CN110339298B - Traditional Chinese medicine composition for external use for treating chloasma, preparation method and preparation - Google Patents
Traditional Chinese medicine composition for external use for treating chloasma, preparation method and preparation Download PDFInfo
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- CN110339298B CN110339298B CN201910678500.3A CN201910678500A CN110339298B CN 110339298 B CN110339298 B CN 110339298B CN 201910678500 A CN201910678500 A CN 201910678500A CN 110339298 B CN110339298 B CN 110339298B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/618—Molluscs, e.g. fresh-water molluscs, oysters, clams, squids, octopus, cuttlefish, snails or slugs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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Abstract
The invention discloses a traditional Chinese medicine composition for treating chloasma, a preparation method and a preparation, and belongs to the technical field of traditional Chinese medicine preparations. A traditional Chinese medicine composition for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 145-155 parts of rhizoma bletillae root, 45-55 parts of ginseng, 95-105 parts of bighead atractylodes rhizome root, 95-105 parts of radix ampelopsis, 95-105 parts of poria cocos, 195-205 parts of white gourd seed, 45-55 parts of salvia miltiorrhiza, 95-105 parts of pearl powder and 5-15 parts of ganoderma sinensis. The Chinese medicinal composition has obvious curative effect on chloasma, and can effectively improve related symptoms of the chloasma; has the effects of inhibiting B16 cell proliferation, tyrosinase activity and melanin, and further shows that the traditional Chinese medicine composition has the effect of improving chloasma. The traditional Chinese medicine composition disclosed by the invention is low in medicine cost, simple in preparation method and suitable for industrial production.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a traditional Chinese medicine composition for external use for treating chloasma, a preparation method and a preparation.
Background
Chloasma is a facial brownish or black pigmented dermatosis formed by increasing skin melanin, and is one of dermatological difficult and complicated diseases.
Modern medicine considers that the pathogenesis of chloasma is related to the stimulation of skin melanocytes by the increase of progesterone and estrogen secretion in bodies, and is related to factors such as ultraviolet irradiation, cosmetics, medicines, pregnancy, diseases, heredity and the like. The brown spot disease has long course and is stubborn and difficult to treat, and the prior instrument and drug therapy needs patients to take effect after long-term adherence, the process is complex, the patients suffer pain, and the curative effect is repeated; the effective medicines for treating the chloasma are few, the effect is not ideal, and the treatment of symptoms and root causes by removing the freckle by the beauty and whitening cosmetics is not permanent. According to long-term clinical experience and the theory of traditional Chinese medicine, the pathogenesis of chloasma is mainly the deficiency of spleen and kidney, qi and blood cannot nourish the head and face, face cannot nourish and generate spots, the traditional Chinese medicine therapy can prepare the medicine into a dosage form which can be used by a patient at home for a long time and is convenient to carry when the patient goes out, and satisfactory curative effect can be obtained after the patient insists on treatment.
Disclosure of Invention
Aiming at the existing problems, the invention provides a traditional Chinese medicine composition for external use for treating chloasma, a preparation method and a preparation, and the obtained traditional Chinese medicine composition or preparation has the effects of inhibiting B16 cell proliferation, tyrosinase activity and melanin, has obvious curative effect on chloasma and can effectively improve related symptoms of the chloasma.
The invention aims to provide a traditional Chinese medicine composition capable of controlling and relieving chloasma caused by facial pigmented spots; another purpose is to provide a preparation method of the traditional Chinese medicine composition; it is a further object to provide a composition for use in formulation.
The technical scheme adopted by the invention is as follows:
a traditional Chinese medicine composition for external use for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 145-155 parts of bletilla striata root, 45-55 parts of ginseng, 95-105 parts of bighead atractylodes rhizome root, 95-105 parts of radix ampelopsis, 95-105 parts of poria cocos, 195-205 parts of white gourd seed, 45-55 parts of salvia miltiorrhiza, 95-105 parts of pearl powder and 5-15 parts of ganoderma sinensis.
Further, the traditional Chinese medicine composition for external use for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 150-155 parts of bletilla striata root, 50-55 parts of ginseng, 95-100 parts of bighead atractylodes rhizome root, 95-100 parts of ampelopsis japonica, 95-100 parts of poria cocos, 200-205 parts of white gourd seed, 50-55 parts of salvia miltiorrhiza, 95-100 parts of pearl powder and 10-15 parts of ganoderma sinensis.
Further, the traditional Chinese medicine composition for external use for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 150 parts of bletilla striata root, 50 parts of ginseng, 100 parts of atractylodes macrocephala root, 100 parts of ampelopsis japonica, 100 parts of poria cocos, 200 parts of wax gourd seed, 50 parts of salvia miltiorrhiza, 100 parts of pearl powder and 10 parts of ganoderma sinensis.
