WO2016095087A1 - Uses of chlorogenic acid in preparation of medicines for treating vitiligo - Google Patents

Uses of chlorogenic acid in preparation of medicines for treating vitiligo Download PDF

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WO2016095087A1
WO2016095087A1 PCT/CN2014/093854 CN2014093854W WO2016095087A1 WO 2016095087 A1 WO2016095087 A1 WO 2016095087A1 CN 2014093854 W CN2014093854 W CN 2014093854W WO 2016095087 A1 WO2016095087 A1 WO 2016095087A1
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chlorogenic acid
preparation
group
model
vitiligo
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PCT/CN2014/093854
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French (fr)
Chinese (zh)
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张洁
朱丽娜
黄望
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四川九章生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate

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  • the present invention relates to the use of chlorogenic acid for the preparation of a medicament for the treatment of vitiligo.
  • Chlorogenic acid also known as coffee tannin, chemically known as 3-0-caffeoylquinic acid, is derived from caffeic acid and quinic acid.
  • Quinic acid consists of a carboxylic acid.
  • Chlorogenic acid is a product of aerobic respiration metabolism in plants. It is the main active ingredient in many Chinese herbal medicines and fruits and vegetables. It has many biological activities, such as cardiovascular protection, anti-oxidation, anti-ultraviolet and anti-radiation effects. Anti-mutagenic and anti-cancer effects, antibacterial effects, antiviral effects, lipid-lowering and hypoglycemic effects, immunomodulatory effects, etc. It has a wide range of applications in the fields of pharmaceutical, chemical and food.
  • the technical solution of the present invention is to provide the use of chlorogenic acid in the preparation of a medicament for treating vitiligo.
  • the invention provides the use of chlorogenic acid for the preparation of a medicament for treating vitiligo.
  • the drug is a drug for reducing the expression of TNF- ⁇ and IL-4 at the lesion.
  • the drug is a drug that promotes melanocyte production.
  • the medicament is prepared by adding chlorogenic acid as an active ingredient, adding a pharmaceutically acceptable adjuvant or an auxiliary component.
  • the preparation is an oral preparation, an injection preparation or a transdermal preparation for external use.
  • the preparation is used in a dose of 1-100 mg/kg. Further preferably, the preparation is administered in a dose of from 1 to 30 mg/kg.
  • the test of the invention shows that chlorogenic acid can improve the pathological changes of the vitiligo model, reduce the expression of TNF- ⁇ and IL-4 in the lesion, promote the formation of melanocytes, and provide a theoretical basis for the clinical treatment of vitiligo.
  • Example 1 In vivo pharmacodynamic study of chlorogenic acid in the treatment of vitiligo
  • Black guinea pig weighing 300-350g, male and female.
  • Black guinea pigs were taken, and the area of the back hair was shaved by electric razor by 5 cm ⁇ 5 cm.
  • the body weight was randomly divided into groups of 10, namely, the blank control group (equal volume saline), the model group (equal volume saline), and the positive control.
  • the group (methicillin tablets 2.8 mg/kg), the chlorogenic acid low dose group (15 mg/kg), the chlorogenic acid medium dose group (30 mg kg), and the chlorogenic acid high dose group (60 mg/kg).
  • the blank control group was applied with 0.05 ml of physiological saline in the depilated area, and the other groups were applied with 5% hydroquinone 0.5 ml in the depilatory area twice a day for 11 days to prepare a vitiligo guinea pig model. On the 11th day, each group was administered daily at a dose of 1 hour after the application of hydroquinone, once a day for 50 days.
  • the pigment distribution in the back skin of guinea pigs was visually observed.
  • Judgment criteria (3cm 2 as the observation unit in the center of the guinea pig medication site): the pigment in the test area (3cm 2 ) basically returned to normal; the pigment area in the test area was >50%; The area is ⁇ 50%; the difference is that the skin in the test area is pale or white spotted, and the total effective rate is superior.
  • Each group of guinea pigs were taken 1 cm ⁇ 1 cm, fixed with 10% formaldehyde, embedded in paraffin, sectioned, and melanin stained with ferrous sulfate (Lillie method) to observe the distribution of melanin in epidermal basal cells and spine cells and guinea pigs with melanin in hair follicles. number.
  • Ten high-power fields were observed for each specimen, and the average number of basal cells containing melanin particles per 100 epidermal basal cells was counted.
  • Degree classification means no melanin; " ⁇ ” means occasional melanin; "+” means 30% to 50% of melanin; "++” means 51% to 85% of melanin; "+++” means 85 More than % of melanin.
  • the color of the epidermis of the guinea pigs in the model group was pale and some hairs were whitish. The difference was significant compared with the normal group. After treatment with chlorogenic acid, the effect was obvious. The skin of the guinea pigs was gradually blackened. The color of each drug group was brownish black, which was closer to the normal group, and there was a small amount of pigmentation, which was significantly different from the positive control group. The results are shown in Table 1.
