treated with methanol and 70% methanol are not greatly different, but are larger than those of the samples treated with other solvents, so that the cost is considered, and 70% methanol is used as a preferred extraction solvent; the extraction method is used for examining the results and displaying: reflux extraction is carried out for 30min and 60min, the peak area of the sample is larger, and the reflux extraction is selected for 30min for the sake of cost consideration.
7. The method for measuring the content of the quercetin is verified and investigated, and the linear investigation result shows that the quercetin sample injection concentration is in good linear relation within the range of 50.013-2500.65 mug/ml; the precision test result shows that the sample is repeatedly injected 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 repeatability test result shows that 6 parts of sample solution is prepared, the peak area of the quercetin is measured, and the RSD value is 1.39%, so that the method for measuring the quercetin has good repeatability; stability test results show that the quercetin is relatively stable within 24 hours.
Drawings
FIG. 1 is a plot of results from different ethanol concentration studies;
FIG. 2 is a plot of the results for different immersion times.
Detailed Description
The following examples are illustrative of the application and are not intended to limit the scope of the application.
Example 1
The formula comprises the following components: 50g of biota orientalis, 30g of semen vaccariae, 30g of garden balsam, 30g of Chinese mahonia, 10g of safflower, 20g of ginger, 20g of polygonum multiflorum and 10g of mulberry leaf.
Example 2
The formula comprises the following components: 20g of biota orientalis, 10g of semen vaccariae, 10g of garden balsam, 10g of Chinese mahonia, 5g of safflower, 10g of ginger, 10g of polygonum multiflorum and 5g of mulberry leaf.
Example 3
The formula comprises the following components: 100g of biota orientalis, 50g of semen vaccariae, 50g of garden balsam, 50g of Chinese mahonia, 15g of safflower, 30g of ginger, 30g of polygonum multiflorum and 15g of mulberry leaf.
Example 4
The formula comprises the following components: 30g of biota orientalis, 20g of semen vaccariae, 20g of garden balsam, 20g of Chinese mahonia, 8g of safflower, 15g of ginger, 15g of polygonum multiflorum and 8g of mulberry leaf.
Example 5
40g of biota orientalis, 25g of semen vaccariae, 25g of garden balsam, 25g of Chinese mahonia, 12g of safflower, 25g of ginger, 25g of polygonum multiflorum and 12g of mulberry leaf.
Example 6
60g of biota orientalis, 35g of semen vaccariae, 35g of garden balsam, 35g of Chinese mahonia, 10g of safflower, 28g of ginger, 28g of polygonum multiflorum and 10g of mulberry leaf.
Example 7
80g of biota orientalis, 20g of semen vaccariae, 30g of garden balsam, 30g of Chinese mahonia, 15g of safflower, 20g of ginger, 20g of polygonum multiflorum and 10g of mulberry leaf.
Example 8
90g of biota orientalis, 40g of semen vaccariae, 30g of garden balsam, 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 each prepared according to any one of the following preparation methods.
Example 9
The preparation method comprises the following steps: pulverizing folium Platycladi, herba Speranskiae Tuberculatae, mahonia, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml of 70% ethanol, soaking for 7 days, filtering, and adding 70% ethanol to adjust total volume to 1000ml to obtain tincture.
Example 10
The preparation method comprises the following steps: pulverizing folium Platycladi, herba Speranskiae Tuberculatae, mahonia, 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, and adding 60% ethanol to adjust total volume to 1000ml to obtain tincture.
Example 11
The preparation method comprises the following steps: pulverizing folium Platycladi, herba Speranskiae Tuberculatae, mahonia, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml of 80% ethanol, soaking for 9 days, filtering, and adding 80% ethanol to adjust total volume to 1000ml to obtain tincture.
Example 12
The preparation method comprises the following steps: pulverizing folium Platycladi, herba Speranskiae Tuberculatae, mahonia, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml of 75% ethanol, soaking for 6 days, filtering, and adding 75% ethanol to adjust total volume to 1000ml to obtain tincture.
The tinctures prepared in examples 9 to 12 were tested according to any one of the following content testing methods.
EXAMPLE 13 Quercetin content detection
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.01% ammonium acetate solution (73:27) was used as mobile phase; the detection wavelength was 265nm.
(2) Preparation of a control solution: taking appropriate amount of quercetin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg per 1 ml.
(3) Preparation of test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 70% methanol 20mL, reflux-extracting for 30min, cooling, supplementing 20mL with 70% methanol, shaking, and filtering to obtain subsequent filtrate.