Further, the preparation method of the traditional Chinese medicine composition for external use for treating chloasma comprises the following steps:
s1, adding water into the traditional Chinese medicine raw materials in parts by weight according to the volume of 8-20 times, soaking for 10-15 min, then performing reflux extraction for 30-120 min, extracting for 1-4 times, filtering, collecting filtrate, performing reduced pressure concentration at 70-80 ℃ to 0.5g/ml, standing at 4 ℃ overnight, and taking supernatant;
s2, adding a flocculating agent into the supernatant at the temperature of 30-60 ℃, flocculating for 20-50 min, filtering the filtrate through a 0.45um membrane to remove impurities, diluting to 0.08-0.12 g/ml to obtain a water extract, and storing the water extract at the temperature of 4-6 ℃;
s3, performing rotary evaporation on the water extract at 80-90 ℃ until the water extract is dried to obtain a water extract sample, and then adding sterile water to prepare a preparation with the concentration of 8-10 mg/mL; filtering with 0.22um fiber filter membrane, and storing at-20 deg.C.
Further, the addition amount of the flocculant is 0.05-0.15% of the mass of the supernatant.
A preparation for external use for treating chloasma comprises the traditional Chinese medicine composition and pharmaceutically acceptable auxiliary materials.
Specifically, the auxiliary materials comprise at least one of a filling agent, a disintegrating agent, a lubricating agent, a suspending agent, a binding agent, a sweetening agent, a flavoring agent and a preservative. Wherein the filler comprises at least one of starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose and sucrose; the disintegrant comprises at least one of starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, and cross-linked sodium carboxymethyl cellulose; the lubricant comprises at least one of magnesium stearate, sodium lauryl sulfate, talcum powder and silicon dioxide; the suspending agent comprises at least one of polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, and hydroxypropyl methylcellulose; the adhesive comprises at least one of starch slurry, polyvinylpyrrolidone and hydroxypropyl methyl cellulose; the sweetener comprises at least one of saccharin sodium, aspartame, sucrose, sodium cyclamate, and glycyrrhetinic acid; the flavoring agent comprises at least one of sweetener and essence; the antiseptic comprises at least one of parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroacetidine acetate, and eucalyptus oil.
The invention has the beneficial effects that: the traditional Chinese medicine composition has obvious curative effect on chloasma in the clinical test process, can effectively improve related symptoms of the chloasma, has no adverse reaction in a case, shows that the traditional Chinese medicine composition has higher safety in treating the chloasma, and is worthy of clinical popularization; through cell culture tests, the pharmaceutical composition has the effects of inhibiting B16 cell proliferation, inhibiting tyrosinase activity and inhibiting melanin, and further shows that the traditional Chinese medicine composition has the effect of improving chloasma; animal experiments show that no obvious erythema and edema, no skin allergy and no allergy are found after the traditional Chinese medicine composition is used. The traditional Chinese medicine composition has simple composition, low medication cost and simple preparation method, and is suitable for industrial production.
Detailed Description
The embodiments of the present invention can be obtained by different substitutions in specific ranges based on the above technical solutions, and therefore, the following embodiments are only preferred embodiments of the embodiments, and any technical substitutions made by the above technical solutions are within the protection scope of the present invention.
Example 1
A traditional Chinese medicine composition for external use for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 145 parts of bletilla striata root, 45 parts of ginseng, 95 parts of atractylodes macrocephala root, 95 parts of ampelopsis japonica, 95 parts of poria cocos, 195 parts of wax gourd seed, 45 parts of salvia miltiorrhiza, 95 parts of pearl powder and 5 parts of ganoderma sinensis.
Example 2
A traditional Chinese medicine composition for external use for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 146 parts of bletilla striata root, 46 parts of ginseng, 96 parts of atractylodes macrocephala root, 96 parts of ampelopsis japonica, 96 parts of poria cocos, 197 parts of wax gourd seed, 47 parts of salvia miltiorrhiza, 97 parts of pearl powder and 7 parts of ganoderma sinensis.
Example 3
A traditional Chinese medicine composition for external use for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 148 parts of bletilla striata root, 48 parts of ginseng, 98 parts of atractylodes macrocephala root, 98 parts of ampelopsis japonica, 98 parts of poria cocos, 198 parts of wax gourd seed, 48 parts of salvia miltiorrhiza, 98 parts of pearl powder and 8 parts of ganoderma sinensis.
Example 4
A traditional Chinese medicine composition for external use for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 150 parts of bletilla striata root, 50 parts of ginseng, 100 parts of atractylodes macrocephala root, 100 parts of ampelopsis japonica, 100 parts of poria cocos, 200 parts of wax gourd seed, 50 parts of salvia miltiorrhiza, 100 parts of pearl powder and 10 parts of ganoderma sinensis.