  • the chlorogenic acid 15mg/kg, 30mg/kg, 60mg/kg could significantly increase the skin melanin of the model guinea pig, and significantly increase the distribution of melanin in the epidermal basal cells and spine cells. And melanin in the hair follicles.
  • Table 4 The results are shown in Table 4.
  • chlorogenic acid can significantly increase the skin melanin of experimental vitiligo model guinea pigs, significantly increase the distribution of melanin in skin epidermal basal cells and spine cells, and can significantly increase the melanin in skin hair follicles, and the tyrosinase content in blood is also significant.
  • Increased sexuality and significantly improved blood rheology indicators indicating that chlorogenic acid can increase the melanin production in experimental guinea pig model skin, reduce skin melanin decomposition and improve blood circulation, and play a better therapeutic effect on vitiligo.
  • Example 2 Chlorogenic acid visceral coefficient and TNF- ⁇ in the lesion area of vitiligo in guinea pig model The effects of IL-10, IL-4 and IFN- ⁇ expression
  • Black guinea pig weighing 300-350g, male and female.
  • An experimental vitiligo guinea pig model was prepared using hydroquinone (hydroquinone). Take healthy black guinea pigs, male and female, shave the back hair area by 5cm ⁇ 5cm with an electric razor, apply 5% hydroquinone 0.5ml twice a day in the hair removal area, and apply for 50 days continuously to prepare a vitiligo guinea pig model.
  • hydroquinone hydroquinone
  • the above model guinea pigs were randomly divided into 5 groups: model group, positive group, chlorogenic acid low dose group (15 mg/kg), chlorogenic acid medium dose group (30 mg/kg), and chlorogenic acid high dose group (60 mg). /kg), 10 in each group; another 10 unmodeled guinea pigs were used as blank control group.
  • the guinea pigs of each drug group started to be administered on the 10th day of modeling, and the guinea pigs in the blank control group and the model group were given an equal volume of physiological saline for 50 days.
  • the guinea pigs were sacrificed, and the skin of the drug site was taken at a center of 1 cm ⁇ 1 cm, fixed in neutral formaldehyde, embedded in paraffin, and sectioned. The thymus and spleen of the guinea pig were weighed and the thymus coefficient was calculated.
  • the slices were conventionally dewaxed to water.
  • Heat-repairing antigen 0.01 M citrate buffer (pH 6.0), heat-burned in an electric furnace or microwave oven until boiling, slice immersion, and PBS was washed 1-2 times after cooling. 30% H 2 O 21 parts + 10 parts of distilled water were mixed, and the endogenous enzyme was inactivated at room temperature for 10 minutes. Wash in PBS for 5 minutes ⁇ 3 times. The normal goat immune serum was blocked, incubated at room temperature for 15 minutes, and the serum was decanted without washing. HMB45 antibody diluted 1:100 was added dropwise at 4 ° C overnight. Wash in PBS for 2 minutes ⁇ 3 times.
  • the horseradish enzyme-labeled streptavidin working solution was added dropwise and incubated at 37 ° C for 10 minutes. Wash in PBS for 5 minutes ⁇ 3 times. DAB color development, control reaction time under the microscope, 20s-80s, to satisfactory color development. Wash with distilled water to stop color development. Hematoxylin mildly counterstained, hydrochloric acid alcohol differentiated, rinsed back to blue for 1 minute. Dehydrated, transparent, and sealed. Microscopic observation.
  • Semi-quantitative scoring was performed based on the degree of staining and the percentage of stained cells.
  • Five high-power fields 200 ⁇ were randomly selected from each slice, and the scores were based on the number of positive cells. The score was: 0 points. ⁇ 25% stained cells; 1 point, 25%-50% stained cells; 2 points, 50%-75% stained cells; 3 points, >75% stained cells.
  • the staining degree was as follows: 0 points, no staining; 1 point, light yellow; 2 points, brownish yellow; 3 points of tan.
  • the staining degree is divided into the percentage of the stained cells to obtain the total score, and the score is divided into 4 grades according to the score: 0, negative (-); 2 points, weak positive (+); 3-4 points, positive (++) 5-6 points, strong positive (+++).
  • Statistics were performed using the rank sum test method (Kruskal-Wallis method).
  • the visceral coefficient of the thymus and spleen of the model group was significantly reduced, and the difference was significant compared with the blank control group.
  • the chlorogenic acid group could increase the thymus and spleen and organ coefficient of the model guinea pig, which was significantly different from the model group. It was shown that chlorogenic acid can enhance the immune function of model guinea pigs. See Table 5.
  • TNF- ⁇ positive cells were mainly distributed in the basal layer of the epidermis, spine layer and granular layer. A few positive cells were distributed in the superficial dermis. IL-4 positive cells were mainly distributed in the basal layer and spinous layer of the epidermis, and the control group showed weak positive expression.
  • the expression levels of TNF- ⁇ and IL-4 in the lesion group were significantly higher than those in the normal control group (P ⁇ 0.01).