(4) Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
EXAMPLE 14 Quercetin content assay
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.01% ammonium acetate solution (60:40) is taken as a mobile phase; the detection wavelength was 265nm.
(2) Preparation of a control solution: taking appropriate amount of quercetin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg per 1 ml.
(3) Preparation of test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 60% methanol 20mL, reflux-extracting for 20 min, cooling, supplementing 20mL with 60% methanol, shaking, and filtering to obtain subsequent filtrate.
(4) Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
EXAMPLE 15 Quercetin content assay
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.01% ammonium acetate solution (80:20) is taken as a mobile phase; the detection wavelength was 265nm.
(2) Preparation of a control solution: taking appropriate amount of quercetin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg per 1 ml.
(3) Preparation of test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 80% methanol 20mL, reflux-extracting for 40 min, cooling, supplementing 20mL with 80% methanol, shaking, and filtering to obtain subsequent filtrate.
(4) Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
EXAMPLE 16 Quercetin content detection
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.01% ammonium acetate solution (65:35) is taken as a mobile phase; the detection wavelength was 265nm.
(2) Preparation of a control solution: taking appropriate amount of quercetin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg per 1 ml.
(3) Preparation of test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 60% methanol 20mL, reflux-extracting for 25 min, cooling, supplementing 20mL with 60% methanol, shaking, and filtering to obtain subsequent filtrate.
(4) Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
EXAMPLE 17 Quercetin content detection
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.01% ammonium acetate solution (75:25) is used as a mobile phase; the detection wavelength was 265nm.
(2) Preparation of a control solution: taking appropriate amount of quercetin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg per 1 ml.
(3) Preparation of test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 75% methanol 20mL, reflux-extracting for 30min, cooling, supplementing 20mL with 75% methanol, shaking, and filtering to obtain the subsequent filtrate.
(4) Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
EXAMPLE 18 Quercetin content detection
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.01% ammonium acetate solution (78:22) is taken as a mobile phase; the detection wavelength was 265nm.
(2) Preparation of a control solution: taking appropriate amount of quercetin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg per 1 ml.
(3) Preparation of test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 70% methanol 20mL, reflux-extracting for 25 min, cooling, supplementing 20mL with 70% methanol, shaking, and filtering to obtain subsequent filtrate.
(4) Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
In order to further verify the effectiveness of the present application, the application performed a series of verification tests, specifically as follows:
1 prescription composition, theoretical basis
1.1 prescription composition
50g of biota orientalis, 30g of semen vaccariae, 30g of garden balsam, 30g of Chinese mahonia, 10g of safflower, 20g of ginger, 20g of polygonum multiflorum and 10g of mulberry leaf.
1.2 theoretical basis
The Chinese arborvitae twig, the lung, the liver and the spleen channels are collected in the pharmaceutical composition, the tuber fleeceflower is used as a monarch, the liver and the kidney are nourished, essence and blood are nourished, hair is blacked and hair is grown, the safflower and the raw cowherb seed are used as main raw materials, the blood is broken, the blood is circulation is promoted, the hair is nourished and grown, the bone is transparent, the wind is dispelling, the collaterals are dredged, the swelling and pain are relieved, the Chinese mahonia is used as a ministerial, the fresh ginger is pungent, the wind is removed, the exterior is relieved, the mulberry leaf is bitter in taste, sweet in taste, cold in nature, the lung is collected, the liver is channel is nourished, the effects of improving eyesight and hair is grown, the wind is dredged, and the effects of nourishing and hair are achieved by the combination of the medicines.
2 study of preparation Process
2.1 ethanol concentration investigation
The extraction rate of the active ingredients of the monarch drug biota orientalis, namely quercetin and the active ingredient of the vaccaria segetalis, namely vaccaria segetalis flavonoid glycoside in the formula is used as an evaluation index, and the concentration of ethanol in the preparation process is optimized.
3 parts of medicinal decoction pieces with the formula of 1/2 are weighed according to the formula proportion of the embodiment 1, 500mL of ethanol with different concentrations is respectively added according to the requirements of the table 3, the mixture is immersed for 7 days, filtered, the content of quercetin and vaccarin in the filtrate is respectively measured, the extraction rate is calculated, and the results are shown in the table 1 and the figure 1.
