Application of pine pollen polysaccharide in preventing and treating atopic eczema

Abstract

The invention discloses the application of pine pollen polysaccharide in preventing and treating atopic eczema, and belongs to the technical field of atopic eczema prevention and treatment. The pine pollen polysaccharide is prepared by the following method: adding broken wall masson pine pollen to petroleum ether, heating and refluxing extraction, collecting filter residue by suction filtration, drying and removing solvent, and obtaining defatted masson pine pollen; heating and refluxing extraction of defatted masson pine pollen with 95% ethanol, collecting filter residue by suction filtration, and drying and removing residual solvent to obtain impurity-removed masson pine pollen; adding water to the impurity-removed masson pine pollen, heating and boiling, repeatedly extracting and combining filtrates, heating and concentrating to obtain a concentrated solution; removing protein from the concentrated solution, precipitating with alcohol, centrifuging and precipitating, and freezing and decompressing to obtain pine pollen polysaccharide. Pine pollen polysaccharide is used to prevent and treat atopic eczema. Pine pollen polysaccharide comes from natural plants. Compared with traditional drugs, pine pollen polysaccharide has higher safety and lower side effects, and is particularly suitable for long-term use and sensitive populations such as infants and young children.

Description

Technical Field

[0001] The invention relates to the technical field of prevention and treatment of atopic eczema, and in particular to application of pine pollen polysaccharide in prevention and treatment of atopic eczema. Background Art

[0002] Atopic eczema is a common chronic, recurrent skin disease that is common in infants and children and may also affect adults. It is characterized by impaired skin barrier function, immune abnormalities, and hypersensitivity to environmental stimuli. The main symptoms of the disease include severe itching, dry skin, erythema, scaling, and secondary infections. The cause of atopic eczema is not yet fully understood and is generally believed to be the result of multiple factors such as genetics, environmental factors, skin barrier defects, and immune system abnormalities. At present, the drugs used to treat atopic eczema mainly include topical glucocorticoids, such as hydrocortisone and triamcinolone, but long-term use may cause side effects such as skin atrophy and capillary dilation; calcineurin inhibitors, such as tacrolimus and pimecrolimus, are suitable for long-term control, but may increase the risk of infection and are expensive; oral antihistamines are used to relieve itching, but the effect is limited in some patients and may cause drowsiness; immunosuppressants and biologics, such as cyclosporine and dupilumab, are suitable for patients with moderate to severe eczema, but have large side effects (such as hepatotoxicity of immunosuppressants) or are expensive, and their long-term safety remains to be verified. Therefore, it is of great significance to develop new drugs with higher safety and suitable for long-term use.

[0003] Pine pollen is the pollen of Pinus massoniana and other plants of the Pinaceae family. Pinus massoniana is a tree species widely distributed in my country, belonging to the gymnosperms, Pinales, Pinaceae. Like other Pinaceae tree species, Pinus massoniana is a monoecious plant, with spore leaves arranged in cones to form female and male cones. Pine pollen is collected from male cones of Pinus massoniana, and is dried and impurities removed. Pine pollen contains a variety of nutrients needed by the human body, such as protein, polysaccharides, fats and some organic acids, vitamins, enzymes, flavonoids, etc. More than 200 types of ingredients have been discovered, and they are reasonably matched. After decades of research, it has been found that pine pollen has antioxidant, anti-fatigue, anti-tumor and immune-regulating effects. In addition, pine pollen also has significant effects in protecting the liver, preventing prostatitis, and delaying aging. Polysaccharides are formed by the condensation and dehydration of multiple monosaccharide molecules. They are a type of carbohydrate substance with complex molecular structure and large size. Polysaccharides have many biological effects such as regulating and enhancing the immunity of animal bodies. There is no report on the use of pine pollen polysaccharides for the prevention and treatment of atopic eczema. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide the use of pine pollen polysaccharide in preventing and treating atopic eczema.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides the use of pine pollen polysaccharide in the preparation of a medicament for preventing and treating atopic eczema.

[0007] Preferably, the pine pollen polysaccharide prevents and treats atopic eczema through at least one of the following pathways:

[0008] (1) Inhibit inflammatory infiltration in skin tissue and abnormal thickening of the epidermis;

[0009] (2) Inhibit programmed cell death in skin tissue;

[0010] (3) Reduce the number of mast cells in skin tissue;

[0011] (4) Reduce the distribution of Th17 cells in skin tissue.