  • the expression levels of TNF- ⁇ and IL-4 in the lesion group were significantly lower than those in the model group. The difference was significant (P ⁇ 0.01).
  • Chlorogenic acid can improve the pathological changes of vitiligo model, reduce the expression of TNF- ⁇ and IL-4 in skin lesions, promote the formation of melanocytes, and provide a theoretical basis for its clinical treatment of vitiligo.

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Abstract

Provides are uses of chlorogenic acid in the preparation of medicines for treating vitiligo. Experiments show that chlorogenic acid can alleviate pathological changes of a vitiligo model, reduce expression of TNF- and IL-4 in damaged skin, promote generation of melanocytes, and provide a theoretical basis for treating vitiligo in clinic by using chlorogenic acid.

Description

绿原酸在制备治疗白癜风的药物中的用途Use of chlorogenic acid in the preparation of a medicament for treating vitiligo 技术领域Technical field
本发明涉及绿原酸在制备治疗白癜风的药物中的用途。The present invention relates to the use of chlorogenic acid for the preparation of a medicament for the treatment of vitiligo.
背景技术Background technique
绿原酸(Chlorogenic acid,CGA)，又名咖啡单宁酸，化学名为3-0-咖啡酰奎尼酸(3-0-caffeoylquinic acid)，是由咖啡酸(Caffeic acid)与奎尼酸(Quinic acid)组成的羧酚酸。Chlorogenic acid (CGA), also known as coffee tannin, chemically known as 3-0-caffeoylquinic acid, is derived from caffeic acid and quinic acid. (Quinic acid) consists of a carboxylic acid.
绿原酸是植物体有氧呼吸代谢的产物，是许多中药材以及水果蔬菜中的主要有效成分，具有多种生物活性，如:心血管保护作用、抗氧化作用、抗紫外及抗辐射作用、抗诱变及抗癌作用、抗菌作用、抗病毒作用、降脂降糖作用、免疫调节作用等。在医药化工和食品等领域都具有广泛的应用。Chlorogenic acid is a product of aerobic respiration metabolism in plants. It is the main active ingredient in many Chinese herbal medicines and fruits and vegetables. It has many biological activities, such as cardiovascular protection, anti-oxidation, anti-ultraviolet and anti-radiation effects. Anti-mutagenic and anti-cancer effects, antibacterial effects, antiviral effects, lipid-lowering and hypoglycemic effects, immunomodulatory effects, etc. It has a wide range of applications in the fields of pharmaceutical, chemical and food.
发明内容Summary of the invention
本发明的技术方案是提供了绿原酸在制备治疗白癜风的药物中的用途。The technical solution of the present invention is to provide the use of chlorogenic acid in the preparation of a medicament for treating vitiligo.
本发明提供了绿原酸在制备治疗白癜风的药物中的用途。The invention provides the use of chlorogenic acid for the preparation of a medicament for treating vitiligo.
其中，所述的药物是降低皮损处TNF-α和IL-4的表达的药物。Wherein the drug is a drug for reducing the expression of TNF-α and IL-4 at the lesion.
其中，所述的药物是促进黑素细胞生成的药物。Among them, the drug is a drug that promotes melanocyte production.
其中，所述的药物是由绿原酸为有效成分，加入药学上可接受的辅料或辅助性成分制备而成的制剂。Wherein, the medicament is prepared by adding chlorogenic acid as an active ingredient, adding a pharmaceutically acceptable adjuvant or an auxiliary component.
其中，所述的制剂是口服制剂、注射制剂或外用透皮给药制剂。Wherein the preparation is an oral preparation, an injection preparation or a transdermal preparation for external use.
其中，所述的制剂的使用剂量为：1-100mg/kg。进一步优选地，所述的制剂的使用剂量为：1-30mg/kg。Wherein, the preparation is used in a dose of 1-100 mg/kg. Further preferably, the preparation is administered in a dose of from 1 to 30 mg/kg.
本发明试验表明，绿原酸能改善白癜风模型的病理变化，降低皮损处TNF-α和IL-4的表达，促进黑素细胞的生成，为其临床治疗白癜风提供了理论依据。The test of the invention shows that chlorogenic acid can improve the pathological changes of the vitiligo model, reduce the expression of TNF-α and IL-4 in the lesion, promote the formation of melanocytes, and provide a theoretical basis for the clinical treatment of vitiligo.
具体实施方式detailed description
实例1：绿原酸治疗白癜风的体内药效学试验研究Example 1: In vivo pharmacodynamic study of chlorogenic acid in the treatment of vitiligo
1.材料Material
1)动物1) Animals
黑色豚鼠，体重300～350g，雌雄各半。Black guinea pig, weighing 300-350g, male and female.