TABLE 1 results of ethanol concentration investigation test
As can be seen from the test results in Table 1 and FIG. 1, when the extract is impregnated with 70% and 90% ethanol, the extraction rates of quercetin and vaccaria segetalis flavonoid glycoside are both significantly higher than 50% ethanol, and the 70% ethanol concentration is selected as the preparation process parameter for saving the production cost because the extraction rates of 70% ethanol concentration and 90% ethanol concentration are equivalent.
2.2 examination of the immersion time
Weighing 4 parts of medicinal decoction pieces with 1/2 prescription dosage according to the formula proportion of the embodiment 1, and respectively adding 70% ethanol
500mL, soaking according to the time requirement of Table 4, filtering, respectively measuring the content of quercetin and vaccarin in the filtrate, and calculating the extraction rate, wherein the results are shown in Table 2 and figure 2.
Table 2 table of impregnating time investigation test results
As can be seen from the test results in Table 2 and FIG. 2, after soaking in 70% ethanol for 7 days, the extraction rates of quercetin and vaccaria segetalis flavonoid glycoside change slowly, which indicates that the extraction rates of soaking for 7 days are equivalent, and the preparation process parameters of the application are selected 7 days for soaking to save the production cost.
3 clinical efficacy evaluation test
3.1 case Source
60 patients with alopecia areata who were diagnosed in hospitals in the Guizhou, and the south of Guizhou, were collected from 2018, 3 months, and 2020, 2 months. Reference to the diagnosis standard of alopecia areata in "traditional Chinese and Western medicine Combined dermatology": the hair flaking occurs in a short period or suddenly, and is single or multiple; the skin at the alopecia part is not atrophic or scarred; the skin at the alopecia part has normal color and no obvious inflammatory reaction.
3.2 methods of treatment
Patients are divided into a control group and an observation group by adopting a random number table method, and the comparison difference of the gender, 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 conventional treatment of western medicine: 50mg of compound glycyrrhizin tablet is orally taken for 3 times/d. Observation group: the tincture prepared in the embodiment 1 of the application is added on the basis of the treatment of a control group, and is uniformly applied to a patient part once a day and in the morning and evening, one month is a treatment course, the treatment effect is comprehensively compared after 3 treatment courses are continuously used, experimental data are analyzed by adopting SPSS23.0 statistical software, metering data are expressed in (+/-s), t-test is adopted, counting data are expressed in an example (%), and χ2 test is adopted. P < 0.05 is statistically significant for the differences.
Efficacy evaluation criteria: the method refers to the "diagnosis and curative effect determination criteria of 5 kinds of skin diseases" established by the skin diseases society of Chinese and Western medicine combination society. And (3) healing: all the hair grows at the alopecia areata, the color, thickness and distribution density of the hair are close to normal, and the hair pulling test is negative; the effect is shown: the new hair growth at the alopecia areata is more than 70%, the color glossiness and the distribution density of the new hair are close to those of the hair in the normal area, more vellus hair becomes final hair, and the result of the light-pull test is negative; the method is effective: the new hair growth at the alopecia areata is 10% -70%, vellus hair growth is carried out, the new hair growth speed is low, coarse hair or white hair exists, and the result of the light-pull test is negative or positive; invalidation: no new occurrence length, or new occurrence length less than 10%, or continued hair loss, and positive result of the light-pull test. Total effective rate= (number of recovery cases + number of significant cases + number of effective cases)/total case number x 100%.
3.3 results
3.3.1 efficacy comparisons, results are shown in Table 3.
Table 3 comparison of clinical efficacy of patients
Note that: p < 0.05 compared to the control group
As can be seen from the results in Table 3, the total effective rate of treatment for the patients in the observation group is 93.3%, which is significantly higher than that of the patients in the control group by 83.3%, and the difference is significant, and the statistical significance (P < 0.05) is achieved. The tincture prepared by the application can improve the treatment effect of alopecia areata.
3.3.2 comparison of IL-12, IL-18 and IFN-gamma levels before and after treatment
Before and after 3 months of treatment, 3mL of fasting peripheral venous blood is extracted from two groups of patients, serum samples are obtained after conventional centrifugation, and the serum albumin-12 (IL-12), interleukin-18 (IL-18) and gamma interferon (IFN-gamma) levels are measured by an ELISA method. The results are shown in Table 4.
TABLE 4 comparison of cytokine levels in serum before and after treatment
Note that P < 0.05 compared to before treatment; p < 0.05 compared to the control group.