[0012] Preferably, the pine pollen polysaccharide is prepared by the following method:

[0013] (1) Add the broken Masson pine pollen to petroleum ether, heat and reflux for extraction at 40-60°C for 2-4 h, collect the residue by filtration, and dry to remove the solvent to obtain defatted Masson pine pollen;

[0014] (2) extracting the defatted Masson pine pollen with 95% ethanol under heating reflux at 60-80°C for 1-3 h, collecting the residue by filtration, and drying to remove the residual solvent to obtain the impurity-free Masson pine pollen;

[0015] (3) Add water to the impurity-removed Masson pine pollen, heat and boil for 0.5-1.5 h, collect the filtrate by suction, repeat the extraction 2-4 times, combine the filtrates, heat and concentrate to obtain a concentrate;

[0016] (4) The concentrate was subjected to the Sevage method to remove protein, and then anhydrous ethanol was added and stirred until the final ethanol concentration was 70-90%. The concentrate was centrifuged and freeze-dried under reduced pressure to obtain pine pollen polysaccharide.

[0017] Preferably, the glycosidic bond type of the pine pollen polysaccharide is β-glycosidic bond.

[0018] Preferably, the pine pollen polysaccharide comprises the following components in molar percentage:

[0019] Arabinose 30.41%, rhamnose 6.52%, galactose 6.49%, glucose 42.89%, xylose 6.56% and galacturonic acid 7.13%.

[0020] Preferably, in step (1), the ratio of the added amount of the broken Masson pine pollen to the added amount of petroleum ether is 1 g: 3-5 mL.

[0021] Preferably, in step (2), the ratio of the added amount of defatted Masson pine pollen to 95% ethanol is 1 g: 3-5 mL.

[0022] Preferably, in step (3), the mass ratio of the removed Masson pine pollen to water is 1:6-10.

[0023] Preferably, in step (4), the centrifugal separation conditions are: centrifugal force is 3000×g, and the centrifugal separation time is 10 min.

[0024] Beneficial effects of the present invention:

[0025] 1. The present invention utilizes pine pollen polysaccharide to prevent and treat atopic eczema. Pine pollen polysaccharide is derived from natural plants. Compared with traditional drugs, pine pollen polysaccharide has higher safety and lower side effects, and is particularly suitable for long-term use and for sensitive groups such as infants and young children.

[0026] 2. Pine pollen polysaccharides intervene in atopic eczema through multiple mechanisms, including inhibiting inflammatory infiltration in skin tissue, reducing epidermal thickening, inhibiting programmed cell death and reducing the number of mast cells. This multi-target effect can effectively alleviate the symptoms of eczema and prevent recurrence of the disease.

[0027] 3. The three-dimensional network porous structure of pine pollen polysaccharide shows that it has good hydrophilicity and moisturizing properties, which can help patients with eczema maintain skin moisture, reduce dryness and peeling problems, and relieve the itching symptoms of eczema; the complex network structure of pine pollen polysaccharide helps to form a natural barrier protection layer to prevent external allergens and pollutants from irritating the skin and reduce the aggravation of eczema symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : Pine pollen polysaccharide content determination diagram;

[0029] Figure 2 : Infrared spectrum of pine pollen polysaccharide;

[0030] Figure 3 : The monosaccharide composition determination diagram of pine pollen polysaccharide and the standard product comparison diagram, wherein A is the standard product comparison diagram; B is the monosaccharide composition determination diagram of the pine pollen polysaccharide prepared in Example 1;

[0031] Figure 4 : Surface morphology of pine pollen polysaccharide;

[0032] Figure 5 : Photos of mouse skin lesions;

[0033] Figure 6: Pathological images of hematoxylin-eosin staining, where A is a representative image of hematoxylin-eosin staining tissue, and B is a histopathological scoring diagram;

[0034] Figure 7 : Mast cell staining images; A is a representative image of mast cell staining tissue, and B is a statistical diagram of mast cell numbers;

[0035] Figure 8 :TUNEL programmed cell death staining images;

[0036] Fig. 9 : Th17 (IL-17A) immunohistochemical staining images. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0039] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0040] The wall-broken Pinus massoniana pollen of the present invention is purchased from Ning'an Yuanhe Agricultural and Sideline Products Co., Ltd., and the wall-broken rate is 99%.

[0041] Example 1: Preparation of pine pollen polysaccharide:

[0042] (1) Add 100 g of broken Masson pine pollen to 400 mL of petroleum ether (boiling point 40-60°C), heat and reflux at 50°C for 3 h, collect the residue by filtration, and dry to remove the solvent to obtain defatted Masson pine pollen;

[0043] (2) Defatted Masson pine pollen was extracted with 95% ethanol under heating reflux at 70°C for 2 h. The ratio of defatted Masson pine pollen to 95% ethanol was 1 g:4 mL. After the extraction was completed, the residue was collected by filtration and then dried at 40±0.1°C to remove the residual solvent to obtain impurity-free Masson pine pollen.

