阅读说明：本技术 一种用于治疗银屑病植物提取物的试验方法及应用 (Test method and application of plant extract for treating psoriasis ) 是由 陈�光 于 2021-08-03 设计创作，主要内容包括：本发明公开了一种用于治疗银屑病植物提取物的试验方法及应用,包括以下步骤：试验动物的选取；试验动物的分组,试验动物随机分为五组,正常对照组雌性小鼠、银屑病模型组雌性小鼠、德国洋甘菊脂溶性提取物外涂组雌性小鼠、德国洋甘菊水溶性提取物灌胃组雌性小鼠、德国洋甘菊脂溶性和水溶性提取物联合处理组雌性小鼠；试验动物的模型制备；银屑病小鼠模型鉴定。本发明通过使用德国洋甘菊脂溶性提取物和德国洋甘菊水溶性提取物的试验方式,与模型组小鼠相比,德国洋甘菊脂溶性提取物外涂组小鼠、德国洋甘菊水溶性提取物灌胃组小鼠、德国洋甘菊脂溶性和水溶性提取物联合处理组小鼠临床症状均有明显改善。(The invention discloses a test method and application of a plant extract for treating psoriasis, which comprises the following steps: selecting a test animal; grouping test animals, wherein the test animals are randomly divided into five groups, namely female mice in a normal control group, female mice in a psoriasis model group, female mice in a German chamomile fat-soluble extract external coating group, female mice in a German chamomile stomach-irrigation group and female mice in a German chamomile fat-soluble and water-soluble extract combined treatment group; preparing a model of a test animal; and identifying a psoriasis mouse model. According to the invention, through a test mode of using the German chamomile fat-soluble extract and the German chamomile water-soluble extract, compared with a model group mouse, the clinical symptoms of the German chamomile fat-soluble extract external application group mouse, the German chamomile water-soluble extract intragastric group mouse and the German chamomile fat-soluble and water-soluble extract combined treatment group mouse are obviously improved.)

1. A test method for the treatment of psoriasis comprising the steps of:

the method comprises the following steps: selecting a test animal, wherein the animal is a female BALB/c mouse;

step two: grouping test animals, wherein the test animals are randomly divided into five groups, namely female mice in a normal control group, female mice in a psoriasis model group, female mice in a German chamomile fat-soluble extract external coating group, female mice in a German chamomile stomach-irrigation group and female mice in a German chamomile fat-soluble and water-soluble extract combined treatment group;

step three: preparing a model of a test animal, shaving hairs of the test animal and processing the hairs by using a medicament;

step four: and identifying the psoriasis mouse model, and comparing relevant indexes of the test animal to obtain a test result.

2. A test method for psoriasis plant extract according to claim 1 wherein:

in the first step, a female BALB/c mouse of 10 weeks old is selected as a test animal, Imiquimod (IMQ) cream, a German chamomile fat-soluble extract, a German chamomile water-soluble extract and vaseline are prepared for later use, and a test apparatus needs to be subjected to aseptic treatment before use.

3. A test method for psoriasis plant extract according to claim 1 wherein:

in the second step, the test mice are divided into 10 normal control groups;

psoriasis model group 12;

the external application group of the German chamomile fat-soluble extract comprises 10 pieces;

the German chamomile water-soluble extract perfuses 10 groups;

the german chamomile fat-soluble and water-soluble extracts were combined in treatment group 10.

4. A test method for psoriasis plant extract according to claim 1 wherein:

in the third step, the experimental animals are treated, the backs of the female mice 42 in the psoriasis model group and the drug treatment group are shaved, and IMQ cream is smeared;

shaving the back of a female mouse, namely removing hairs in a 2 x 3cm area on the back of the female mouse by using an electric hair cutter, removing fine hairs by using a single-head electric shaver, and raising the shaved mouse for 3 days without any treatment so as to eliminate the damage and influence of the shaved hair on the back skin;

mice were then given 62.5 mg/mouse of IMQ cream at 5%, 1 time/day, for 7 consecutive days;

uniformly smearing IMQ on the exposed area of the back of the mouse, covering a preservative film of 3 multiplied by 3cm, continuously and lightly pressing with fingers for 60 times, carrying out twelve o' clock treatment at noon every day, taking a picture and recording before each administration, removing new fine villi with a single-head electric shaver, and always keeping the exposed area of the back of the mouse in a hairless state;

the external application of the German chamomile fat-soluble extract to 10 female mice is carried out with drug treatment: model mice, which were externally coated with IMQ cream, were treated on the same day as follows: coating the model mouse with a German chamomile fat-soluble extract at eight points in the morning and six points in the evening at 200 muL/time and 2 times/day for 7 days continuously, and simultaneously irrigating 200 mul/10 g of physiological saline;

