An external use Bletilla striata polysaccharide preparation for preventing and treating eczema and pruritus, its preparation method and application

Abstract

The present invention relates to the technical field of new drug indications, and specifically discloses an external use preparation of bletilla striata polysaccharide for preventing and treating eczema and pruritus, its preparation method and application. The present invention proves through scientific experiments that the external use preparation of bletilla striata polysaccharide can promote the rapid regression of acute and chronic eczema symptoms induced by dinitrofluorobenzene (DNFB) in rats within a short period of time, and its prevention and treatment effect is significantly better than the commonly used control drugs in clinical practice of traditional Chinese and Western medicine (compound phellodendron liquid, Binghuang Fule ointment, compound dexamethasone acetate cream), and can also rapidly inhibit the pruritus signs in mice induced by the combined use of histamine and 4-aminopyridine, and its prevention and treatment effect is also close to the commonly used control drugs in clinical practice of traditional Chinese and Western medicine (Qingpeng ointment, compound dexamethasone acetate cream).

Description

Technical Field

[0001] The present invention relates to the technical field of new drug indications, and particularly to an external preparation of bletilla striata polysaccharide for preventing and treating eczema and pruritus, a preparation method thereof, and an application thereof. Background Art

[0002] Eczema is a skin disease characterized by local polymorphic rashes and exudation tendency or infiltration and thickening in the superficial epidermis and dermis caused by various internal factors (such as chronic digestive system diseases, endocrine disorders, mental stress, insomnia, excessive fatigue, mood changes, etc.) and external factors (such as environment, climate change, sunlight, and various animal furs, plants, cosmetics, etc.). It is accompanied by severe pruritus and is prone to recurrence. Among all skin diseases, eczema has the highest incidence rate. At the same time, eczema can cause skin damage. Acute eczema produces large areas of skin lesions and papulovesicles with an exudation tendency; the affected skin of chronic eczema will be significantly thickened, showing lichenification, and is prone to recurrence, which greatly affects the daily work and life of patients.

[0003] At present, Western medicine clinically classifies eczema as an allergic disorder, and the treatment principle is to relieve pruritus as soon as possible, control skin inflammation, and restore the skin barrier function, so as to improve the quality of life of patients. Western medicine mostly adopts the internal and external combined treatment method. Patients are often advised to take antihistamines such as chlorpheniramine, cetirizine, loratadine, olopatadine hydrochloride, terfenadine, etc. orally, and at the same time, combined with topical glucocorticoids (such as prednisone, compound dexamethasone acetate cream, etc.) and calcium modulin inhibitors and calcium preparations (such as calcium lactate, calcium gluconate, etc.) of non-hormonal treatment drugs, or cooperate with calamine lotion (used when there is no exudate at the initial stage) and 3% boric acid solution (used when there is more exudation or erosion) for local external washing. Although the use of the above-mentioned therapies and drugs is simple, their actual clinical treatment effects are not ideal. Long-term repeated use may cause adverse reactions such as skin atrophy and pigmentation, and it is easy to relapse after stopping the drug, and even cause endocrine and metabolic disorders, which are difficult to control well.

[0004] Therefore, actively searching for a drug that can reduce the recruitment of inflammatory cells and promote the restoration of skin barrier function, and finding a breakthrough treatment method for eczema have become the focus of attention of many medical scholars and the main purpose of the present invention. Summary of the Invention

[0005] Aiming at the problem of the lack of specific drugs for preventing and treating eczema and pruritus in the above-mentioned existing technologies:

[0006] The first object of the present invention is to provide an external preparation of bletilla striata polysaccharide for effectively preventing and treating eczema and pruritus.

[0007] The second object of the present invention is to provide a preparation method of the above-mentioned external preparation of bletilla striata polysaccharide for preventing and treating eczema and pruritus.

[0008] The third object of the present invention is to provide the medical use of the above-mentioned external Bletilla striata polysaccharide preparation for preventing and treating eczema and pruritus.

[0009] The inventive concept of the present invention is as follows:

[0010] Eczema is a skin disease characterized by local polymorphic rashes and exudation tendency or infiltration and thickening in the superficial epidermis and dermis caused by various internal factors (such as chronic digestive system diseases, endocrine disorders, mental stress, insomnia, over-fatigue, mood changes, etc.) and external factors (such as environment, climate change, sunlight, and various animal furs, plants, cosmetics, etc.), accompanied by severe pruritus and prone to recurrence. Among all skin diseases, eczema has the highest incidence rate.

[0011] From the perspective of the pathological changes in the tissue structure of local skin, in patients with acute eczema, the affected epidermis shows spongiotic edema, with blisters in the stratum spinosum and subcorneal layer, and limited lymphocyte and neutrophil infiltration can be seen; there is dilation of small blood vessels in the superficial dermis and mild inflammatory cell infiltration mainly composed of lymphocytes around the blood vessels. Once it deteriorates into subacute or even chronic eczema, the epidermis will show thickening, hyperkeratosis, obvious spongiotic edema, obvious inflammatory cell infiltration around small blood vessels in the superficial dermis, an increase in the number of capillaries, and even abnormal changes such as endothelial cell swelling and hyperplasia.

[0012] Currently, the clinical treatment of eczema in Western medicine is based on the principle of quickly relieving pruritus, controlling skin inflammation, and restoring the skin barrier function to improve the quality of life of patients. Western medicine mostly adopts the combined internal and external treatment method, and often advises patients to take antihistamines such as chlorpheniramine, cetirizine, loratadine, terfenadine orally, and at the same time combines topical glucocorticoids and calcium modulin inhibitors and calcium preparations of non-hormonal treatment drugs, or cooperates with calamine lotion, 3% boric acid solution, etc. for local external washing. Although the use of the above-mentioned therapies and drugs is simple, their actual clinical treatment effects are not ideal, and even various adverse reactions occur, and the condition is prone to recurrence and is difficult to control well.

[0013] Of course, from the definition of eczema, it can be known that skin pruritus is one of its important clinical manifestations, and it is also the most important sign that seriously affects the quality of life of patients and urgently requires timely and effective treatment.