2)试剂与试验药物2) Reagents and test drugs
对苯二酚、甲氧沙林片、绿原酸；酪氨酸酶、胆碱酯酶(CHE)、单胺 氧化酶(MAO)、丙二醛(MDA)测定试剂盒Hydroquinone, methoxamer tablets, chlorogenic acid; tyrosinase, cholinesterase (CHE), monoamine Oxidase (MAO), malondialdehyde (MDA) assay kit
2.实验方法2. Experimental methods
1)模型制备及分组给药1) Model preparation and group administration
取黑色豚鼠，用电动剃毛刀剃取背部毛面积5cm×5cm，按体重随机分组，每组10只，即空白对照组(等容量生理盐水)、模型组(等容量生理盐水)、阳性对照组(甲氧沙林片2.8mg/kg)、绿原酸小剂量组(15mg/kg)、绿原酸中剂量组(30mgkg)、绿原酸大剂量组(60mg/kg)。其中空白对照组在脱毛区涂抹生理盐水0.05ml，其余各组均在脱毛区涂抹5％氢醌0.5ml，每天2次，连续涂抹11天，制备白癜风豚鼠模型。第11天开始各组分别每天在涂抹氢醌过后1小时按剂量分别给药，每天1次，连续给药50天。Black guinea pigs were taken, and the area of the back hair was shaved by electric razor by 5 cm × 5 cm. The body weight was randomly divided into groups of 10, namely, the blank control group (equal volume saline), the model group (equal volume saline), and the positive control. The group (methicillin tablets 2.8 mg/kg), the chlorogenic acid low dose group (15 mg/kg), the chlorogenic acid medium dose group (30 mg kg), and the chlorogenic acid high dose group (60 mg/kg). The blank control group was applied with 0.05 ml of physiological saline in the depilated area, and the other groups were applied with 5% hydroquinone 0.5 ml in the depilatory area twice a day for 11 days to prepare a vitiligo guinea pig model. On the 11th day, each group was administered daily at a dose of 1 hour after the application of hydroquinone, once a day for 50 days.
2)观察指标及测定2) Observation indicators and determination
①疗效判定1 efficacy judgment
实验结束后，肉眼观察豚鼠背部皮肤色素分布。疗效判断标准(以豚鼠用药部位中心3cm2为一观察单位)：优为受试区(3cm2)色素基本恢复正常；良为受试区出现色素面积＞50％；中为受试区出现色素面积＜50％；差为受试区皮肤呈苍白或白斑状，总有效率以优加良计。At the end of the experiment, the pigment distribution in the back skin of guinea pigs was visually observed. Judgment criteria (3cm 2 as the observation unit in the center of the guinea pig medication site): the pigment in the test area (3cm 2 ) basically returned to normal; the pigment area in the test area was >50%; The area is <50%; the difference is that the skin in the test area is pale or white spotted, and the total effective rate is superior.
②血液流变学测定2 blood rheology determination
末次给药后40min，于腹部主动脉取血，离心得血浆，用于CHE、MAO、MDA、TYR活性的测定，测血液流变学指标(采用YDA-IU血液流变仪测定)。40 min after the last administration, blood was taken from the abdominal aorta, and plasma was centrifuged for measurement of CHE, MAO, MDA, TYR activity, and blood rheology index (measured by YDA-IU blood rheometer).
③皮肤黑色素的测定3 Determination of skin melanin
将各组豚鼠取皮肤1cm×1cm，用10％甲醛固定，常规石蜡包埋切片，用硫酸亚铁(Lillie法)进行黑色素染色，观察表皮基底细胞和棘细胞黑色素分布和毛囊有黑色素的豚鼠个数。每个标本观察10个高倍视野，计算每100个表皮基底细胞中含黑素颗粒的基底细胞的平均数量。程度分级：“-”表示无黑色素；“±”表示偶见黑色素；“+”表示30％～50％有黑色素；“++”表示51％～85％有黑色素；“+++”表示85％以上有黑色素。Each group of guinea pigs were taken 1 cm×1 cm, fixed with 10% formaldehyde, embedded in paraffin, sectioned, and melanin stained with ferrous sulfate (Lillie method) to observe the distribution of melanin in epidermal basal cells and spine cells and guinea pigs with melanin in hair follicles. number. Ten high-power fields were observed for each specimen, and the average number of basal cells containing melanin particles per 100 epidermal basal cells was counted. Degree classification: "-" means no melanin; "±" means occasional melanin; "+" means 30% to 50% of melanin; "++" means 51% to 85% of melanin; "+++" means 85 More than % of melanin.
3.统计学方法3. Statistical methods
所有数据均用SPSS 19.0统计软件处理。All data was processed using SPSS 19.0 statistical software.
4.结果4. Results
1)疗效观察1) Observation of curative effect
肉眼观察模型组豚鼠表皮颜色均明显苍白，部分毛发也发白，与正常组比较差异有显著性，经绿原酸各剂量组治疗后，疗效明显，肉眼观察豚鼠皮肤也逐渐黑化，绿原酸各给药组颜色呈棕黑色，比较接近正常组，并有少量色素沉着，与阳性对照组比较有显著性差异，结果见表1。 The color of the epidermis of the guinea pigs in the model group was pale and some hairs were whitish. The difference was significant compared with the normal group. After treatment with chlorogenic acid, the effect was obvious. The skin of the guinea pigs was gradually blackened. The color of each drug group was brownish black, which was closer to the normal group, and there was a small amount of pigmentation, which was significantly different from the positive control group. The results are shown in Table 1.