Example 5
A traditional Chinese medicine composition for external use for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 152 parts of bletilla striata root, 52 parts of ginseng, 102 parts of atractylodes macrocephala root, 102 parts of ampelopsis japonica, 102 parts of poria cocos, 203 parts of wax gourd seed, 53 parts of salvia miltiorrhiza, 102 parts of pearl powder and 12 parts of ganoderma sinensis.
Example 6
A traditional Chinese medicine composition for external use for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 153 parts of bletilla striata root, 54 parts of ginseng, 97 parts of atractylodes macrocephala root, 97 parts of ampelopsis japonica, 97 parts of poria cocos, 203 parts of wax gourd seed, 53 parts of salvia miltiorrhiza, 97 parts of pearl powder and 13 parts of ganoderma sinensis.
Example 7
A traditional Chinese medicine composition for external use for treating chloasma comprises the following traditional Chinese medicine raw materials in parts by weight: 155 parts of bletilla striata root, 55 parts of ginseng, 105 parts of atractylodes macrocephala root, 105 parts of ampelopsis japonica, 105 parts of poria cocos, 205 parts of wax gourd seed, 55 parts of salvia miltiorrhiza, 105 parts of pearl powder and 15 parts of ganoderma sinensis.
Example 8
A preparation method of a traditional Chinese medicine composition for external use for treating chloasma comprises the following steps:
s1, taking 150g of bletilla striata root, 50g of ginseng, 100 g of bighead atractylodes rhizome root, 100 g of ampelopsis japonica, 100 g of poria cocos, 200 g of white gourd seed, 50g of salvia miltiorrhiza, 100 g of pearl powder and 10g of ganoderma sinensis, adding 11kg of water, soaking for 12min, then performing reflux extraction for 60min, extracting for 3 times, filtering, collecting filtrate, concentrating under reduced pressure at 70 ℃ to 0.5g/ml, standing at 4 ℃ overnight, and taking supernatant;
s2, adding a flocculating agent into the supernatant at 50 ℃, wherein the adding amount of the flocculating agent is 0.09% of the mass of the supernatant, flocculating for 40min, filtering the filtrate through a 0.45um membrane to remove impurities, diluting to 0.1g/ml to obtain a water extract, and storing the water extract at 4 ℃;
s3, carrying out rotary evaporation on the water extract at 80 ℃, and carrying out rotary drying to obtain a water extract sample, and then adding sterile water to prepare a preparation with the concentration of 10 mg/mL; filtering with 0.22um fiber filter membrane, and storing at-20 deg.C.
Example 9
A preparation method of a traditional Chinese medicine composition for external use for treating chloasma comprises the following steps:
s1, taking 60g of bletilla striata root, 20 g of ginseng, 40 g of bighead atractylodes rhizome root, 40 g of ampelopsis japonica, 40 g of poria cocos, 80 g of white gourd seed, 20 g of salvia miltiorrhiza, 40 g of pearl powder and 4g of ganoderma sinensis, adding 5kg of water, soaking for 10min, then performing reflux extraction for 80min, extracting for 2 times, filtering, collecting filtrate, concentrating under reduced pressure at 75 ℃ to 0.5g/ml, standing at 4 ℃ overnight, and taking supernatant;
s2, adding a flocculating agent into the supernatant at 30 ℃, wherein the adding amount of the flocculating agent is 0.05 percent of the mass of the supernatant, flocculating for 50min, filtering the filtrate through a 0.45um membrane to remove impurities, diluting to 0.08g/ml to obtain water extract, and storing the water extract at 5 ℃;
s3, carrying out rotary evaporation on the water extract at 85 ℃ until the water extract is dried to obtain a water extract sample, and then adding sterile water to prepare a preparation with the concentration of 8 mg/mL; filtering with 0.22um fiber filter membrane, and storing at-20 deg.C.
Example 10
A preparation method of a traditional Chinese medicine composition for external use for treating chloasma comprises the following steps:
s1, taking 145g of bletilla striata root, 45g of ginseng, 95 g of bighead atractylodes rhizome root, 95 g of Japanese ampelopsis root, 95 g of poria cocos, 195 g of white gourd seed, 45g of salvia miltiorrhiza, 95 g of pearl powder and 5g of ganoderma sinensis, adding 16kg of water, soaking for 15min, then performing reflux extraction for 120min, extracting for 4 times, filtering, collecting filtrate, concentrating under reduced pressure at 80 ℃ to 0.5g/ml, standing at 4 ℃ overnight, and taking supernatant;
s2, adding a flocculating agent into the supernatant at 60 ℃, wherein the adding amount of the flocculating agent is 0.15 percent of the mass of the supernatant, flocculating for 20min, filtering the filtrate through a 0.45um membrane to remove impurities, diluting to 0.12g/ml to obtain water extract, and storing the water extract at 6 ℃;
s3, carrying out rotary evaporation on the water extract at 90 ℃ until the water extract is dried to obtain a water extract sample, and then adding sterile water to prepare a preparation with the concentration of 9 mg/mL; filtering with 0.22um fiber filter membrane, and storing at-20 deg.C.