10 female mice in the gastric lavage group of the water-soluble extract of German chamomile are treated with the following medicaments: performing intragastric treatment on the water-soluble extract of the German chamomile 7 days before the preparation of the model, performing intragastric treatment on the water-soluble extract of the German chamomile eight times in the morning and six times in the evening every day, coating the IMQ cream outside the mice after 7 days to prepare the model, continuously performing intragastric treatment on the water-soluble extract of the German chamomile for 7 days, performing continuous treatment for 14 days, and continuously coating vaseline outside the mice for 7 days;

female mice in the german chamomile fat-soluble and water-soluble extract combination treatment group were treated with 10 drugs: the method is characterized in that the mouse is processed by superposing according to the processing method of the mouse with the German chamomile water-soluble extract gavage group and the processing method of the mouse with the German chamomile fat-soluble extract externally coated;

shaving the normal control group female mice according to the treatment method of the psoriasis model group female mice, and smearing Vaseline.

5. A test method and use of a plant extract for the treatment of psoriasis according to claim 1 wherein:

in the fourth step, in order to evaluate the severity of the back skin inflammation, a scoring system is established on the basis of clinical PASI scoring in reference documents;

the erythema, the scaling and the thickening degree are independently scored according to the score of 0-4, and the scoring rule is as follows:

0= none, 1= mild, 2= moderate, 3= overt, 4= very overt, used to indicate the degree of skin inflammation;

and (3) detecting the histopathology of the skin: on day 8 after model preparation, mice were decapped and sacrificed, 0.5 × 1 cm-sized skin lesion tissues were taken, subcutaneous fat was removed, placed in formalin fixing solution, and HE staining was performed to observe skin lesion degree and detect thickness of spinous layer.

6. Use of a plant extract for the treatment of psoriasis according to claims 1 to 5 in the testing of the effectiveness of lipid-soluble and water-soluble extracts of Matricaria chamomilla in psoriasis.

Technical Field

The invention relates to the field of psoriasis, in particular to a test method and application of a psoriasis treatment plant extract.

Background

Psoriasis, like most autoimmune diseases, is an immune skin disease affected by genetic and epigenetic modification, is clinically manifested as chronic, inflammatory and easy recurrence, and the skin damage of patients is often manifested as scales on the basis of erythema with severe pruritus, thus greatly damaging the physical and mental health of the patients. Psoriasis is a very common disease, the prevalence rate accounts for about 2% -4% of the world population, the treatment of psoriasis is very difficult, the patient's condition is often lingering, the physical and mental health is often damaged due to difficult pruritus, impaired appearance and the like, the quality of life is seriously reduced, and the long-term treatment causes heavy economic burden, so that the specific molecular mechanism of the formation and maintenance of the inflammatory response of psoriasis is further deeply researched, more potential treatment targets are favorably discovered, more targeted molecular targeted drugs are helped to be developed, and the clinical treatment problem of psoriasis is solved.

Disclosure of Invention

The invention aims to provide a test method and application of a plant extract for treating psoriasis, so as to solve the problems in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a test method and use of a plant extract for treating psoriasis comprising the steps of:

the method comprises the following steps: selecting a test animal, wherein the animal is a female BALB/c mouse;

step two: grouping test animals, wherein the test animals are randomly divided into five groups, namely female mice in a normal control group, female mice in a psoriasis model group, female mice in a German chamomile fat-soluble extract external coating group, female mice in a German chamomile stomach-irrigation group and female mice in a German chamomile fat-soluble and water-soluble extract combined treatment group;

step three: preparing a model of a test animal, shaving hairs of the test animal and processing the hairs by using a medicament;

step four: and identifying the psoriasis mouse model, and comparing relevant indexes of the test animal to obtain a test result.

Preferably, in the first step, female BALB/c mice of 10 weeks old are selected as test animals, IMQ cream, German chamomile fat-soluble extract, German chamomile water-soluble extract and vaseline are prepared for standby, and the test instruments are subjected to sterile treatment before use.

Preferably, in the second step, the test mice are divided into 10 normal control groups;

psoriasis model group 12;

the external application group of the German chamomile fat-soluble extract comprises 10 pieces;

the German chamomile water-soluble extract perfuses 10 groups;

the german chamomile fat-soluble and water-soluble extracts were combined in treatment group 10.