表1 绿原酸对白癜风豚鼠模型的疗效观察(3cm2，n＝10)Table 1 Effect of chlorogenic acid on vitiligo guinea pig model (3cm 2 , n=10)
Figure PCTCN2014093854-appb-000001
Figure PCTCN2014093854-appb-000001
注：与模型组比较＊P＜0.05，＊＊P＜0.01Note: Compared with the model group *P<0.05, **P<0.01
2)绿原酸对模型豚鼠血液流变学各指标的影响2) Effects of chlorogenic acid on various indexes of blood rheology in model guinea pigs
造模后，模型组豚鼠全血粘度和血浆粘度明显升高，绿原酸15mg/kg，30mg/kg，60mg/kg能明显降低模型豚鼠的全血粘度和血浆粘度。绿原酸各给药组降低模型豚鼠的全血粘度和血浆粘度明显优于阳性组，结果见表2 After modeling, the whole blood viscosity and plasma viscosity of the model group guinea pigs were significantly increased. Chlorogenic acid 15 mg/kg, 30 mg/kg, 60 mg/kg could significantly reduce the whole blood viscosity and plasma viscosity of the model guinea pigs. The chlorogenic acid treatment group reduced the whole blood viscosity and plasma viscosity of the model guinea pigs significantly better than the positive group. The results are shown in Table 2.
表2  绿原酸对白癜风豚鼠模型的血液流变学各指标的影响
Figure PCTCN2014093854-appb-000002
Figure PCTCN2014093854-appb-000003
Table 2 Effect of chlorogenic acid on various indexes of hemorheology in vitiligo model of vitiligo
Figure PCTCN2014093854-appb-000002
Figure PCTCN2014093854-appb-000003
Figure PCTCN2014093854-appb-000004
Figure PCTCN2014093854-appb-000004
注：与模型组比较＊P＜0.05，＊＊P＜0.01Note: Compared with the model group *P<0.05, **P<0.01
3)绿原酸对模型豚鼠血浆中MAO、CHE、MDA、TYR指标的影响3) Effects of chlorogenic acid on MAO, CHE, MDA and TYR in plasma of model guinea pigs
造模后，模型组豚鼠血浆MAO、CHE及MDA含量明显增加，酪氨酸酶含量明显减少，绿原酸15mg/kg、30mg/kg、60mg/kg豚鼠血浆中MAO、CHE及MDA的含量均明显降低，绿原酸15mg/kg、30mg/kg、60mg/kg能明显增加皮肤黑色素中酪氨酸酶的含量。结果见表3。 After modeling, the contents of MAO, CHE and MDA in the model group were significantly increased, and the content of tyrosinase was significantly decreased. The contents of MAO, CHE and MDA in guinea pig plasma of chlorogenic acid 15mg/kg, 30mg/kg and 60mg/kg were all decreased. Significantly reduced, chlorogenic acid 15mg / kg, 30mg / kg, 60mg / kg can significantly increase the content of tyrosinase in skin melanin. The results are shown in Table 3.
表3  绿原酸对白癜风豚鼠模型的血浆中MAO、CHE、MDA、TYR水平的影响
Figure PCTCN2014093854-appb-000005
Table 3 Effects of chlorogenic acid on the levels of MAO, CHE, MDA and TYR in plasma of vitiligo guinea pig model
Figure PCTCN2014093854-appb-000005
Figure PCTCN2014093854-appb-000006
Figure PCTCN2014093854-appb-000006
注：与模型组比较＊P＜0.05，＊＊P＜0.01Note: Compared with the model group *P<0.05, **P<0.01
4)绿原酸对模型豚鼠皮肤黑色素的影响4) Effect of chlorogenic acid on melanin in model guinea pig skin
造模后，模型组基底层和棘层的黑色明显缺失，绿原酸15mg/kg、30mg/kg、60mg/kg能明显增加模型豚鼠的皮肤黑色素，明显增加皮肤表皮基底细胞和棘细胞黑色素分布和皮肤毛囊中黑色素。结果见表4。After modeling, the black color of the basal layer and the spinous layer of the model group was obviously lost. The chlorogenic acid 15mg/kg, 30mg/kg, 60mg/kg could significantly increase the skin melanin of the model guinea pig, and significantly increase the distribution of melanin in the epidermal basal cells and spine cells. And melanin in the hair follicles. The results are shown in Table 4.