Example 11
A preparation method of a traditional Chinese medicine composition for external use for treating chloasma comprises the following steps:
s1, taking 60g of bletilla striata root, 20 g of ginseng, 40 g of bighead atractylodes rhizome root, 40 g of ampelopsis japonica, 40 g of poria cocos, 80 g of white gourd seed, 20 g of salvia miltiorrhiza, 40 g of pearl powder and 4g of ganoderma sinensis, adding 2.75kg of water, soaking for 10min, then performing reflux extraction for 30min, filtering, collecting filtrate, concentrating under reduced pressure at 75 ℃ to 0.5g/ml, standing at 4 ℃ overnight, and taking supernatant;
s2, adding a flocculating agent into the supernatant at 30 ℃, wherein the adding amount of the flocculating agent is 0.05 percent of the mass of the supernatant, flocculating for 50min, filtering the filtrate through a 0.45um membrane to remove impurities, diluting to 0.08g/ml to obtain water extract, and storing the water extract at 5 ℃;
s3, carrying out rotary evaporation on the water extract at 85 ℃ until the water extract is dried to obtain a water extract sample, and then adding sterile water to prepare a preparation with the concentration of 8 mg/mL; filtering with 0.22um fiber filter membrane, and storing at-20 deg.C.
Example 12
A preparation for external use for treating chloasma comprises the following traditional Chinese medicine raw materials: 150g of bletilla striata root, 50g of ginseng, 100 g of atractylodes macrocephala root, 100 g of ampelopsis japonica, 100 g of poria cocos, 200 g of wax gourd seed, 50g of salvia miltiorrhiza, 100 g of pearl powder and 10g of ganoderma sinensis; the preparation method of the traditional Chinese medicine composition is the same as that in the embodiment 8, and pharmaceutically acceptable auxiliary materials are added into the prepared traditional Chinese medicine composition to obtain the preparation.
Example 13
A preparation for external use for treating chloasma comprises the following traditional Chinese medicine raw materials: taking 60g of bletilla striata root, 20 g of ginseng, 40 g of atractylodes macrocephala root, 40 g of ampelopsis japonica, 40 g of poria cocos, 80 g of wax gourd seed, 20 g of salvia miltiorrhiza, 40 g of pearl powder and 4g of ganoderma sinensis; the preparation method of the traditional Chinese medicine composition is the same as that in example 9, and pharmaceutically acceptable auxiliary materials are added into the prepared traditional Chinese medicine composition to obtain the preparation.
Example 14
A preparation for external use for treating chloasma comprises the following traditional Chinese medicine raw materials: taking 145g of bletilla striata root, 45g of ginseng, 95 g of atractylodes macrocephala root, 95 g of ampelopsis japonica, 95 g of poria cocos, 195 g of wax gourd seed, 45g of salvia miltiorrhiza, 95 g of pearl powder and 5g of ganoderma sinensis; the preparation method of the traditional Chinese medicine composition is the same as that in the embodiment 10, and pharmaceutically acceptable auxiliary materials are added into the prepared traditional Chinese medicine composition to obtain the preparation.
Example 15
In order to investigate the clinical efficacy and safety of the drug of the invention, the study takes the traditional Chinese medicine composition obtained in the examples as the drug research object, namely the drug; the safety evaluation is carried out by animal experiments.
(1) Acute oral toxicity test
30 KM mice were selected and used for male and female.
Before the test: the experimental animals were fasted overnight without restriction of drinking water.
And (3) formal test: weighing animals, randomly grouping, and dividing into normal control group, once-daily administration group, and three-daily administration group, and orally intragastrically administering.
The administration mode comprises the following steps: the administration was carried out once by gavage at a dose of 0.4ml of the preparation per 10g of body weight.
Recording and observing: recording the toxic manifestations and death status of each animal; and observing for 14d, weighing the stored live animals three times per week in the observation period, weighing the live animals at the end of the observation period, and performing necropsy after the animals are sacrificed.
As a result: the mice administered with the drug have no toxic reaction and death; after the observation period, all mice had no pathological changes in organs. The maximum tolerance of the acute oral administration of the medicament is more than 120ml of medicament per kg of body weight, and the toxicity level of the medicament can be judged to be actually nontoxic. The weight gain of the mice in the observation period was not different from that of the normal control group, and is shown in Table 1.
(2) Acute transdermal toxicity test
20 guinea pigs were divided into normal control group and administration group.