[0014] For the treatment of pruritus, Western medicine clinical first emphasizes avoiding contact with allergens, stopping suspected drugs, and effectively treating the primary disease. At the same time, it mainly uses antihistamine H1 and H2 receptor antagonists, glucocorticoids (for the pediatric population, there is no standard protocol for topical glucocorticoids), immune regulation, etc., but often due to adverse reactions, it cannot be used for a long time. As a result, after the treatment is interrupted, the pruritus recurs quickly.

[0015] Bletilla striata polysaccharide has been reported to have the effects of immunizing and repairing chemical liver injury, and has also been found to have the effect of repairing the skin barrier. However, whether Bletilla striata polysaccharide can prevent and treat eczema and pruritus remains to be scientifically studied. Therefore, the applicant has carried out a large number of scientific experiments to verify. By establishing animal models of acute and chronic eczema and pruritus respectively, the therapeutic effect of the prepared Bletilla striata polysaccharide on the disease through topical administration was studied. The research results confirmed that Bletilla striata polysaccharide can not only promote the acute and chronic eczema symptoms of rats induced by dinitrofluorobenzene (DNFB), but also quickly inhibit the pruritus signs of mice induced by the combined use of histamine and 4-aminopyridine, and quickly promote the repair of the skin tissue structure of the wound surface of each model animal; in terms of preventing and treating eczema, its effect is significantly better than the commonly used control drugs in Western medicine and traditional Chinese medicine clinics (① anti-inflammatory Western medicine: topical compound dexamethasone acetate cream; ② traditional Chinese patent medicine with the effects of clearing heat and drying dampness, promoting blood circulation and expelling wind, relieving itching and reducing inflammation: topical compound Phellodendron amurense liquid, Binghuang Fule ointment, Qizheng Qingpeng ointment).

[0016] To achieve the first object, the present invention adopts the following technical measures:

[0017] An external medicine of Bletilla striata polysaccharide for preventing and treating eczema and pruritus, wherein the concentration of Bletilla striata polysaccharide in the external medicine is 1 mg / ml.

[0018] To achieve the second object, the present invention adopts the following technical measures:

[0019] A preparation method of the above-mentioned external Bletilla striata polysaccharide preparation for preventing and treating eczema and pruritus, which makes Bletilla striata polysaccharide into any dosage form acceptable for skin external medicine.

[0020] A relatively simple preparation method can take the following steps:

[0021] Dissolve the dry powder of Bletilla striata polysaccharide in ultrapure water containing Tween 80 and glycerol to prepare an aqueous phase, dissolve egg yolk lecithin in edible vegetable oil to prepare an oil phase, drop the oil phase into the aqueous phase according to the volume ratio of the oil phase to the aqueous phase of 1:4, stir, and then perform high-speed shearing and high-pressure homogenization to obtain a Bletilla striata polysaccharide solution. The specific operation steps are as follows:

[0022] Preparation steps of the Bletilla striata polysaccharide solution: ① Dissolve the dry powder of Bletilla striata polysaccharide in ultrapure water, and add Tween 80 and glycerol to the obtained solution to obtain an aqueous phase containing Bletilla striata polysaccharide (where the final concentration of Tween 80 is 1.25% V / V; the final concentration of glycerol is 2.81% V / V); ② Dissolve egg yolk lecithin in edible vegetable oil to prepare an oil phase (the final concentration of egg yolk lecithin is 6% W / V, g / ml). ③ Under the condition of a constant temperature water bath at 60 °C, drop the oil phase into the aqueous phase according to the volume ratio of the oil phase to the aqueous phase of 1:4, and stir for 10 min under the condition of 1500 r / min. ④ Perform high-speed shearing for 2 min under the condition of 20000 rpm, and then perform high-pressure homogenization 3 times under the condition of 1000 bar to obtain the Bletilla striata polysaccharide solution, and the content of Bletilla striata polysaccharide in the obtained Bletilla striata polysaccharide solution is equal to 1 mg / ml.

[0023] The edible vegetable oil is selected from at least one of sesame oil, peanut oil, coconut oil, soybean oil, linseed oil, castor oil and olive oil, preferably at least one of sesame oil and coconut oil.

[0024] In order to achieve the third object of the present invention, the present invention also adopts the following technical measures:

[0025] The present invention also provides the application of the above-mentioned external Bletilla striata polysaccharide preparation in the preparation of drugs for preventing and treating eczema and pruritus.

[0026] The eczema and pruritus are caused by sensitizing and / or inflammatory drugs.

[0027] Bletilla striata is one of the precious traditional Chinese herbs in China. Because of its good effects of astringing to stop bleeding, moisturizing, delaying skin aging, detumescence and promoting granulation, it has high medicinal value in promoting the repair of skin cracks and damaged skin. Bletilla striata polysaccharide is a glucomannan extracted from the traditional Chinese medicine Bletilla striata, and is found to have effects such as anti-tumor, hypoglycemic, antioxidant, and promoting gastric digestion. It is a low-toxic and safe polymer with great development value. Whether the Bletilla striata polysaccharide derived from Bletilla striata can effectively prevent and treat skin problems such as eczema and pruritus remains to be scientifically proven by a large amount of evidence.

[0028] Compared with the prior art, the present invention has the following advantages and effects:

[0029] The research results show that after topically administering the external Bletilla striata polysaccharide drug of the present invention, the sufferings of eczema and pruritus can be quickly eliminated. Its prevention and treatment effects are significantly better than or close to the control drugs commonly used in Western medicine and traditional Chinese medicine clinics (① Western medicine for anti-inflammatory and anti-allergic: compound dexamethasone acetate cream; ② Traditional Chinese medicine with the effects of clearing heat and drying dampness, promoting blood circulation and expelling wind, and relieving itching and inflammation: external compound Phellodendron amurense liquid, Binghuang Fule Ointment, Qizheng Qingpeng Ointment).

[0030] The Bletilla striata polysaccharide external medicine in the present invention has the following advantages in preventing and treating eczema and pruritus: ① low drug cost; ② convenient for external use, quick in taking effect and good in effect. Description of the Drawings

[0031] Figure 1 It is a scoring chart of the influence of the test drug on the skin of the affected part of acute eczema and its damage degree. A is the chart of the influence of Bletilla striata extract, Bletilla striata polysaccharide (BSP), compound dexamethasone acetate cream (DXMS) and compound Huangbai liquid (HBXJ) on the appearance of the skin of the affected part of experimental rats; B is its scoring chart. In the figure, *, ** and *** respectively indicate that compared with the model group, the P value is less than 0.05, 0.01 and 0.001. The scale in the figure is 1 cm.