表4  绿原酸对模型豚鼠皮肤黑色素的影响
Figure PCTCN2014093854-appb-000007
Table 4 Effect of chlorogenic acid on melanin in model guinea pig skin
Figure PCTCN2014093854-appb-000007
Figure PCTCN2014093854-appb-000008
Figure PCTCN2014093854-appb-000008
5.结论5 Conclusion
上述实验结果表明，绿原酸可明显增加实验性白癜风模型豚鼠的皮肤黑色素，明显增加皮肤表皮基底细胞和棘细胞黑色素分布，能使皮肤毛囊中黑色素显著增加，血液中酪氨酸酶含量也显著性增加，并且显著性改善血液流变学各项指标；说明绿原酸能增加实验性白癜风模型豚鼠皮肤黑色素生成，减少皮肤黑色素分解和改善血液循环，对白癜风起到较好的治疗作用。The above experimental results show that chlorogenic acid can significantly increase the skin melanin of experimental vitiligo model guinea pigs, significantly increase the distribution of melanin in skin epidermal basal cells and spine cells, and can significantly increase the melanin in skin hair follicles, and the tyrosinase content in blood is also significant. Increased sexuality and significantly improved blood rheology indicators; indicating that chlorogenic acid can increase the melanin production in experimental guinea pig model skin, reduce skin melanin decomposition and improve blood circulation, and play a better therapeutic effect on vitiligo.
实例2：绿原酸对白癜风豚鼠模型免疫器官脏器系数及皮损区TNF-α、 IL-10、IL-4、IFN-γ表达的影响Example 2: Chlorogenic acid visceral coefficient and TNF-α in the lesion area of vitiligo in guinea pig model The effects of IL-10, IL-4 and IFN-γ expression
1.材料Material
1)动物1) Animals
黑色豚鼠，体重300～350g，雌雄各半。Black guinea pig, weighing 300-350g, male and female.
2)试剂与试验药物2) Reagents and test drugs
对苯二酚、甲氧沙林片、绿原酸Hydroquinone, methoxacin tablets, chlorogenic acid
2.实验方法2. Experimental methods
1)实验性白癜风豚鼠模型制备1) Preparation of experimental vitiligo guinea pig model
采用对苯二酚(氢醌)制备实验性白癜风豚鼠模型。取健康黑色豚鼠，雌雄各半，用电动剃毛刀剃取背部毛面积5cm×5cm，每天在脱毛区涂抹5％的氢醌0.5ml，每天2次，连续涂抹50天，制备白癜风豚鼠模型。An experimental vitiligo guinea pig model was prepared using hydroquinone (hydroquinone). Take healthy black guinea pigs, male and female, shave the back hair area by 5cm × 5cm with an electric razor, apply 5% hydroquinone 0.5ml twice a day in the hair removal area, and apply for 50 days continuously to prepare a vitiligo guinea pig model.
2)动物分组及给药2) Animal grouping and administration
将上述模型豚鼠随机分为5组，分别为模型组、阳性组、绿原酸小剂量组(15mg/kg)、绿原酸中剂量组(30mg/kg)、绿原酸大剂量组(60mg/kg)，每组10只；另取10只未造模豚鼠作为空白对照组。各药物组豚鼠分别于造模第10日起开始给药，空白对照组和模型组豚鼠分别给予等容量的生理盐水，连续给药50天。The above model guinea pigs were randomly divided into 5 groups: model group, positive group, chlorogenic acid low dose group (15 mg/kg), chlorogenic acid medium dose group (30 mg/kg), and chlorogenic acid high dose group (60 mg). /kg), 10 in each group; another 10 unmodeled guinea pigs were used as blank control group. The guinea pigs of each drug group started to be administered on the 10th day of modeling, and the guinea pigs in the blank control group and the model group were given an equal volume of physiological saline for 50 days.
3)动物处理3) Animal treatment
治疗结束后，处死豚鼠，取用药部位中心1cm×1cm皮肤，中性甲醛固定，常规石蜡包埋、切片。并取豚鼠胸腺、脾脏称重，计算胸腺系数。After the end of the treatment, the guinea pigs were sacrificed, and the skin of the drug site was taken at a center of 1 cm × 1 cm, fixed in neutral formaldehyde, embedded in paraffin, and sectioned. The thymus and spleen of the guinea pig were weighed and the thymus coefficient was calculated.
4)免疫组化法观察IL-4、TNF-α4) Observation of IL-4 and TNF-α by immunohistochemistry
切片常规脱蜡至水。热修复抗原：0.01M枸橼酸盐缓冲液(pH6.0)，电炉或微波炉加热至沸腾后断电，切片浸入，冷却后PBS洗涤1-2次。30％H2O21份+蒸馏水10份混合，室温10分钟以灭活内源性酶。PBS洗5分钟×3次。正常山羊免疫血清封闭，室温孵育15分钟，倾去血清，不洗。滴加按1:100稀释的HMB45抗体，4℃过夜。PBS洗2分钟×3次。滴加辣根酶标记链霉卵白素工作液，37℃孵育10分钟。PBS洗5分钟×3次。DAB显色，镜下控制反应时间，20s-80s，至显色满意。蒸馏水洗涤，终止显色。苏木素轻度复染，盐酸酒精分化，流水冲洗返蓝1分钟。脱水，透明，封片。显微镜观察。The slices were conventionally dewaxed to water. Heat-repairing antigen: 0.01 M citrate buffer (pH 6.0), heat-burned in an electric furnace or microwave oven until boiling, slice immersion, and PBS was washed 1-2 times after cooling. 30% H 2 O 21 parts + 10 parts of distilled water were mixed, and the endogenous enzyme was inactivated at room temperature for 10 minutes. Wash in PBS for 5 minutes × 3 times. The normal goat immune serum was blocked, incubated at room temperature for 15 minutes, and the serum was decanted without washing. HMB45 antibody diluted 1:100 was added dropwise at 4 ° C overnight. Wash in PBS for 2 minutes × 3 times. The horseradish enzyme-labeled streptavidin working solution was added dropwise and incubated at 37 ° C for 10 minutes. Wash in PBS for 5 minutes × 3 times. DAB color development, control reaction time under the microscope, 20s-80s, to satisfactory color development. Wash with distilled water to stop color development. Hematoxylin mildly counterstained, hydrochloric acid alcohol differentiated, rinsed back to blue for 1 minute. Dehydrated, transparent, and sealed. Microscopic observation.