After 3 months of treatment, the serum IL-12, IL-18 and IFN-gamma levels were significantly lower in both groups than before treatment, and the observed IL-12, IL-18 and IFN-gamma levels were lower in both groups than in the control group, and the differences were statistically significant (P < 0.05). The tincture combined with the compound glycyrrhizin tablet can improve the serum IL-12, IL-18 and IFN-gamma levels of patients and improve the treatment effect.
Investigation of 4 Quercetin content determination method
4.1 method source, refer to the detection standard of cacumen Platycladi in the pharmacopoeia of 2020 edition.
Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; methanol-0.01 mol/L potassium dihydrogen phosphate solution-glacial acetic acid (40:60:1.5) is taken as a mobile phase; the detection wavelength was 254nm. The theoretical plate number should be not less than 1500 calculated as quercetin peak.
Preparation of a control solution: taking appropriate amount of quercetin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg per 1 ml.
Preparation of test solution: taking 1mL of the tincture of the application, precisely weighing, placing into a conical flask with a plug, precisely adding 20mL of methanol, sealing, weighing, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing the reduced weight with methanol, shaking uniformly, filtering, and taking the subsequent filtrate.
Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
Results: the problems of poor separation degree, poor peak shape, poor accuracy, poor stability and the like of the detection result are found, and the mobile phase, the proportion, the wavelength, the extraction solvent, the extraction method and the like in the quercetin content method are examined according to the problems, and the specific steps are as follows:
4.2 mobile phase investigation
Precisely measuring 1mL of tincture, placing into a conical flask with a plug, precisely adding 20mL of methanol, sealing, weighing, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing the reduced weight with methanol, shaking, filtering, and collecting the subsequent filtrate to obtain the mobile phase in Table 5, and measuring according to chromatographic conditions under item "4.1", wherein the result is as follows:
table 5 mobile phase investigation table
As is clear from Table 5, the results of the mobile phase detection using "acetonitrile-0.01% ammonium acetate solution (73:27)" show that the separation degree, peak shape and peak purity are good, the reproducibility is good, and the method is suitable for the inspection of tincture quercetin.
4.3 detection wavelength investigation
Precisely measuring 1mL of tincture, placing into a conical flask with a plug, precisely adding 20mL of methanol, sealing, weighing, performing ultrasonic treatment for 30 minutes, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, filtering, collecting the subsequent filtrate, and recording the absorption spectrum in the range of 190-400 nm.
As a result, the tincture of quercetin has maximum absorption at about 265nm, high peak response and stable baseline, so that "265nm" is selected as the preferred detection wavelength.
4.4 extraction solvent investigation
Screening of the extraction solvent was performed with a mobile phase of "acetonitrile-0.01% ammonium acetate solution (60:40)", wavelength of "265 nm":
respectively precisely measuring 1mL of tincture, placing in conical flask with plug, respectively treating test sample with 20mL of methanol, 70% methanol, acetonitrile, ethyl acetate and glacial acetic acid as solvents, sealing, weighing, performing ultrasonic treatment for 30min, cooling, weighing again, respectively supplementing the reduced weight with corresponding solvents, shaking, filtering, collecting filtrate, and detecting according to method under item "4.1", and the results are shown in Table 6.
TABLE 6 solvent review Table for quercetin content method
Results: the peak areas were not very different for samples treated with methanol and 70% methanol, but were larger than for samples treated with other solvents. Therefore, in terms of cost, "70% methanol" is the preferred extraction solvent.
4.5 investigation of solvent extraction method and extraction time
The tincture was precisely measured by taking 1mL of the mobile phase of acetonitrile-0.01% ammonium acetate solution (73:27), "265nm" as the extraction solvent and "70% methanol", and extracted by the extraction method of Table 7, and the results are shown in Table 7 under the chromatographic conditions of "4.1".
TABLE 7 extraction method of quercetin content investigation table
Results: reflux extraction is carried out for 30min and 60min, the peak area of the sample is larger, and the reflux extraction is selected for 30min for the sake of cost consideration.
4.6 conclusion: through the above investigation experiments, the preferred method for measuring the content of the quercetin is as follows:
chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.01% ammonium acetate solution (73:27) was used as mobile phase; the detection wavelength was 265nm.
Preparation of a control solution: taking appropriate amount of quercetin reference substance, precisely weighing, and adding methanol to obtain solution containing 50 μg per 1 ml.
Preparation of test solution: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 70% methanol 20mL, reflux-extracting for 30min, cooling, supplementing 20mL with 70% methanol, shaking, and filtering to obtain subsequent filtrate.
Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
4.7 methodological validation investigation
4.7.1 Linear test
Precisely taking quercetin reference substance solutions with concentrations of 50.013 mug/ml, 100.026 mug/ml, 500.130 mug/ml, 1000.26 mug/ml and 2500.65 mug/ml, and carrying out regression analysis on the corresponding concentrations by using the chromatographic peak area of the standard working solution to obtain a linear equation of y=147.74x+2025.5, R 2 =0.999, showing that the quercetin sample injection concentration is in good linear relationship within the range of 50.013-2500.65 mug/ml, and the resultSee table 8.
TABLE 8 Linear test results of quercetin
4.7.2 Instrument precision test
Taking a quercetin reference substance solution with the concentration of 50.013 mug/ml, repeatedly injecting the sample for 5 times under the chromatographic condition of '4.6', measuring the peak area of the quercetin, and calculating the RSD% value to be 1.40%, wherein the instrument precision is good, and the measurement result is shown in Table 8.
Table 8 results of instrument precision test
4.7.3 repeatability test
Precisely measuring 1mL of tincture, preparing 6 parts of sample solution according to the preparation method of the sample solution under the item of 4.6, precisely sucking 10 mu L of each sample solution, injecting the sample solution into a liquid chromatograph, analyzing according to chromatographic conditions, and determining the peak area of the quercetin, wherein the RSD value is 1.39%, which indicates that the determination method of the quercetin has good repeatability; the results are shown in Table 9.
TABLE 9 repeatability test results
4.7.4 stability test
Taking one part of tincture sample solution, standing at room temperature, and respectively injecting samples according to chromatographic conditions of '4.6' under the conditions of 0,4,8, 12, 12, 16 and 24 hours, and determining peak area, wherein RSD% is 1.40%, which shows that quercetin is relatively stable within 24 hours. The stability test results are shown in Table 10.
Table 10 stability test results
While the application has been described in detail in the foregoing general description, with reference to specific embodiments and experiments, it will be apparent to one skilled in the art that modifications or improvements can be made thereto, and it is therefore intended that the application as defined in the appended claims be construed as broadly as possible without departing from the spirit of the application.
Claims (4)
1. The content detection method for the alopecia areata tincture medicine comprises the following steps of: 50g of biota orientalis, 30g of semen vaccariae, 30g of garden balsam, 30g of Chinese mahonia, 10g of safflower, 20g of ginger, 20g of polygonum multiflorum and 10g of mulberry leaf; the preparation method of the medicine comprises the following steps: pulverizing folium Platycladi, herba Speranskiae Tuberculatae, mahonia, rhizoma Zingiberis recens, polygoni Multiflori radix, and folium Mori into coarse powder, mixing with semen Vaccariae and Carthami flos, adding 1000ml of 70% ethanol, soaking for 7 days, filtering, and adding 70% ethanol to adjust total volume to 1000ml to obtain tincture; the content detection method is characterized by comprising the following steps of:
(1) Chromatographic conditions and system suitability test: octadecylsilane chemically bonded silica is used as a filler; the proportion is 60-80: acetonitrile-0.01% ammonium acetate solution of 20-40% is mobile phase; the detection wavelength is 265nm;
(2) Preparation of a control solution: taking a proper amount of quercetin reference substance, precisely weighing, and adding methanol to prepare a solution containing 50 mug per 1 ml;
(3) Preparation of test solution: precisely measuring 1mL of tincture, placing in conical flask with plug, precisely adding 60-80% methanol 20mL, reflux extracting for 20-40 min, cooling, supplementing 20mL with 60-80% methanol, shaking, and filtering to obtain filtrate;
(4) Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.
2. The content detection method according to claim 1, wherein the chromatographic condition and system applicability test in step (1) is: octadecylsilane chemically bonded silica is used as a filler; the proportion is 70-75:25-30 acetonitrile-0.01% ammonium acetate solution as mobile phase; the detection wavelength was 265nm.
3. The content detection method according to claim 2, wherein the chromatographic condition and system applicability test in step (1) is: octadecylsilane chemically bonded silica is used as a filler; the proportion is 73:27 in acetonitrile-0.01% ammonium acetate as mobile phase; the detection wavelength was 265nm.
4. The content detection method according to claim 1, wherein the preparation of the sample solution in step (3) is: precisely measuring 1mL of tincture, placing in a conical flask with a plug, precisely adding 70% methanol 20mL, reflux-extracting for 30min, cooling, supplementing 20mL with 70% methanol, shaking, and filtering to obtain subsequent filtrate.
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