[0032] Figure 2 It is a scoring chart of the influence of the test drug on the pathological structure of the skin of the affected part of chronic eczema and its pathological score. A is the chart of the influence of Bletilla striata extract, Bletilla striata polysaccharide (BSP), compound dexamethasone acetate cream (DXMS) and Binghuang Fule (BHFL) on the pathological tissue of the skin of the affected part of experimental rats (HE staining result); B is its scoring chart. In the figure, the white line segment represents the epidermal layer of the thickened skin tissue of the rat; the white arrow represents the infiltration of inflammatory cells; the white hollow arrow represents the subcutaneous fibrous tissue; in the figure, *, ** and *** respectively indicate that compared with the model group, the P value is less than 0.05, 0.01 and 0.001. The scale in the figure is 200 microns (objective lens: ×4) and 50 microns (objective lens: ×20).

[0033] Figure 3 It is a scoring chart of the influence of the test drug on the skin of the affected part of chronic eczema and its damage degree. A is the chart of the influence of Bletilla striata extract, Bletilla striata polysaccharide (BSP), compound dexamethasone acetate cream (DXMS) and Binghuang Fule (BHFL) on the appearance of the skin of the affected part of experimental rats; B is its scoring chart. In the figure, *, ** and *** respectively indicate that compared with the model group, the P value is less than 0.05, 0.01 and 0.001. The scale in the figure is 1 cm.

[0034] Figure 4It is a diagram showing the effect of the test drug on the skin pathological structure of the affected part of chronic eczema and its pathological score. Figure A is a diagram showing the effect of Bletilla striata extract, Bletilla striata polysaccharide (BSP), compound dexamethasone acetate cream (DXMS), and Binghuang Fule (BHFL) on the pathological tissues of the affected skin of experimental rats (HE staining results); Figure B is its scoring diagram. The white line segments in the figure represent the epidermal layer of the thickened skin tissue of the rats; the white arrows represent inflammatory cell infiltration; the white hollow arrows represent subcutaneous fibrous tissue; in the figure, *, **, and *** respectively indicate that compared with the model group, the P value is less than 0.05, 0.01, and 0.001. The scale bars in the figure are 200 microns (objective lens: ×4) and 50 microns (objective lens: ×20).

[0035] Figure 5 It is a diagram showing the effect of Bletilla striata extract, Bletilla striata polysaccharide (BSP), compound dexamethasone acetate cream (DXMS), and Qingpeng ointment (QP) on the scratching frequency of experimental mice. The "-1" on the horizontal axis indicates the test result before modeling; *, **, *** indicate P < 0.05, 0.01, and 0.001, and respectively represent the comparison of differences between the test drug group and the model group at the corresponding time points.

[0036] Figure 6 It is a diagram showing the effect of Bletilla striata extract, Bletilla striata polysaccharide (BSP), compound dexamethasone acetate cream (DXMS), and Qingpeng ointment (QP) on the pathological changes of the skin structure at the scratching site of experimental mice (HE staining diagram and the pathological scoring diagram in the lower right corner). In the HE staining diagram, in the HE pathological diagram, the white line segments represent epidermal thickening; the white solid arrows represent inflammatory cell infiltration; the white hollow arrows represent subcutaneous fibrous tissue. **, *** indicate P < 0.01 and 0.001, and respectively represent the comparison of differences between the test drug group and the model group at the corresponding time points. The scale bars in the figure are 100 microns (objective lens: ×4) and 50 microns (objective lens: ×20). Detailed implementation manners

[0037] Next, the applicant will further elaborate on the technical solution of the present invention in combination with specific embodiments, aiming to enable those skilled in the art to have a clearer understanding and recognition of this application.

[0038] Example 1: A preparation method of an external Bletilla striata polysaccharide for preventing and treating eczema and itching, the steps are as follows.

[0039] Preparation steps of Bletilla striata polysaccharide solution: ① Dissolve the dry powder of Bletilla striata polysaccharide (purchased from Sichuan Victy Bio-Tech Co., Ltd., with a purity of ≥90%) in ultrapure water, and add Tween 80 and glycerol to the resulting solution to obtain an aqueous phase containing Bletilla striata polysaccharide (where the final concentration of Tween 80 is 1.25% V / V; the final concentration of glycerol is 2.81% V / V); ② Dissolve egg yolk lecithin in sesame oil to prepare an oil phase (the final concentration of egg yolk lecithin is 6% W / V, g / mL). ③ Under the condition of a constant temperature water bath at 60°C, drop the oil phase into the aqueous phase according to the volume ratio of the oil phase to the aqueous phase of 1:4, and then stir for 10 min under the condition of 1500 r / min. ④ High-speed shear for 2 min under the condition of 20000 rpm, and then perform high-pressure homogenization 3 times under the condition of 1000 bar to obtain the Bletilla striata polysaccharide solution, and the content of Bletilla striata polysaccharide in the obtained Bletilla striata polysaccharide solution is equal to 1 mg / ml.

[0040] Preparation of Bletilla striata extract: Place 1.62 L of sesame oil in a container, then add 20 g of the dry powder of Bletilla striata, and stir evenly; place it in a colloid mill and grind it cyclically at room temperature for 5 h. After stopping, perform high-speed shearing 3 times (5 min each time) at 20000 rpm through a flash extraction device; filter under negative pressure through a 5000-mesh sieve, collect the filtered oil, obtain the external-use Bletilla striata extract, and store it in a sealed manner after dispensing into 100 ml sterilized bottles.

[0041] Example 2 Influence of the Bletilla striata polysaccharide solution prepared in Example 1 on the DNCB-induced rat contact acute eczema model

[0042] The test animals were 24 male SPF-grade SD rats at 6 weeks old (provided by Liaoning Changsheng Biotechnology Co., Ltd.). They had free access to water and food, and the temperature was maintained at 25 ± 1°C during the breeding period, and the light-dark cycle was 12 hours.