3.统计学方法及分析3. Statistical methods and analysis
所有数据均用SPSS 19.0统计软件处理。All data was processed using SPSS 19.0 statistical software.
IL-4、TNF-α的表达：Expression of IL-4 and TNF-α:
根据染色程度以及着色细胞的百分比进行半定量评分。每个切片随机选取5处高倍视野(200×)，根据阳性细胞数进行评分，评分标准为：0分， ＜25％着色细胞；1分，25％-50％着色细胞；2分，50％-75％着色细胞；3分，>75％着色细胞。染色程度评分标准为：0分，无染色；1分，浅黄色；2分，棕黄色；3分棕褐色。将染色程度分加上着色细胞百分比分得出总积分，按照积分分为4个等级：0分，阴性(-)；2分，弱阳性(+)；3-4分，阳性(++)；5-6分，强阳性(+++)。统计学采用秩和检验方法(Kruskal-Wallis法)。Semi-quantitative scoring was performed based on the degree of staining and the percentage of stained cells. Five high-power fields (200×) were randomly selected from each slice, and the scores were based on the number of positive cells. The score was: 0 points. <25% stained cells; 1 point, 25%-50% stained cells; 2 points, 50%-75% stained cells; 3 points, >75% stained cells. The staining degree was as follows: 0 points, no staining; 1 point, light yellow; 2 points, brownish yellow; 3 points of tan. The staining degree is divided into the percentage of the stained cells to obtain the total score, and the score is divided into 4 grades according to the score: 0, negative (-); 2 points, weak positive (+); 3-4 points, positive (++) 5-6 points, strong positive (+++). Statistics were performed using the rank sum test method (Kruskal-Wallis method).
4.结果4. Results
1)绿原酸对模型豚鼠免疫器官脏器系数的影响1) Effect of chlorogenic acid on organ coefficient of immune organs in model guinea pigs
模型组豚鼠胸腺和脾脏的脏器系数明显减小，与空白对照组比较差异显著，用药后，绿原酸各剂量组能增加模型豚鼠胸腺和脾脏和脏器系数，与模型组比较差异显著。表明，绿原酸能够增强模型豚鼠的免疫的功能。见表5。The visceral coefficient of the thymus and spleen of the model group was significantly reduced, and the difference was significant compared with the blank control group. After treatment, the chlorogenic acid group could increase the thymus and spleen and organ coefficient of the model guinea pig, which was significantly different from the model group. It was shown that chlorogenic acid can enhance the immune function of model guinea pigs. See Table 5.
表5  绿原酸对模型豚鼠免疫器官脏器系数的影响
Figure PCTCN2014093854-appb-000009
Table 5 Effect of chlorogenic acid on organ coefficient of immune organs in model guinea pigs
Figure PCTCN2014093854-appb-000009
Figure PCTCN2014093854-appb-000010
Figure PCTCN2014093854-appb-000010
注：与正常对照组比较，*P＜0.05，**P＜0.01；与模型组比较，#P＜0.05，##P＜0.01Note: Compared with the normal control group, *P<0.05, **P<0.01; compared with the model group, #P<0.05,##P<0.01
2)绿原酸对白癜风豚鼠模型皮损区TNF-α、IL-10、IL-4、IFN-γ表达的影响2) Effects of chlorogenic acid on the expression of TNF-α, IL-10, IL-4 and IFN-γ in the lesion area of vitiligo guinea pig model
TNF-α阳性细胞主要分布于表皮基底层、棘层、颗粒层，少数阳性细胞分布在真皮浅层；IL-4阳性细胞主要分布于表皮基底层、棘层，对照组呈弱阳性表达。模型组皮损区TNF-α和IL-4表达水平明显高于正常对照组，差异有显著性(P<0.01)；治疗组皮损区TNF-α和IL-4表达水平明显低于模型组，差异有显著性(P<0.01)。TNF-α positive cells were mainly distributed in the basal layer of the epidermis, spine layer and granular layer. A few positive cells were distributed in the superficial dermis. IL-4 positive cells were mainly distributed in the basal layer and spinous layer of the epidermis, and the control group showed weak positive expression. The expression levels of TNF-α and IL-4 in the lesion group were significantly higher than those in the normal control group (P<0.01). The expression levels of TNF-α and IL-4 in the lesion group were significantly lower than those in the model group. The difference was significant (P<0.01).