Before the test: 24h before the start of the test, the hair of the infected area of the guinea pig trunk back is shaved, and the length of the hair is about 46 cm2～54 cm2。
And (3) formal test: animals were weighed, randomly divided into normal control groups, and once daily dosing groups.
The administration mode comprises the following steps: the skin smearing mode is characterized in that the medicine is evenly coated on a skin poisoning area on the back of a guinea pig and then covered by a thin film, and the medicine is fixed by a non-irritant adhesive plaster to prevent animals from licking.
And (4) observation: observing the animal for 14 days, weighing the stored living animals every week during the observation period, weighing the stored living animals after the observation period is finished, killing the animals, and performing necropsy.
As a result: no toxic reaction and death condition exist when the guinea pigs are administrated; after the observation period, all guinea pigs were necropsied and there was no pathological change in organs. The maximum acute percutaneous tolerance of the medicament is more than 2.0ml medicament/kg body weight, and the toxicity level of the medicament can be judged to be actually nontoxic. The weight gain of the guinea pigs was not different from that of the normal control group during the observation period, as shown in Table 2.
(3) Skin irritation/Corrosion test
Acute skin irritation test
8 white rabbits were bred in a single cage and divided into a control group and a control group.
Before the test: adaptation to the experimental animal room environment for 3 d; the hair on both sides of the spine of the experimental animal is cut off about 24 hours before the experiment, the epidermis cannot be damaged, and the hair removing range is about 3cm multiplied by 3cm on the left and the right respectively.
The administration mode comprises the following steps: applying 0.5ml of the preparation directly onto skin, covering with two layers of gauze (2.5 cm × 2.5 cm) and a layer of cellophane or the like, and fixing with non-irritating adhesive plaster and bandage. The other side of the skin served as a control.
And (3) testing and observing: sealing test is adopted, and the application time is 4 h; and observing skin reactions of the smearing parts at 1h, 24h, 48h and 72h after the medicament is removed, grading the skin reactions, comprehensively evaluating by using the average value of the integrals of the tested animals, and judging the skin irritation intensity according to the highest integral average value of observation time points of 24h, 48h and 72 h.
As a result: the result of the test of acute irritation of the medicament to the rabbit skin shows that the rabbit skin of 4 sample groups has no erythematous inflammation and edema reaction, which indicates that the medicament has no acute skin irritation.
② multiple skin irritation test
8 white rabbits were bred in a single cage and divided into a control group and a control group.
Before the test: adaptation to the experimental animal room environment for 3 d; the hair on both sides of the spine of the experimental animal is cut off about 24 hours before the experiment, the epidermis cannot be damaged, and the hair removing range is about 3cm multiplied by 3cm on the left and the right respectively.
The administration mode comprises the following steps: the application area is 2.5cm × 2.5cm, and 0.5ml of the preparation is applied to one side of skin, and the other side is coated with distilled water as control, and applied for 14 days continuously for 1 time per day. Shearing hair from the next day before each smearing, and removing residual medicament with water; the results were observed after one hour, integrated; the control group and the test group were treated in the same manner.
As a result: the skin of 4 rabbits in the sample group shows slight erythema reaction without edema inflammatory reaction, and the average stimulation reaction integral mean value of each animal per day is 0.125, which indicates that the medicament has no skin irritation.
(4) Acute eye irritation/corrosivity test
3 white rabbits were bred in a single cage.
Before the test: adaptation to the experimental animal room environment for 3 d; the lower eyelid of the rabbit's eye was gently pulled open, and 0.1 mL (100 mg) of the drug was dropped into the conjunctival sac to passively close the upper and lower eyelids for 1 s. The other eye was not treated for self-control. The eyes are not washed within 24 hours after the medicine is dropped. The eyes of the animals were examined 1h, 24h, 48h, 72h, and 4d and 7d after instillation of the test substance. If no stimulus response occurs for 72 hours, the test is terminated.
As a result: no damage to cornea, iris and conjunctiva was seen in eyes of 3 rabbits, indicating that the preparation has no acute eye irritation.
(5) Skin allergy test
20 white guinea pigs, half male and half female, were taken and animals were randomly divided into test and control groups.
Before the test: acclimating in a laboratory environment for at least 3 days; the hair of the left back of guinea pig is removed 24h before the test, and the hair removing range is 4cm2～6cm2。
And (3) induction contact: applying 0.2ml of the preparation on the skin of the hair-removed area, covering with two layers of gauze and one layer of cellophane, sealing and fixing with non-irritating adhesive plaster for 6h, and repeating the same method at 7d and 14 d.