[0043] 1) Grouping, modeling and drug administration

[0044] Randomly divide the 24 rats into 6 groups evenly: blank group, model group, Bletilla striata extract group, Bletilla striata polysaccharide group, compound dexamethasone acetate cream group, compound Phellodendron liquid group.

[0045] After 7 days of adaptive feeding, the hair on the back of each rat was roughly cut off close to the skin with a razor, exposing an area of about 3 cm × 3 cm on the skin. Then, depilatory cream was used for depilation again. After depilation, it was immediately and thoroughly washed with warm water to expose the skin at the experimental site for standby. One day after depilation, except for the blank group which was not treated with any injury, all the rats in the other groups were anesthetized by inhaling ether and placed on a fixing rack. Then, 100 μL of 7% DNCB acetone solution was externally applied to the exposed back skin of the rats for sensitization. Two days after depilation, the same method was used for intensive application once again for re-sensitization. Seven days after depilation, 20 μL of 1% DNCB acetone solution was externally applied to the exposed back skin of the rats with a pipette for challenge. The successful modeling was determined by the appearance of obvious exudation, papules, erythema, etc. in the back area of the rats.

[0046] On the day of drug administration, except for the blank group, 0.25 ml of normal saline was applied to the wound site of the rats in the model group; for the Bletilla striata extract group and the Bletilla striata polysaccharide group, 0.25 ml of Bletilla striata extract and Bletilla striata polysaccharide (1 mg / ml; Bletilla striata polysaccharide solution prepared in Example 1) were respectively applied to the wound sites of the rats in each group; 0.25 ml of compound dexamethasone acetate cream (China Resources Sanjiu Medical & Pharmaceutical Co., Ltd.) was applied to the wound sites of the rats in each group in the compound dexamethasone acetate cream group; 0.25 ml of compound Phellodendron liquid (Shandong Hanfang Pharmaceutical Co., Ltd.) was applied to the wound sites of the rats in each group in the compound Phellodendron liquid group. The drug was administered once a day for 3 consecutive days.

[0047] 2) Gross observation of the skin of the test rats before and after acute eczema modeling

[0048] Select four time periods: the day of successful modeling, 1 day after drug administration, 2 days after drug administration, and 3 days after drug administration. Use a Sony single-lens reflex macro camera to record the wound on the back of the rats to obtain the skin change pictures of acute eczema rats at each time period. According to the skin sensitization reaction experimental scoring standard table, the severity of the skin lesions on the back of the rats in each group (Table 1) was scored. During the evaluation process, to avoid subjective factors, at least two people (3 people in this example) were required to conduct independent evaluations simultaneously, and the results were averaged.

[0049] Table 1. Skin sensitization reaction experimental scoring standard table.

[0050]

[0051] 3) HE staining and pathological analysis of the wound tissues of the test rats

[0052] Three days after drug administration, all the test animals were sacrificed by anesthetization method. The skin tissue at the wound site was removed, wrapped with gauze, and fixed in formalin solution for 24 h. Then, the skin tissue was successively rinsed with running water, dehydrated, embedded in paraffin, followed by tissue sectioning and routine HE staining, and observed and photographed under an optical microscope.

[0053] Pathological scoring of rat skin lesions: Under an optical microscope, the rat skin tissues were pathologically scored according to the following inflammatory scoring table (Table 2), and the results were expressed as mean±SEM.

[0054] Table 2. Pathological scoring of rat skin lesions.

[0055]

[0056] 4) Experimental results

[0057] (1) Gross observation of the skin of rats with acute eczema induced by DNCB

[0058] As Figure 1 shown, from the day when the acute eczema model of rats was successfully induced by DNCB to 3 days after administration, it could be observed that the skin of the rats in the blank group was light red, delicate, smooth, soft in texture, and the skin texture was clearly visible.

[0059] a) On the day when the model was successfully established, severe erythema, severe edema, accompanied by exudation and eschar formation appeared on the wound surface of the rats in the model group; 1 day after administration, the eschar area on the wound surface of the rats in the model group became smaller, there were still large areas of scales, and the degree of erythema and edema was slightly reduced; 2 days after administration, the skin wound surface of the rats in the model group further decreased, the eschar still existed, and the color of the eschar became darker, but the erythema and edema were less severe than those 1 day after administration; 3 days after administration, there was still a small part of eschar on the wound surface, and obvious erythema and edema were accompanied.

[0060] b) Compared with the model group, the compound dexamethasone acetate cream group and compound phellodendron liquid group that simulated clinical medication (there were still large areas of eschar with a darker color of the eschar 1 day after administration; there was still a small part of eschar accompanied by erythema and edema 2 days after administration; the eschar still did not completely fall off 3 days after administration) had certain improvements.

[0061] c) Compared with the model group, the skin of the rats in the bletilla striata extract group scabbed and the color of the eschar became darker 1 day after administration; part of the eschar fell off 2 days after administration, and there was still edema and erythema around the eschar surface; there was still a small part of eschar and erythema 3 days after administration.

[0062] d) Compared with the recovery effect of the bletilla striata extract group, the bletilla striata polysaccharide group showed better performance: the color of the eschar became darker and the area of the eschar became smaller 1 day after administration; most of the eschar and scales fell off 2 days after administration, leaving erythema and edema; the erythema and edema disappeared 3 days after administration, leaving a small area of scales and eschar that did not fall off.

[0063] From the day when the experimental model was successfully established to 3 days after administration, according to the scoring standard table of the skin sensitization reaction experiment, the severity of the skin lesions on the backs of the rats in each group was scored (the full score was 14 points; the scoring table is shown in Table 1). The score of the rats in the blank group was recorded as 0 points. According to the appearance scoring diagram of the rats with acute eczema (Figure 1 )It can be found that:

[0064] (1) The overall score of the rats in the model group showed a relatively slow change trend from the day of successful modeling to 3 days after drug administration;

[0065] (2) Compared with the model group, there were significant differences in the score changes among the test drug groups:

[0066] One day after drug administration, compared with the model group (12.5 ± 0.3; P < 0.001), the appearance scores of the compound dexamethasone acetate cream group (11.3 ± 0.5) and the compound phellodendron liquid group (12.3 ± 0.3) were both improved to a certain extent;

[0067] Two days after drug administration, compared with the model group (10.0 ± 0.4), the appearance scores of the compound dexamethasone acetate cream group (6.0 ± 0.4; P < 0.01) and the compound phellodendron liquid group (8.0 ± 0.7) were both improved to a certain extent;

[0068] Three days after drug administration, compared with the model group (8.5 ± 0.3), although there were still scabs and swelling in the skin of the rats in the compound dexamethasone acetate cream group (4.0 ± 0.4; P < 0.01) and the compound phellodendron liquid group (3.5 ± 0.5; P < 0.01), it showed good skin repair ability and therapeutic effect.