[0044] (3) Add 8 times the mass of water to the impurity-free Masson pine pollen, heat and boil for 1 h, repeat the extraction 3 times, combine the 3 filtrates, heat and concentrate to 200 mL to obtain a concentrated solution;

[0045] (4) Sevage protein removal solution was prepared by mixing chloroform and butanol in a volume ratio of 4:1. The Sevage solution and the concentrate obtained in step (3) were used to remove the protein in the concentrate in a volume ratio of 3:1. The polysaccharide solution layer was taken out, and then anhydrous ethanol was added and stirred until the final ethanol concentration was 80%. The solution was centrifuged at 3000×g for 10 min to separate the precipitate, and the pine pollen polysaccharide was obtained by freeze-drying under reduced pressure.

[0046] Test example:

[0047] 1. The pine pollen polysaccharide content prepared in Example 1 was tested using the phenol-sulfuric acid method.

[0048] like Figure 1 As shown, the results show that the purity of the pine pollen polysaccharide prepared in Example 1 is 84.68%.

[0049] 2. The functional group type of the pine pollen polysaccharide prepared in Example 1 was determined using an infrared spectrometer.

[0050] like Figure 2 As shown in the infrared spectrum, 3410 cm −1 The signal at 2931 cm is due to the stretching vibration of OH. −1 The signal at 1631 cm is caused by CH, which is the characteristic absorption peak of polysaccharides; the asymmetric and symmetric vibrations of the COO structure correspond to 1631 cm −1 and 1401 cm −1 The absorption peak at 1241 cm −1 、1042cm −1 shows the OH vibration, 1042 cm −1 The strong absorbance at 762 cm −1 There is a weak absorbing bond at the pyranose ring, which is consistent with the backbone bending of the pyranose ring.

[0051] 3. The monosaccharide composition of the pine pollen polysaccharide prepared in Example 1 was determined using a mass spectrometer.

[0052] Depend on Figure 3 It can be seen that pine pollen polysaccharide is a heteropolysaccharide composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), and galacturonic acid (Gal-UA), with molar percentages of 6.52%, 30.41%, 6.49%, 42.89%, 6.56%, and 7.13%, respectively, among which arabinose and glucose are the main monosaccharides.

[0053] 4. The surface morphology of the pine pollen polysaccharide prepared in Example 1 was tested using a scanning electron microscope.

[0054] Depend on Figure 4 It can be seen that the surface morphology of pine pollen polysaccharide was obtained by scanning electron microscopy technology. The image shows that the surface of pine pollen polysaccharide is relatively rough, with a three-dimensional network porous structure, similar to a sponge, indicating that it has good hydrophilicity, moisture retention and biocompatibility.

[0055] Experimental Example 1: Effect of pine pollen polysaccharide on atopic eczema model in mice:

[0056] 1. Experimental groups:

[0057] Fifty 7-week-old Kunming mice were randomly divided into 5 groups, including a normal group, an atopic eczema model group, a 2% pine pollen polysaccharide treatment group, a 5% pine pollen polysaccharide treatment group and a positive drug treatment group (dexamethasone treatment group), with 10 mice in each group.

[0058] 2. Establishment of mouse atopic eczema model:

[0059] All mice had their back hair shaved before the experiment and were depilated with a depilatory cream. Except for the normal group, the rest of the mice were sensitized by applying 100 μL of 0.5% DNCB solution (the matrix solution was acetone: olive oil = 1:3) to the depilatory area on the back on the 1st, 4th, and 7th days of the experiment. On the 14th, 17th, 20th, 23rd, 26th, and 29th days, 100 μL of 0.5% DNCB solution was applied again to the left ear and depilatory area on the back of the mice for stimulation. The model was successfully established when typical clinical symptoms appeared on the back of the mice: erythema, edema / papules, epidermal exfoliation / scratches, and scaling.

[0060] The normal group was smeared with an equal amount of matrix solution (the matrix solution was acetone: olive oil = 1:3).

[0061] Starting from the 14th day, the drug was administered every day. The administration of drugs in different treatment groups is as follows:

[0062] Normal group: white vaseline was evenly applied on the hair removal area on the back of mice once a day.

[0063] Atopic eczema model group: White vaseline was evenly applied to the hair-depleted area on the back of mice once a day.