And (3) exciting contact: 14-28 days after the last induction, 0.2ml of the medicament is applied to a hair removal area of 2cm multiplied by 2cm on the right side of the back of the guinea pig (24 hours before contact for hair removal), then two layers of gauze and a layer of cellophane are used for covering, and then the guinea pig is fixed by a non-irritant adhesive plaster for 6 hours. The negative control group was coated with distilled water alone as a control at the time of induction contact and with the test substance at the time of challenge contact. Skin reactions were observed 24h and 48h after challenge contact and scored.
As a result: no obvious erythema and edema were observed on the skin of 20 guinea pigs in the test group, indicating no skin allergy and no allergy to the drug.
Example 16
In order to investigate the clinical efficacy and safety of the drug of the invention, the study takes the traditional Chinese medicine composition obtained in the examples as the drug research object, namely the drug; the clinical efficacy of the composition is evaluated by animal experiments.
According to clinical diagnosis and curative effect standard of chloasma (revised draft in 2003) adopted by pigment pathology group of the professional society for skin diseases of Chinese medical society of Chinese and western medicine in 2003 through years of clinical practice and discussion.
The chloasma can be diagnosed when the composition meets the following five conditions:
(1) the face is light brown to dark brown, and the clear spot is generally distributed symmetrically without inflammation and scales; (2) no obvious subjective symptoms; (3) women are frequent, mainly after puberty; (4) the disease condition can be seasonal, and the disease condition is severe in summer and mild in winter; (5) excluding pigmentation caused by other diseases (such as naevus bruguinus, Riehl melanosis, pigmented actinic lichen planus, etc.).
The patients who included the cases: the patients with the chloasma characteristics, female patients with the age of 20-45 years old, patients with primary diseases such as cardiovascular diseases, cerebrovascular diseases, liver diseases, kidney diseases, hemopoietic systems and the like, psychotic patients, malignant tumor patients, allergic constitution, patients allergic to various medicines and patients who have been systemically treated with pigmented spots within 3 months are excluded.
All the cases in the study are chloasma crowds, the included cases meet the diagnosis standard of chloasma patients, all the patients obtain complete follow-up visits, and 20 female patients are counted, wherein the minimum age is 22 years old, the maximum age is 39 years old, and the average age is 27.7 +/-4.39 years old.
The test method comprises the following steps: the medicine is used after cleaning the facial skin in the morning and evening, and is directly smeared on the face.
The evaluation method of the curative effect comprises the following steps: the comprehensive integral before and after treatment of the test group is compared, the comprehensive integral reduction rate is calculated to be used as a curative effect evaluation index, and the clinical curative effect evaluation is carried out according to 4 grades of clinical recovery, obvious effect, effective effect and ineffective effect. Integral of skin loss = integral of skin loss area + integral of skin loss color degree
Therapeutic efficacy evaluation index (nimodipine method) = (before treatment integrated point-after treatment integrated point) ÷ before treatment integrated point × 100%
According to the pigment pathology group of the skin disease professional committee of the Chinese society of Chinese and western medicine, "clinical diagnosis and curative effect standard of chloasma (revision of 2003"), the clinical recovery is as follows: the area of the visual stain is faded by more than 90 percent by naked eyes, the color basically disappears, and the curative effect evaluation index is more than or equal to 0.8; the effect is shown: the area of the eye-color spot is faded to be more than 60 percent, the color is obviously lightened, and the curative effect evaluation index is more than or equal to 0.5; the method has the following advantages: the area of the eye-color spot is faded by more than 30 percent, the color is lightened, and the curative effect evaluation index is more than or equal to 0.3; and (4) invalidation: the area of the eye-color spot is reduced by less than 30%, the color change is not obvious, and the evaluation index of the curative effect is less than 0.3.
And (3) safety evaluation: level 1: safe and has no adverse reaction; and 2, stage: is safe, has mild adverse reaction, and can continue treatment without any treatment; and 3, level: has the problems of safety and moderate adverse reaction, and can continue to treat the disease after being treated.
The statistical method comprises the following steps: statistical analysis was performed in a sps 20. All data are mean ± sd ((S))
) Showing that the measured data are compared by using paired T test; with P>0.5 is no statistical significance for the difference, P<A difference of 0.5 is statistically significant.
Clinical observation indexes and scoring standards: refer to the pigment disease Committee of the national institute of Chinese and western medicine (Gentle for skin diseases) group, "clinical diagnosis and therapeutic criteria for chloasma (revised draft in 2003)"; the therapeutic effect index was calculated by observing the degree of facial pigmentation spots and the area of skin lesions, and was recorded 1 time each at 4 weeks, 8 weeks, and 12 weeks before treatment, and the therapeutic effects are shown in tables 3, 4, and 5.