[0069] One day after drug administration, compared with the appearance score of the rats in the model group (12.5 ± 0.3), the skin appearance scores of the bletilla striata extract group (10.7 ± 0.2; P < 0.05) and the bletilla striata polysaccharide group (10.2 ± 0.3; P < 0.01) were smaller;

[0070] Two days after drug administration, compared with the appearance score of the rats in the model group (10.0 ± 0.4), the skin appearance scores of the bletilla striata extract group (6.0 ± 0.4; P < 0.01) and the bletilla striata polysaccharide group (5.7 ± 0.2; P < 0.01) were closer;

[0071] Three days after drug administration, compared with the appearance score of the rats in the model group (8.5 ± 0.3), it was found by comparison that the scores of the bletilla striata extract group (3.3 ± 0.5; P < 0.01) and the bletilla striata polysaccharide group (2.8 ± 0.4; P < 0.001) were closer.

[0072] (2) HE staining observation and pathological analysis of the wound tissues of DNCB-induced acute eczema rats

[0073] Three days after drug administration (the 10th day of the experiment), all the test rats were sacrificed by anesthesia, and the skin tissues at the wound sites were taken. After HE staining, the pathological analysis results were as Figure 2 shown.

[0074] It can be seen that the cells of the epidermis and dermis of the blank group rats are arranged normally without edema, and a large number of hair follicles and other accessory organs can be seen, and no abnormal pathological morphological changes are observed.

[0075] a) Compared with the blank control group, the skin tissue of the rats in the model group showed the following: ① the epidermal structure was incomplete, the ratio of the epidermis to the dermis was unbalanced, the stratum corneum and granular layer were thickened, the stratum spinosum was thickened, and the epidermal processes were widened and elongated; ② a large number of sweat gland hyperplasia and inflammatory cell infiltration were observed in the dermis; ③ the pathological score of the skin tissue (2.7±0.5; P<0.001) was significantly lower than that of the blank group (10.0±0.0).

[0076] b) Compared with the model group, the efficacy of the compound dexamethasone acetate cream group was not very obvious: ① The structure of the epidermis was disordered and rough, the epidermis was over-keratinized, the thickening of the stratum corneum and granular layer was not significantly improved compared with the model group, and the epidermal protrusions were widened and elongated; ② Hair follicles and other skin appendages were observed in the dermis, accompanied by a large number of inflammatory cell infiltration and red blood cell extravasation; ③ Its skin tissue pathology score (4.7±0.5) was slightly higher than that of the model group.

[0077] c) At the same time, compared with the model group, the therapeutic effect of the compound phellodendron liquid group was not ideal: ① Although the structure of the epidermis was intact, there was no significant improvement in the thickening of the stratum corneum and the hypertrophy of the stratum spinosum, and the epidermal processes were widened and elongated; ② In the dermis, the growth of skin appendages such as hair follicles was observed, accompanied by inflammatory cell infiltration and red blood cell extravasation; ③ The pathological score of its skin tissue (6.0±0.4; P<0.01) was higher than that of the model group.

[0078] d) Skin tissue of rats in the Bletilla striata extract group: ① The skin structure was intact, with obvious keratinization in the epidermis and thickening of the stratum corneum and stratum granulosum; ② In the dermis, hair follicles and other skin appendages were observed to grow, accompanied by moderate inflammatory cell infiltration, and interstitial congestion and edema; ③ The pathological score of the skin tissue (6.0±0.4; P<0.001) was higher than that in the model group.

[0079] e) The effect of the Bletilla striata polysaccharide group was more obvious: ① The structure of the entire skin layer was intact, the skin stratum corneum was thinner than that of the model group, and the epidermal processes were slightly widened and elongated; ② A large number of hair follicles and other skin appendages were observed in the dermis, and no obvious inflammatory cell infiltration and red blood cell extravasation were observed; ③ Its skin tissue pathology score (6.5±0.6; P<0.001) was significantly higher than that of the model group.

[0080] Example 3 Effect of the Bletilla striata polysaccharide solution prepared in Example 1 on the DNCB-induced rat contact chronic eczema model

[0081] The test animals were 24 male 6-week-old SPF SD rats (provided by Liaoning Changsheng Biotechnology Co., Ltd.), with free access to water and food, and the temperature was maintained at 25±1°C during the feeding period, with a light-dark cycle of 12 hours.

[0082] 1) Sub - group modeling and drug administration

[0083] Twenty - four rats were randomly and evenly divided into 6 groups: blank group, model group, Bletilla striata extract group, Bletilla striata polysaccharide group, Compound Dexamethasone Acetate Cream group, and Binghuang Fule Ointment group.

[0084] After 7 days of adaptive feeding, the back hair of each rat was roughly cut off close to the skin with a razor, exposing an area of about 3 cm × 3 cm on the back for the experiment. Then, depilatory cream was used for depilation again. After depilation, it was immediately and thoroughly washed with warm water to expose the skin at the experimental site for standby. On the 1st day after depilation, except for the blank group without any injury treatment, all the rats in the other groups were anesthetized by inhaling ether and placed on a fixing rack. Then, 100 μL of 7% DNCB acetone solution was applied externally to the exposed back skin of the rats for sensitization. One week later, the hair in the shaved area on the rats' backs was removed again, and the next day, 50 μL of 5% DNCB acetone solution was applied externally for the second sensitization. Thereafter, sensitization continued, once every 5 days, for 3 consecutive times. The success of the model was determined by the appearance changes of the skin on the rats' backs, such as skin thickening, surface roughness, papules, and swelling.