[0064] 2% pine pollen polysaccharide treatment group: 0.2 g of the pine pollen polysaccharide prepared in Example 1 was added to 9.8 g of white vaseline, and the mixture was stirred at 50°C for 1 h to obtain a 2% pine pollen polysaccharide cream, which was evenly applied to the depilated area on the back of the mice, once a day;

[0065] 5% pine pollen polysaccharide treatment group: 0.5 g of the pine pollen polysaccharide prepared in Example 1 was added to 9.5 g of white vaseline, and the mixture was stirred at 50°C for 1 h to obtain a 5% pine pollen polysaccharide cream, which was evenly applied to the depilated area on the back of the mice, once a day;

[0066] Positive drug (dexamethasone) treatment group: 5 mg of dexamethasone was added to 9.995 g of white vaseline, stirred at 50 °C for 1 h to obtain 0.05% dexamethasone cream, which was evenly applied to the depilated area on the back of the mice once a day;

[0067] The mice were killed on day 30 for subsequent experiments.

[0068] 3. Test indicators:

[0069] The clinical symptoms on the back of the mice were observed (photographs were taken), and the skin tissue was subjected to pathological staining (hematoxylin-eosin staining), mast cell staining (toluidine blue staining), apoptosis (TUNEL staining), and Th17 staining (IL-17A immunohistochemical staining).

[0070] 4. Test results:

[0071] pass Figure 5 It can be seen that pine pollen polysaccharide can effectively alleviate the symptoms of atopic eczema mice (damage and bleeding on the back skin).

[0072] pass Figure 6 It can be seen that the results of hematoxylin-eosin pathological tissue staining showed that pine pollen polysaccharide can inhibit inflammatory infiltration and abnormal thickening of the epidermis in the skin tissue of mice with atopic eczema.

[0073] Mast cells are the main cells that release allergic mediators. They are mainly distributed around the microvessels under the skin and visceral mucosa. Figure 7 It can be seen that the toluidine blue staining results showed that pine pollen polysaccharide can significantly reduce the number of mast cells in the skin tissue of atopic eczema mice.

[0074] pass Figure 8 It can be seen that the TUNEL apoptosis staining results show that pine pollen polysaccharide can inhibit programmed cell death in the skin tissue of atopic eczema mice, especially the programmed cell death of skin epidermal cells.

[0075] pass Fig. 9 It can be seen that the IL-17A immunohistochemistry results showed that pine pollen polysaccharide can inhibit the expression of Th17 cells in the skin tissue of mice with atopic eczema.

[0076] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of pine pollen polysaccharide in the preparation of a medicament for preventing and treating atopic eczema, characterized in that: The pine pollen polysaccharide is prepared by the following method: (1) Add 100 g of broken Masson pine pollen into 400 mL of petroleum ether, heat and reflux at 50 °C for 3 h, collect the residue by filtration, and dry to remove the solvent to obtain defatted Masson pine pollen; (2) Defatted Masson pine pollen was extracted with 95% ethanol under heating reflux at 70°C for 2 h. The ratio of defatted Masson pine pollen to 95% ethanol was 1 g:4 mL. After the extraction was completed, the residue was collected by filtration and then dried at 40±0.1°C to remove the residual solvent to obtain impurity-free Masson pine pollen. (3) Add 8 times the mass of water to the impurity-free Masson pine pollen, heat and boil for 1 h, repeat the extraction 3 times, combine the filtrates from the 3 times, heat and concentrate to 200 mL to obtain a concentrated solution; (4) Sevage protein removal solution was prepared by mixing chloroform and butanol in a volume ratio of 4:

1. The Sevage solution and the concentrate obtained in step (3) were used to remove the protein in the concentrate in a volume ratio of 3:

1. The polysaccharide solution layer was taken out, and then anhydrous ethanol was added and stirred until the final ethanol concentration was 80%. The solution was centrifuged at 3000×g for 10 min to separate the precipitate, and the pine pollen polysaccharide was obtained by freeze-drying under reduced pressure.

2. The use according to claim 1, characterized in that: The pine pollen polysaccharide prevents and treats atopic eczema through at least one of the following pathways: (1) Inhibit inflammatory infiltration in skin tissue and abnormal thickening of the epidermis; (2) Inhibit programmed cell death in skin tissue; (3) Reduce the number of mast cells in skin tissue; (4) Reduce the distribution of Th17 cells in skin tissue.

3. The use according to claim 1, characterized in that: The glycosidic bond type of the pine pollen polysaccharide is β-glycosidic bond.

4. The use according to claim 1, characterized in that: The pine pollen polysaccharide comprises the following components in molar percentage: Arabinose 30.41%, rhamnose 6.52%, galactose 6.49%, glucose 42.89%, xylose 6.56% and galacturonic acid 7.13%.

Images