The P value is less than 0.05 in the aspect of comprehensive integral improvement score of the chloasma skin on the face by external treatment of the medicine for 12 weeks, and statistical differences exist, which shows that the traditional Chinese medicine composition has obvious curative effect on chloasma and can better improve related symptoms of the chloasma. In the experimental process, adverse reactions do not appear in 20 observed cases, which shows that the traditional Chinese medicine composition has higher safety in treating the disease and is worthy of clinical popularization.
Example 17
The research on the in-vitro whitening effect of the traditional Chinese medicine composition is carried out by adopting a cell culture mode; the sample determination operation is carried out under the aseptic condition, and all the used instruments are aseptic; the traditional Chinese medicine composition is used as a medicine sample.
Cell culture: the cells were purchased from the institute of Chinese academy of technology; the culture medium is DMEM high-sugar culture medium 90% (containing 1 × non-essential amino acids, 2mmol/L glutamine, 1mmol/L sodium pyruvate, 1.5 g/L sodium bicarbonate) + fetal calf serum 10%; sucking the original culture solution of B16 cells in logarithmic phase, adding 0.05% trypsin-0.53 mmol/L EDTA for digestion, adding fresh culture solution for repeatedly beating after the cells are blown off by the culture solution for 2-5 min, mixing, and passaging at a ratio of 1: 4.
Cell plating: sucking the cells in logarithmic phase out of the original culture solution, adding 0.05% trypsin-0.53 mmol/L EDTA for digestion, and waiting for 2-5 min until the cells can be blown off by the culture solution. Adding fresh culture solution, repeatedly beating, mixing, and transferring into penicillin bottle. Add 20. mu.L of cell suspension to the EP tube, add 180. mu.L of Trypan blue solution, and mix well. And (5) microscopic examination and counting under a microscope.
Taking a proper amount of cell suspension to a 50mL glass bottle, filling the volume to 18mL by using fresh culture solution, adding 180 mu L of diluted cell suspension into each hole of a 96-hole cell culture plate to ensure that the number of cells in each hole is 6 multiplied by 103Cell plate placed with CO2Incubate overnight in the incubator.
Effect on B16 cell proliferation
Taking 2 mu L of sample, thawing at room temperature, shaking for dissolution, mixing uniformly to obtain a sample mother solution, setting 5 sample concentration gradients, and repeating each gradient for 3 times. Samples were added as designed and supplemented to 20 μ L with PBS in 96 well cell culture plates to give final concentrations of 12.5, 25, 50, 75, 100 μ g/mL, respectively. Adding 20 μ LPBS into blank control well, mixing, placing CO on cell plate2Incubating for 48h in an incubator; 4 hours before the cell culture, the cell plate was removed, the culture medium was aspirated, 100. mu.L of PBS was added, 10. mu.L of MTT solution (5 mg/mL) was added, the mixture was mixed well, and CO was added2After incubation for 4-6 h in an incubator, 100. mu.L of SDS solution is added into each hole, and the cell plate is placed in CO2Incubate overnight in the incubator. OD values were measured at 570nm using a microplate reader, and the results are shown in Table 6.
Effect on tyrosinase Activity of B16 cells
After cell plating, adding theophylline with the final concentration of 1mmol/L into each hole of cells for induction, taking 2uL of sample, unfreezing at room temperature, shaking for dissolution, and uniformly mixing to obtain a sample, setting 5 sample concentration gradients, and repeating each gradient for 3 times. Samples were added as designed and supplemented to 20 μ L with PBS in 96 well cell culture plates to give final concentrations of 12.5, 25, 50, 75, 100 μ g/mL, respectively. Add 20. mu.L PBS to blank control well, mix well, Place cell plate in CO2Incubate in incubator for 48 h. After the cell culture is finished, taking out the cell culture plate, sucking out the culture solution, adding 100uL of 1% Triton X-100 solution, and standing at-80 ℃ for 60 min. The cell plate is taken out of the cell plate,after returning to room temperature, 10uL of the suspension was used for protein content determination by BCA method, and another 80uL of the suspension was added with 50uL of 0.1% L-Dopa solution and incubated at 37 ℃ for 2 h. OD was measured at 490nm using a microplate reader. The results are shown in Table 7.
Effect on melanogenesis in B16 cells
After cell plating, adding theophylline with the final concentration of 1mmol/L into each hole of cells, taking 2uL of sample, unfreezing at room temperature, shaking for dissolution, and mixing uniformly to obtain a sample, setting a concentration gradient of 3 samples, adding the sample into a 96-hole cell culture plate, and supplementing the sample to 20 mu L with PBS so that the final concentration of the sample is 25, 50 and 75 mu g/ml respectively. Adding 20 μ LPBS into blank control well, mixing, placing CO on cell plate2Incubate in incubator for 72 h. After the cell culture was completed, the cell plate was taken out, the culture solution was aspirated, washed twice with PBS, and 50. mu.l of 1mol/L NaOH was added thereto, followed by standing at 37 ℃ for 60 min. OD was measured at 460nm using a microplate reader. The results are shown in Table 8.