[0085] On the 3rd day after the successful establishment of the modeling experiment, except for the blank group, 0.25 ml of normal saline was applied to the wound site of the rats in the model group; 0.25 ml of Bletilla striata extract and Bletilla striata polysaccharide (1 mg / ml; Bletilla striata polysaccharide solution prepared in Example 1) were respectively applied to the wound sites of the rats in the Bletilla striata extract group and Bletilla striata polysaccharide group; 0.25 ml of Compound Dexamethasone Acetate Cream was applied to the wound site of the rats in the Compound Dexamethasone Acetate Cream group; 0.25 ml of Binghuang Fule Ointment (Tibet Hairong Tangguo Pharmaceutical Co., Ltd.) was applied to the wound site of the rats in the Binghuang Fule Ointment group. Once a day, for 3 consecutive days of drug administration.

[0086] 2) Gross observation of the skin of the rats under test before and after chronic eczema modeling

[0087] Select three time periods: the day of successful modeling, 1 day after drug administration, and 3 days after drug administration. The back wounds of the rats were recorded with a Sony single - lens reflex macro camera to obtain the skin change pictures of the rats with chronic eczema at each time period. According to the skin sensitization reaction experimental scoring standard table (Table 1), the severity of the skin lesions on the backs of the rats in each group was scored. During the evaluation process, in order to avoid subjective factors, two people were used to conduct independent evaluations simultaneously, and the results were averaged.

[0088] 3) HE staining and pathological analysis of the wound tissues of the rats under test

[0089] Three days after administration, all the test animals were sacrificed by anesthesia after taking pictures. The skin tissues at the wound site were removed, wrapped with gauze, and fixed in formalin solution for 24 h. Then the skin tissues were rinsed with running water, dehydrated, embedded in paraffin, followed by tissue sectioning, routine HE staining, and observed and photographed under an optical microscope.

[0090] Pathological scoring of rat skin lesions: Under an optical microscope, the rat skin tissues were pathologically scored according to the inflammatory scoring table (Table 2), and expressed as mean±SEM.

[0091] 4) Experimental results

[0092] (1) Gross observation of the skin of rats with DNCB-induced chronic eczema

[0093] a) As Figure 3 shown, during the period from successful establishment of the model of DNCB-induced chronic eczema in rats to 3 days after administration, it could be observed that the skin of rats in the blank group was light red, delicate, smooth, soft in texture, and the skin texture was clearly visible.

[0094] b) After successful establishment of the model, the wound surface of rats in the model group had severe erythema, severe edema, and was accompanied by severe exfoliation and scales: 1 day after administration, the scale and exfoliation area of the wound surface skin of rats in the model group became smaller, and the degree of erythema and edema was slightly reduced; 3 days after administration, the skin wound surface of rats in the model group further decreased, scales and exfoliation still existed, and there were still erythema and edema.

[0095] c) Compared with the model group, the compound dexamethasone acetate cream group and Binghuang Fule Ointment group that simulated clinical medication (there were still large areas of scales and the scab color was darker 1 day after administration; there were still a small part of scabs not fallen off, accompanied by erythema and edema 3 days after administration) had certain curative effects.

[0096] d) The skin of rats in the Bletilla striata extract group scabbed and the scab color became darker 1 day after administration, and there were erythema and edema around the scab surface; 3 days after administration, part of the scab fell off, leaving a small part of the scab, and there were erythema and edema around the scab.

[0097] e) Compared with the recovery effect of the Bletilla striata extract group, the Bletilla striata polysaccharide group showed better performance: the scab surface area became smaller 1 day after administration; most of the scales fell off 3 days after administration, leaving a small part of the scab, accompanied by a small area of erythema.

[0098] During the period from successful establishment of the experimental model to 3 days after administration, according to the scoring standard table of skin sensitization reaction experiment, the severity of skin lesions on the backs of rats in each group was scored (the full score was 12 points), and the score of rats in the blank group was recorded as 0 points.

[0099] a) According to the appearance scoring diagram of rats with chronic eczema ( Figure 3) It can be found that the overall score of the rats in the model group changed slowly from the day of successful modeling to 3 days after drug administration;

[0100] b) The positive control group scores changed significantly:

[0101] After one day of administration, the skin appearance scores of rats in the compound dexamethasone acetate cream group (8.8±0.5) and the Binghuangfule ointment group (6.8±0.5; P<0.05) changed less than those in the model group (10.0±0.4; P<0.001).

[0102] Three days after administration, the skin of rats in the compound dexamethasone acetate cream group (4.0±0.4) and the Binghuangfule ointment group (3.5±0.6) still had scabs and redness and swelling compared with the model group (6.7±0.6).

[0103] d) Compared with the model group, the Bletilla striata extract group and Bletilla striata polysaccharide group also showed good repair effects:

[0104] One day after administration, the skin appearance scores of rats in the Bletilla striata extract group (6.8±0.3; P<0.001) and the Bletilla striata polysaccharide group (6.0±0.4; P<0.01) were lower than those in the model group (10.3±0.3).

[0105] After 3 days of administration, the appearance score of the rats in the model group was 7.3±0.4. Through comparison among the groups, it was found that the score of the Bletilla striata polysaccharide group was the lowest (3.3±0.3; P<0.01), and the effect of the Bletilla striata extract group (3.8±0.3; P<0.01) was slightly weaker than that of the Bletilla striata polysaccharide group.

[0106] 2) HE staining observation and pathological analysis of wound tissues in rats with chronic eczema induced by DNCB

[0107] Three days after administration, all the rats were anesthetized and killed, and the skin tissues at the wound sites were taken for HE staining to obtain the pathological analysis results. Figure 4 As shown in the figure, it can be seen that the cells of the epidermis and dermis of the rats in the blank group are arranged normally without edema, and a large number of hair follicles and other accessory organs can be seen without any abnormal pathological morphological changes.

[0108] a) Compared with the blank control group, the skin tissue of the rats in the model group showed: ① the epidermal structure was blurred, the ratio of the epidermis to the dermis was unbalanced, obvious keratinization was observed, the stratum corneum and granular layer were thickened, the stratum spinosum was thickened, and the epidermal processes were widened and elongated; ② inflammatory cell infiltration in the dermis; ③ the pathological score of the skin tissue (1.5±0.3; P<0.001) was significantly lower than that of the blank group (10.0±0.0).