Preferably, in step three, the female mice 42 of the experimental animal treatment, the psoriasis model group and the drug treatment group are only shaved on the back, and smearing IMQ cream, shaving hair on the back of female mouse by electric hair clipper in area of 2 × 3cm, shaving fine hair with single-head electric shaver, feeding shaved mouse without any treatment for 3 days, the mouse is given 62.5 mg/mouse of IMQ cream with the concentration of 5%, 1 time/day and continuously treated for 7 days so as to eliminate the damage and influence of shaving hair on back skin, the IMQ cream is uniformly coated on the exposed area of the back of the mouse, a preservative film with the concentration of 3 multiplied by 3cm is coated on the exposed area, fingers continuously and lightly press the exposed area for 60 times, twelve o' clock treatment is carried out every day, photographing record is carried out before each administration, new fine villi are removed by a single-head electric shaver, and the exposed area of the back of the mouse is always kept in a hairless state;

the external application of the German chamomile fat-soluble extract to 10 female mice is carried out with drug treatment: model mice, which were externally coated with IMQ cream, were treated on the same day with the following treatments: coating the model mouse with a German chamomile fat-soluble extract at eight points in the morning and six points in the evening at 200 muL/time and 2 times/day for 7 days continuously, and simultaneously irrigating 200 mul/10 g of physiological saline;

10 female mice in the gastric lavage group of the water-soluble extract of German chamomile are treated with the following medicaments: performing intragastric treatment on the water-soluble extract of the German chamomile 7 days before the preparation of the model, performing intragastric treatment on the water-soluble extract of the German chamomile eight times in the morning and six times in the evening every day, coating the IMQ cream outside the mice after 7 days to prepare the model, continuously performing intragastric treatment on the water-soluble extract of the German chamomile for 7 days, performing continuous treatment for 14 days, and continuously coating vaseline outside the mice for 7 days;

when 10 female mice in the combined treatment group of the German chamomile fat-soluble and water-soluble extracts are treated by the medicine, the female mice are treated by superposing according to the treatment method of the mice in the gastric perfusion group of the German chamomile water-soluble extracts and the treatment method of the mice in the external coating group of the German chamomile fat-soluble extracts;

shaving the normal control group female mice according to the treatment method of the psoriasis model group female mice, and smearing Vaseline.

Preferably, in step four, in order to evaluate the severity of the back skin inflammation, a scoring system is established on the basis of clinical PASI scores in reference documents;

the erythema, the scaling and the thickening degree are independently scored according to the score of 0-4, and the scoring rule is as follows:

0= none, 1= mild, 2= moderate, 3= overt, 4= very overt, used to indicate the degree of skin inflammation;

and (3) detecting the histopathology of the skin: on day 8 after model preparation, mice were decapped and sacrificed, 0.5 × 1 cm-sized skin lesion tissues were taken, subcutaneous fat was removed, placed in formalin fixing solution, and HE staining was performed to observe skin lesion degree and detect thickness of spinous layer.

Preferably, a psoriasis treatment plant extract is applied to the test of psoriasis efficacy of the German chamomile fat-soluble and water-soluble extract.

Drawings

FIG. 1 is a schematic representation of the clinical manifestations of the psoriasis-treated mice of the invention.

FIG. 2 is a schematic view of the observation of HE staining of the skin of psoriasis mice.

FIG. 3 is a schematic diagram of the thickness of the spinous layer of the skin of a psoriasis mouse.

The invention has the technical effects and advantages that:

according to the invention, through a test mode of using the German chamomile fat-soluble extract and the German chamomile water-soluble extract, compared with a model group mouse, the clinical symptoms of the German chamomile fat-soluble extract external application group mouse, the German chamomile water-soluble extract intragastric group mouse and the German chamomile fat-soluble and water-soluble extract combined treatment group mouse are obviously improved. Compared with a model group, the skin damage degree of mice is obviously reduced after the mice are treated by the external application group of the German chamomile fat-soluble extract, the intragastric group of the German chamomile water-soluble extract and the combined treatment group of the German chamomile fat-soluble and water-soluble extracts; the granular layer, the thorn layer and the matrix layer have complete structures; the phenomena of stratum corneum thickening, parakeratosis and hyperkeratosis are obviously improved. Compared with the mice with the German chamomile fat-soluble extract externally coated group, the stomach-drenching treatment group of the German chamomile water-soluble extract and the combined treatment group of the German chamomile fat-soluble extract and the water-soluble extract have better effects.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a test method and application of a plant extract for treating psoriasis as shown in figures 1-3, which comprises the following steps:

the method comprises the following steps: selecting a test animal, wherein the animal is a female BALB/c mouse;

step two: grouping test animals, wherein the test animals are randomly divided into five groups, namely female mice in a normal control group, female mice in a psoriasis model group, female mice in a German chamomile fat-soluble extract external coating group, female mice in a German chamomile stomach-irrigation group and female mice in a German chamomile fat-soluble and water-soluble extract combined treatment group;

step three: preparing a model of a test animal, shaving hairs of the test animal and processing the hairs by using a medicament;

step four: and identifying the psoriasis mouse model, and comparing relevant indexes of the test animal to obtain a test result.