TABLE 1 drug acute oral toxicity test mice weight gain
TABLE 2 medicament acute transdermal toxicity test weight gain in guinea pigs
TABLE 3 comparison of the before-treatment and after-treatment 4-week skin lesions integration score: (
） n=20
By T test, P is more than 0.05, and the difference has no statistical significance.
TABLE 4 comparison of pre-treatment and post-treatment 8-week skin lesion score improvement scores: (
） n=20
By T test, P is more than 0.05, and the difference has no statistical significance.
TABLE 5 comparison of the pre-treatment and post-treatment 12-week skin lesion score improvement scores: (
） n=20
By T test, P is less than 0.05, and the difference has statistical significance.
TABLE 6 inhibitory Effect (%) of the agents on B16 cell proliferation
TABLE 7 inhibition of B16 cell tyrosinase Activity (%)
TABLE 8 inhibitory Effect (%) of the agents on melanin production by B16 cells
Through three experimental studies of inhibiting B16 cell proliferation, inhibiting tyrosinase activity and inhibiting melanin generation, the results show that the traditional Chinese medicine composition has a certain effect of inhibiting B16 cell proliferation, can reduce B16 cell tyrosinase activity and inhibit melanin generation, and has a good whitening effect.
The invention extends to any novel feature or any novel combination of features disclosed in this specification and any novel method or process steps or any novel combination of features disclosed.
Claims (6)
1. A traditional Chinese medicine composition for external use for treating chloasma is characterized by comprising the following traditional Chinese medicine raw materials in parts by weight: 145-155 parts of bletilla striata root, 45-55 parts of ginseng, 95-105 parts of bighead atractylodes rhizome root, 95-105 parts of radix ampelopsis, 95-105 parts of poria cocos, 195-205 parts of white gourd seed, 45-55 parts of salvia miltiorrhiza, 95-105 parts of pearl powder and 5-15 parts of ganoderma sinensis.
2. The traditional Chinese medicine composition for external use for treating chloasma as claimed in claim 1, which is prepared from the following traditional Chinese medicine raw materials in parts by weight: 150-155 parts of bletilla striata root, 50-55 parts of ginseng, 95-100 parts of bighead atractylodes rhizome root, 95-100 parts of ampelopsis japonica, 95-100 parts of poria cocos, 200-205 parts of white gourd seed, 50-55 parts of salvia miltiorrhiza, 95-100 parts of pearl powder and 10-15 parts of ganoderma sinensis.
3. The traditional Chinese medicine composition for external use for treating chloasma as claimed in claim 1, which is prepared from the following traditional Chinese medicine raw materials in parts by weight: 150 parts of bletilla striata root, 50 parts of ginseng, 100 parts of atractylodes macrocephala root, 100 parts of ampelopsis japonica, 100 parts of poria cocos, 200 parts of wax gourd seed, 50 parts of salvia miltiorrhiza, 100 parts of pearl powder and 10 parts of ganoderma sinensis.
4. The preparation method of the traditional Chinese medicine composition for external use for treating chloasma as claimed in any one of claims 1 to 3, comprising the following steps:
s1, adding water into the traditional Chinese medicine raw materials in parts by weight according to the volume of 8-20 times, soaking for 10-15 min, then performing reflux extraction for 30-120 min, extracting for 1-4 times, filtering, collecting filtrate, performing reduced pressure concentration at 70-80 ℃ to 0.5g/ml, standing at 4 ℃ overnight, and taking supernatant;
s2, adding a flocculating agent into the supernatant at the temperature of 30-60 ℃, flocculating for 20-50 min, filtering the filtrate through a 0.45um membrane to remove impurities, diluting to 0.08-0.12 g/ml to obtain a water extract, and storing the water extract at the temperature of 4-6 ℃;
s3, performing rotary evaporation on the water extract at 80-90 ℃ until the water extract is dried to obtain a water extract sample, and then adding sterile water to prepare a preparation with the concentration of 8-10 mg/mL; filtering with 0.22um fiber filter membrane, and storing at-20 deg.C.
5. The preparation method of the traditional Chinese medicine composition for external use for treating chloasma according to claim 4, wherein the addition amount of the flocculating agent is 0.05-0.15% of the mass of the supernatant.
6. A preparation for external use for treating chloasma, which is characterized by comprising the traditional Chinese medicine composition of claim 1 and pharmaceutically acceptable auxiliary materials.
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| 黄褐斑那么多该怎样去掉?分享去黄褐斑的有效方法;赤坂泽;《http://k.sina.com.cn/article_5282079824_13ad6245000100299l.html》;20171220;第1页，尤其是第4-8行 * |
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