[0109] b) Compared with the model group, the effect of Compound Dexamethasone Acetate Cream group (3.8 ± 0.5; P < 0.05) was not very obvious: ① The structure of the epidermal layer was blurred, with hyperkeratosis, and the stratum corneum and granular layer were thickened, showing no obvious improvement compared with the model group, and acanthosis was present; ② The dermis was thickened, with excessive proliferation of fibers and moderate infiltration of inflammatory cells; ③ The pathological score of its skin tissue was slightly higher than that of the model group.

[0110] c) At the same time, compared with the model group, the treatment effect of Binghuangfulao Ointment group (5.2 ± 0.4; P < 0.01) was not ideal either: ① The thickening of the epidermal layer showed no obvious improvement compared with the model group, with hyperkeratosis, thickening of cell gaps, and no obvious reduction in acanthosis, and epidermal papillae were widened and elongated; ② The dermis was thickened, with excessive proliferation of fibers accompanied by highly infiltrated inflammatory cells and extravasation of red blood cells; ③ The pathological score of its skin tissue was higher than that of the model group.

[0111] Compared with the model group, the Bletilla striata extract group and the Bletilla striata polysaccharide group also showed certain repair effects:

[0112] d) The skin tissue of the rats in the Bletilla striata extract group: ① The structure of the epidermal layer was not very clear, the epidermal layer was thickened, and the cell gaps were thickened; ② There was infiltration of inflammatory cells in the dermis, and the stroma was congested and edematous; ③ The pathological score of its skin tissue (5.0 ± 0.4; P < 0.001) was higher than that of the model group.

[0113] e) While the skin tissue of the Bletilla striata polysaccharide group showed better performance: ① The epidermal layer was clear and complete, the stratum corneum of the skin was thinner than that of the model group, and the keratinized substances were slightly thickened; ② There was a small amount of neutrophil infiltration in the dermis; ③ The pathological score of its skin tissue (6.7 ± 0.2; P < 0.001) was significantly higher than that of the model group.

[0114] Example 4 Effect of the Bletilla striata polysaccharide solution prepared in Example 1 on the pruritus model induced by combined administration of histamine and 4-aminopyridine

[0115] The test animals were 30 male SPF-grade KM mice at 6 weeks old (provided by Liaoning Changsheng Biotechnology Co., Ltd.). They had free access to water and food, and the temperature was maintained at 25 ± 1 °C during the breeding period, with a light-dark cycle of 12 hours.

[0116] 1) Grouping, modeling and drug administration

[0117] Thirty healthy SPF-grade KM mice were randomly divided into 6 groups: blank control group (Control), model group (Model), Bletilla striata extract group, Bletilla striata polysaccharide group (BSP), compound dexamethasone acetate cream group (DXMS), and Qizheng Qingpeng ointment group (QP). After 7 days of adaptive feeding, the hair on the back of the neck and behind the ears of each mouse was roughly cut off close to the skin with a razor, and the exposed skin area was about a circular area with a diameter of about 1.0 cm. Then, depilatory cream was used for depilation again. Immediately after depilation, it was thoroughly washed with warm water to expose the skin of the experimental site for standby. One day after depilation, the itching frequency of each group of mice before drug administration was recorded by video. Two days after depilation, except for the control group which was not given subcutaneous injection, histamine (2 mg / ml, 50 μl) and 4-aminopyridine (0.8 mg / ml, 50 μl) were subcutaneously injected into the neck and back of all mice in the remaining groups to induce scratching behavior in mice.

[0118] During the modeling process, except for the blank control group, histamine (2 mg / ml, 50 μl) and 4-aminopyridine (0.8 mg / ml, 50 μl) were subcutaneously injected into the animals in the model group, Bletilla striata extract group, Bletilla striata polysaccharide group, compound dexamethasone acetate cream group, and Qizheng Qingpeng ointment group. Immediately after injection, they were placed in a transparent observation box and observed for 30 min, and the scratching latency and total number of scratches were recorded with a digital video camera. When the second injection of the modeling drug was given, except for the control group and the model group, the shaved areas of each mouse in the remaining groups were smeared with Bletilla striata extract, Bletilla striata polysaccharide (1 mg / ml; Bletilla striata polysaccharide solution prepared in Example 1), compound dexamethasone acetate cream (China Resources Sanjiu Medical & Pharmaceutical Co., Ltd.), and Qizheng Qingpeng ointment (Tibet Qizheng Tibetan Medicine Co., Ltd.) for 4 days until the end of the experiment.

[0119] 2) Ethological observation and itching score of scratching behavior in the test mice

[0120] The severity of itching among groups was observed through the number of scratching behaviors of the mice. After subcutaneous injection of histamine and 4-aminopyridine into the neck and back of the mice, the mice were immediately placed in a transparent observation cage and photographed for 30 min in a quiet environment without people, and the number of scratching behaviors of the mice was recorded. When a mouse lifted its hind paw to scratch the shaved area and then put the paw back on the floor or into its mouth until it stopped, it was recorded as 1 scratch (continuous scratching was also recorded as 1 time). The itching frequency of each group of mice was recorded ( Figure 5 ).

[0121] 3) HE staining and pathological analysis of the wound tissues of the test mice

[0122] Once a day, 4 days after drug administration (i.e., the end day of the experiment), all the test animals were sacrificed by anesthesia. The skin tissues at the wound site were removed, wrapped with gauze, and then fixed in formalin solution for 24 h. Then the skin tissues were rinsed with running water, dehydrated, embedded in paraffin, followed by tissue sectioning, routine HE staining, and observed under an optical microscope. Pathological scoring of the skin tissues was performed ( Figure 6 ).

[0123] 4) Experimental results

[0124] (1) Case analysis of itchy mice

[0125] During the planned 6 recording time periods (1 day before administration of the modeling drug, the day of only administration of the modeling drug, 1 day, 2 days, 3 days, and 4 days after administration of the treatment drug), the scratching times of mice with itching caused by the combined use of histamine and 4-aminopyridine were recorded for 30 min in a transparent observation cage (the results are as Figure 5 shown).