In the first step, a test animal selects a female BALB/c mouse with the age of 10 weeks, IMQ cream, a German chamomile fat-soluble extract, a German chamomile water-soluble extract and vaseline are prepared for later use, and a test apparatus needs to be subjected to aseptic treatment before use.

In the second step, the test mice are divided into 10 normal control groups;

psoriasis model group 12;

the external application group of the German chamomile fat-soluble extract comprises 10 pieces;

the German chamomile water-soluble extract perfuses 10 groups;

the german chamomile fat-soluble and water-soluble extracts were combined in treatment group 10.

In the third step, the experimental animals are treated, the backs of the female mice 42 in the psoriasis model group and the drug treatment group are shaved, the IMQ cream is smeared, the backs of the female mice are shaved, namely, hairs in the area of 2 x 3cm of the backs of the mice are removed by electric hair clippers, fine hairs are removed by a single-head electric shaver, the shaved mice are raised for 3 days without any treatment to eliminate the damage and the influence of the shaved hairs on the back skins, the mice are given 62.5 mg/mouse of 5% of the IMQ cream for 1 time/day for 7 days after continuous treatment, the IMQ is uniformly smeared on the exposed areas of the backs of the mice, a preservative film of 3 x 3cm is covered, fingers are continuously and lightly pressed for 60 times, twelve noon treatment is carried out every day, recording is carried out before each administration, new fine hairs are removed by a single-head electric shaver, and the exposed areas of the backs of the mice are always kept in a hairless state;

the external application of the German chamomile fat-soluble extract to 10 female mice is carried out with drug treatment: model mice, which were externally coated with IMQ cream, were treated on the same day with the following treatments: coating the model mouse with a German chamomile fat-soluble extract at eight points in the morning and six points in the evening at 200 muL/time and 2 times/day for 7 days continuously, and simultaneously irrigating 200 mul/10 g of physiological saline;

10 female mice in the gastric lavage group of the water-soluble extract of German chamomile are treated with the following medicaments: performing intragastric treatment on the water-soluble extract of the German chamomile 7 days before the preparation of the model, performing intragastric treatment on the water-soluble extract of the German chamomile eight times in the morning and six times in the evening every day, coating the IMQ cream outside the mice after 7 days to prepare the model, continuously performing intragastric treatment on the water-soluble extract of the German chamomile for 7 days, performing continuous treatment for 14 days, and continuously coating vaseline outside the mice for 7 days;

when 10 female mice in the combined treatment group of the German chamomile fat-soluble and water-soluble extracts are treated by the medicine, the female mice are treated by superposing according to the treatment method of the mice in the gastric perfusion group of the German chamomile water-soluble extracts and the treatment method of the mice in the external coating group of the German chamomile fat-soluble extracts;

shaving the normal control group female mice according to the treatment method of the psoriasis model group female mice, and smearing Vaseline.

In the fourth step, in order to evaluate the severity of the back skin inflammation, a scoring system is established on the basis of clinical PASI scoring in reference documents;

the erythema, the scaling and the thickening degree are independently scored according to the score of 0-4, and the scoring rule is as follows:

0= none, 1= mild, 2= moderate, 3= overt, 4= very overt, used to indicate the degree of skin inflammation;

and (3) detecting the histopathology of the skin: on day 8 after model preparation, mice were decapped and sacrificed, 0.5 × 1 cm-sized skin lesion tissues were taken, subcutaneous fat was removed, placed in formalin fixing solution, and HE staining was performed to observe skin lesion degree and detect thickness of spinous layer.

A plant extract for treating psoriasis is applied to the test of the curative effect of a German chamomile fat-soluble and water-soluble extract on psoriasis.