表6  绿原酸对皮损区TNF-α表达的影响(阳性例数)(秩和检验)Table 6 Effect of chlorogenic acid on the expression of TNF-α in the lesion area (positive cases) (rank sum test)
组别Group 例数Number of cases -- ++ ++++ ++++++
空白对照组Blank control group 1010 44 55 11 00
模型组Model group 1010 00 00 22 88
绿原酸组Chlorogenic acid group 1010 44 44 22 00
注：模型组与正常对照组比较差异有显著性(P<0.01)；治疗组与模型 组比较差异有显著性(P<0.01)Note: There was a significant difference between the model group and the normal control group (P<0.01); the treatment group and the model The difference in group comparison was significant (P<0.01).
表7  绿原酸对皮损区IL-4表达的影响(阳性例数)(秩和检验)Table 7 Effect of chlorogenic acid on IL-4 expression in lesion area (positive cases) (rank sum test)
组别Group 例数Number of cases -- ++ ++++ ++++++
空白对照组Blank control group 1010 44 44 22 00
模型组Model group 1010 00 00 33 77
绿原酸组Chlorogenic acid group 1010 33 55 22 00
注：模型组与正常对照组比较差异有显著性(P<0.01)；治疗组与模型组比较差异有显著性(P<0.01)Note: There was significant difference between the model group and the normal control group (P<0.01). There was significant difference between the treatment group and the model group (P<0.01).
5.结论5 Conclusion
绿原酸能改善白癜风模型的病理变化，降低皮损处TNF-α和IL-4的表达，促进黑素细胞的生成，为其临床治疗白癜风提供了理论依据。 Chlorogenic acid can improve the pathological changes of vitiligo model, reduce the expression of TNF-α and IL-4 in skin lesions, promote the formation of melanocytes, and provide a theoretical basis for its clinical treatment of vitiligo.

Claims (7)

  1. 绿原酸在制备治疗白癜风的药物中的用途。The use of chlorogenic acid in the preparation of a medicament for treating vitiligo.
  2. 根据权利要求1所述的用途，其特征在于：所述的药物是降低皮损处TNF-α和IL-4的表达的药物。The use according to claim 1, characterized in that the drug is a drug which reduces the expression of TNF-α and IL-4 at the lesion.
  3. 根据权利要求1所述的用途，其特征在于：所述的药物是促进黑素细胞生成的药物。The use according to claim 1, wherein the drug is a drug that promotes melanocyte production.
  4. 根据权利要求1-3任意一项所述的用途，其特征在于：所述的药物是由绿原酸为有效成分，加入药学上可接受的辅料或辅助性成分制备而成的制剂。The use according to any one of claims 1 to 3, characterized in that the medicament is a preparation prepared by adding chlorogenic acid as an active ingredient and adding a pharmaceutically acceptable adjuvant or auxiliary ingredient.
  5. 根据权利要求4所述的用途，其特征在于：所述的制剂是口服制剂、注射制剂或外用透皮给药制剂。The use according to claim 4, characterized in that the preparation is an oral preparation, an injection preparation or a transdermal administration preparation for external use.
  6. 根据权利要求5所述的用途，其特征在于：所述的制剂的使用剂量为：1-100mg/kg。The use according to claim 5, characterized in that the preparation is used in a dose of from 1 to 100 mg/kg.
  7. 根据权利要求6所述的用途，其特征在于：所述的制剂的使用剂量为：1-30mg/kg。 The use according to claim 6, characterized in that the preparation is used in a dose of from 1 to 30 mg/kg.
PCT/CN2014/093854 2014-12-15 2014-12-15 Uses of chlorogenic acid in preparation of medicines for treating vitiligo WO2016095087A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09151130A (en) * 1995-12-01 1997-06-10 Yakurigaku Chuo Kenkyusho:Kk Medicine for treating trichopoliosis and leukoplakia
WO2012017551A1 (en) * 2010-08-06 2012-02-09 Yuasa Makoto Therapeutic agent for disease
CN104434902A (en) * 2014-12-15 2015-03-25 四川九章生物科技有限公司 Application of chlorogenic acid in preparing medicines for treating leucoderma

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH09151130A (en) * 1995-12-01 1997-06-10 Yakurigaku Chuo Kenkyusho:Kk Medicine for treating trichopoliosis and leukoplakia
WO2012017551A1 (en) * 2010-08-06 2012-02-09 Yuasa Makoto Therapeutic agent for disease
CN104434902A (en) * 2014-12-15 2015-03-25 四川九章生物科技有限公司 Application of chlorogenic acid in preparing medicines for treating leucoderma

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