[0126] a) It can be seen from Figure 5 that the modeling scheme of this experiment was very successful. The scratching frequency of each group of mice increased significantly after administration of the modeling drug on the day of modeling, and the scratching frequency was generally in the range of 30 - 40 times within 30 min. The scratching times of the blank group did not change significantly 1, 2, 3, and 4 days after administration (1.2 ± 0.3, 1.4 ± 0.2, 2.0 ± 0.5, 1.6 ± 0.4 respectively);

[0127] b) Compared with the blank group, the scratching frequencies of the mice in the model group were significantly increased 1, 2, 3, and 4 days after administration (37.8 ± 1.8, P < 0.001; 36.6 ± 1.8, P < 0.001; 35.0 ± 1.3, P < 0.001; 34.2 ± 1.5, P < 0.001 respectively). During the experiment, the itching frequencies of the mice in the blank group and the model group remained relatively stable, indicating that this modeling scheme was successful.

[0128] c) Compared with the model group, the scratching times of the mice in the compound dexamethasone acetate cream group (28.6 ± 3.1, P < 0.01; 16.0 ± 0.7, P < 0.001; 15 ± 1.3, P < 0.001; 16.4 ± 2.3, P < 0.001) also showed a significant decrease over time after administration, and there were also significant differences. The results indicate that compound dexamethasone acetate cream also has a good therapeutic effect on itching caused by the combined use of histamine and 4-aminopyridine.

[0129] d) Compared with the model group, the scratching times of the mice in the Qizheng Qingpeng Ointment group after 1, 2, 3, and 4 days of drug administration (25.8 ± 0.9, P < 0.001; 22.8 ± 1.4, P < 0.001; 21.0 ± 2.2, P < 0.001; 17.3 ± 2.0, P < 0.001) also showed a significant decrease over time, with significant differences, indicating that Qingpeng Ointment also showed good therapeutic effects on itching caused by the combined use of histamine and 4-aminopyridine.

[0130] e) Compared with the model group, the scratching times of the mice in the Bletilla striata polysaccharide group after 1, 2, 3, and 4 days of drug administration (37.4 ± 1.9; 20.4 ± 1.5, P < 0.001; 23.8 ± 3.9, P < 0.01; 22.2 ± 1.3, P < 0.001) also showed a significant decrease over time, and there were significant differences after 2, 3, and 4 days of drug administration. The results showed that Bletilla striata polysaccharide had a certain alleviating effect on itching caused by the combined use of histamine and 4-aminopyridine.

[0131] f) Compared with the model group, the scratching times of the mice in the Bletilla striata extract group after 1, 2, 3, and 4 days of drug administration (30.8 ± 1.9; 28.6 ± 1.8; 22.2 ± 2.4, P < 0.001; 20.8 ± 1.8, P < 0.001) also showed a significant decrease over time, and there were significant differences after 3 and 4 days of drug administration. The results showed that Bletilla striata extract had a certain therapeutic effect on itching caused by the combined use of histamine and 4-aminopyridine.

[0132] (2) Results of histopathological experiments

[0133] After all the experimental mice were sacrificed by cervical dislocation (4 days after modeling), the pathological analysis results of the skin at the affected part after HE staining were as Figure 6 shown.

[0134] a) As Figure 6 seen from A, the epidermal layer structure of the mice in the blank group was complete, and the epithelial cells were arranged in polarity; a large number of hair follicles and other appendages could be seen under the skin, and no abnormal pathological morphological changes such as edema and infiltration of inflammatory cell nests were observed.

[0135] b) Compared with the blank control group, the skin of the scratching part of the mice in the model group: ① The epidermal layer was partially missing and the structure was disordered, and the epidermal layer was significantly thickened; ② Obvious edema and a large number of inflammatory cell nests were infiltrated in the dermis layer; ③ The pathological score of its skin tissue (2.0 ± 0.3, P < 0.001) was significantly less than that of the blank group (9.0 ± 0.3).

[0136] c) Compared with the model group, the skin of the scratched area of ​​mice in the compound dexamethasone acetate cream group showed: ① the epidermis was intact and slightly thickened; ② hair follicle hyperplasia and mild edema as well as a small amount of inflammatory cell infiltration were observed in the dermis; ③ its skin tissue pathology score (6.8±0.3; P<0.001) was significantly higher than that in the model group.

[0137] d) Compared with the model group, the skin of the scratched area of ​​the mice in the Qingpeng ointment group showed: ① the epidermis was intact and slightly thickened; ② a small amount of hair follicle hyperplasia and a small amount of inflammatory cell infiltration were visible in the dermis; ③ its skin tissue pathology score (5.4±0.2; P<0.001) was significantly higher than that of the model group.

[0138] e) Compared with the model group, the skin of the scratched area of ​​mice in the Bletilla striata extract group showed: ① the epidermis was intact and slightly thickened, with acceptable arrangement of epithelial cells; ② the dermis showed proliferation of skin appendages such as hair follicles; ③ its skin tissue pathology score (4.6±0.2, P<0.001) was higher than that of the model group.

[0139] f) Compared with the model group, the skin of the scratched area of ​​mice in the Bletilla striata polysaccharide group showed: ① partial loss of the epidermal structure; ② proliferation of skin appendages such as hair follicles and focal inflammatory cell infiltration in the dermis; ③ its skin tissue pathology score (3.4±0.2, P<0.01) was higher than that of the model group.

Claims

1. Use of Bletilla striata polysaccharide preparation in the preparation of drugs for preventing and treating eczema, characterized in that, Dissolve the dried powder of Bletilla striata polysaccharide in ultrapure water containing Tween 80 and glycerol to prepare an aqueous phase, dissolve egg yolk lecithin in edible vegetable oil to prepare an oil phase, drop the oil phase into the aqueous phase according to the volume ratio of the oil phase to the aqueous phase of 1:4, stir, and then perform high-speed shearing and high-pressure homogenization to obtain the Bletilla striata polysaccharide preparation. The concentration of Bletilla striata polysaccharide in the Bletilla striata polysaccharide preparation is 1 mg / mL; The eczema is caused by a sensitizing drug.

2. The use according to claim 1, characterized in that, In the aqueous phase: the final concentration of Tween 80 is 1.25% V / V, and the final concentration of glycerol is 2.81% V / V; in the oil phase: the final concentration of egg yolk lecithin is 6% W / V, g / mL.

3. The use according to claim 1, characterized in that, The edible vegetable oil is selected from at least one of sesame oil, peanut oil, coconut oil, soybean oil, linseed oil, castor oil, and olive oil.

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