Fig. 1 is a schematic diagram showing the observation of clinical performance of psoriasis mice, wherein a is a normal control group, B is a psoriasis model group, C is a german chamomile fat-soluble extract external application group, D is a german chamomile water-soluble extract gastric lavage group, E is a german chamomile fat-soluble and water-soluble extract combined treatment group, 0D is the state of female BALB/C mice before the test, and 7D is the state of female BALB/C mice after the test, so that the model group mice can be visually observed to have obvious erythema, scaling and psoriasis-like lesions on the back compared with the normal control group. Compared with a model group mouse, the mouse with the German chamomile fat-soluble extract externally coated, the mouse with the German chamomile water-soluble extract intragastrically filled and the mouse with the German chamomile fat-soluble and water-soluble extract combined treatment group have the advantages that the back skin damage is obviously improved, and the erythema, the scale and the psoriasis-like lesion are obviously reduced. Compared with the mice with the German chamomile fat-soluble extract externally coated group, the mice with the German chamomile water-soluble extract intragastrically coated group and the German chamomile fat-soluble and water-soluble extract combined treatment group have more obvious improvement degree on the skin damage on the back.

In fig. 2, HE staining observation is performed on psoriasis mouse skin, a is a normal control group, B is a psoriasis model group, C is a german chamomile fat-soluble extract external application group, D is a german chamomile water-soluble extract intragastric group, E is a german chamomile fat-soluble and water-soluble extract combined treatment group, and the results are shown under an HE staining microscope: the skin epidermis of the mice in the blank control group is complete, the stratum corneum is about 2 layers and the cell structure is normal, the granular layer, the spinous layer and the matrix layer can be seen to be complete structures, the boundary is clear, and obvious single-layer columnar cells which are seen in the matrix layer are free from obvious lymphocyte infiltration. The epidermis of the mice in the psoriasis model group protrudes downwards, the stratum corneum is thickened, partial cell hyperkeratosis and parakeratosis do not have obvious granular layers, the spinous layer can be obviously thickened, and the dermal layer can be infiltrated by neutrophils and lymphocytes. Compared with a model group, the mouse treated by the external application of the German chamomile fat-soluble extract, the mouse treated by the intragastric administration of the German chamomile water-soluble extract and the mouse treated by the combined treatment of the German chamomile fat-soluble and water-soluble extracts obviously reduces the skin damage degree of the mouse, the granular layer, the acantho layer and the matrix layer have complete structures, and the phenomena of stratum corneum thickening, parakeratosis and hyperkeratosis are obviously improved. Compared with the German chamomile fat-soluble extract treatment group, the German chamomile water-soluble extract treatment group has more obvious improvement degree on the skin damage of mice. The thickness of the spinous layer is slightly increased, the basal layer structure is complete, and the boundary is clear.

In fig. 3, the thickness of the spinous layer is measured after HE staining of the skin of the psoriasis mouse, a is a normal control group, B is a psoriasis model group, C is an external application group of a fat-soluble extract of german chamomile, D is a gastric lavage group of a water-soluble extract of german chamomile, and E is a combined treatment group of the fat-soluble extract and the water-soluble extract of german chamomile, so that the thickness of the spinous layer of the back skin of the mouse in the model group is obviously increased (compared with the normal control group), (the thickness of the spinous layer of the back skin of the mouse in the model group is obviously increased by the thickness of the spinous layer of the back skin of the mouse in the normal control group: (P<0.0001). Compared with the model group, the fat-soluble extract of the German chamomile is coated on the mice of the group, the water-soluble extract of the German chamomile is perfused on the mice of the group, and the fat-soluble and water-soluble extracts of the German chamomile are treated on the mice of the group in a combined wayThe thickness of the spinous layer of the skin of the mouse is obviously reduced after treatmentP<0.01). There was no significant difference in thickness of dorsal spinous skin layer in the three drug-treated mice.

In the table is the psoriasis mouse skin lesion PASI score table (' X ± S). It can be found that: compared with the normal control group, the skin damage PASI score of the model control group is obviously increased compared with that of the normal control group. Compared with a model group, the mouse of the German chamomile fat-soluble extract external coating group, the mouse of the German chamomile water-soluble extract intragastric group and the mouse of the German chamomile fat-soluble and water-soluble extract combined treatment group have obviously reduced scores, and the difference has statistical significance (P < 0.05). There was no significant difference in the skin lesion PASI scores of the three drug-treated groups of mice.

Group of	Example number (n)	PASI score
			Group A	10	0
Group B	12	6.4±0.79
			Group C	10	4.3±0.43*
Group D	10	3.31±0.35*
			Group E	10	3.94±0.44*

Note: model groups were compared to three drug treatment groupsP< 0.05。

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.