Application and reagents of Hedysari polysaccharide

Abstract

The invention relates to the field of medicines, and discloses the use of spatholobi polysaccharide as an active ingredient of a medicine for preventing and treating allergic rhinitis. Spatholobi polysaccharide is used to inhibit the levels of histamine and IgE in serum, improve nasal mucosal tissue damage, alleviate mast cell and eosinophil infiltration and goblet cell hyperplasia in the nasal mucosa, reduce the content of IL-4, IL-5 and IL-17A in nasal lavage fluid, weaken nasal mucosal cell apoptosis, and increase the expression of PINK1, Parkin and LC3II proteins in nasal mucosal tissue, thereby playing a role in treating allergic rhinitis. In addition, a reagent is also disclosed.

Description

Technical Field

[0001] The invention relates to the field of medicines, and in particular to uses and reagents of scutellaria polysaccharide. Background Art

[0002] Allergic rhinitis is a specific immune response produced by the body after allergens stimulate the body. According to epidemiological surveys, the incidence of allergic rhinitis has been increasing year by year in recent years worldwide, and it has become a major chronic inflammatory disease of the respiratory system, which not only has a serious impact on the quality of life of patients, but also brings a large burden to the society and family economy. The clinical manifestations of allergic rhinitis include sneezing and itching. It is an inflammation mediated by immunoglobulins such as serum immunoglobulin E (IgE). The inflammatory process involves infiltration of mast cells and eosinophils and proliferation of goblet cells, causing excessive production of allergic mediators and mucus. At the same time, when stress occurs, the body will selectively clear and phagocytose damaged mitochondria. Studies have shown that impaired mitochondrial structure and function are closely related to allergic diseases. The activated PINK1 / Parkin pathway can weaken tissue cell apoptosis and inflammation induced by mitochondrial damage. Therefore, regulating the PINK1 / Parkin mitochondrial autophagy pathway may be an important target for the treatment of allergic rhinitis.

[0003] Chinese patent 201610792466.9 discloses a pharmaceutical composition for treating rhinitis, sinusitis and allergic rhinitis, which is made of the following raw materials in parts by weight: 110 to 120 parts of Xanthium sibiricum aqueous extract, 80 to 90 parts of siler chromone, 140 to 150 parts of astragalus polysaccharide, 80 to 90 parts of Angelica dahurica total coumarins, 80 to 90 parts of magnolia volatile oil, 80 to 90 parts of Atractylodes macrocephala aqueous extract, and 20 to 30 parts of peppermint volatile oil.

[0004] This scheme uses a composite composition of Xanthium sibiricum water extract, Saposhnikovia divaricata chromone, Astragalus polysaccharide, Angelica dahurica total coumarins, Magnolia odorata volatile oil, Atractylodes macrocephala water extract, and Mentha volatile oil as the active ingredient for treating rhinitis, sinusitis and allergic rhinitis, thereby playing a therapeutic role on rhinitis, sinusitis and allergic rhinitis, and can inhibit the proliferation of granulation tissue after inflammation, and has an improving effect on pain throughout the body.

[0005] Chinese patent 201911053655.4 discloses a PLGA nanoparticle root canal filling material for deciduous teeth and a preparation method thereof, wherein the components are PLGA 50% to 99% by mass, and the drug contained is 1% to 50% by mass, wherein the PLGA is the main scaffold material, and the drug contained is a regulating material for regulating the degradation rate of the material and increasing the therapeutic effect. The other antibacterial and anti-inflammatory drugs are nano-sized granular drugs, which can be selected from one of nano-silver, soluble drugs, Chinese herbal medicines, and Chinese patent medicines. The soluble drugs can be selected from vitamin C and lidocaine hydrochloride, which have antibacterial and analgesic drugs that promote tissue healing; the Chinese herbal medicines can be selected from drugs such as epimedium and two-sided needles that have the effects of promoting blood circulation, inhibiting bacteria, and promoting tissue growth; the Chinese patent medicine can be selected from one of tetracycline, amoxicillin, metronidazole, rhubarb, puerarin, scutellaria polysaccharide, and lactoferrin.

[0006] This scheme uses Hedysari polysaccharide to make a soluble drug with antibacterial and anti-inflammatory capabilities, and uses the drug as a loading drug for PLGA as the main scaffold material, thereby improving the therapeutic effect of PLGA nanoparticle root canal filling materials for deciduous teeth.

[0007] "Progress in Comparative Studies on the Immunomodulatory and Antioxidant Effects of Red Astragalus and Astragalus", Chinese Journal of Traditional Chinese Medicine, Vol. 39, No. 4, April 2021, records: Red Astragalus and Astragalus belong to the same family but different genera, have the same efficacy and effects, are widely used in clinical practice, and are one of the most commonly used qi-tonifying medicines. Through a summary and comparative analysis of comparative studies on the immunomodulatory and antioxidant effects of red astragalus and astragalus in the past 10 years, the results showed that both red astragalus and astragalus have significant immunomodulatory and antioxidant effects, and the immunomodulatory effect of red astragalus is stronger than that of astragalus, and the antioxidant effect is similar to that of astragalus. This shows that red astragalus and astragalus can be used interchangeably in clinical practice in terms of antioxidant effects, and the former may have certain advantages over the latter in terms of clinical efficacy in immunomodulation, and the efficacy of red astragalus in tonifying the middle and replenishing qi may be stronger than that of astragalus.

[0008] Some studies have investigated the in vitro antioxidant activity of water extracts of Astragalus membranaceus and Astragalus membranaceus using 1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical scavenging method, hydroxyl free radical (-OH) scavenging method, and potassium ferricyanide reduction method. The results show that both water extracts have good in vitro antioxidant activity and are dose-dependent. Based on the D-galactose-induced oxidative stress mouse model, the in vivo antioxidant activity of water extracts of Astragalus membranaceus and Astragalus membranaceus was studied by measuring the activity of T-SOD, GSH-Px and MDA levels in mouse serum, liver and brain tissues. The results show that the effect of Astragalus membranaceus is similar to that of Astragalus membranaceus. Other studies have found that water extracts of Astragalus membranaceus and Astragalus membranaceus can significantly increase the SOD activity of brain tissues of aging model mice and significantly reduce the MDA content, and the effect of Astragalus membranaceus is slightly stronger than that of Astragalus membranaceus. The above literature reports show that water extracts of Astragalus membranaceus and Astragalus membranaceus have significant antioxidant activity, and the antioxidant activity of the former is slightly better than that of the latter.

[0009] This paper focuses on reviewing and comparing the immunomodulatory and antioxidant effects of red astragalus and astragalus. The above review analysis shows that the tonic compound, water extract and polysaccharide of red astragalus and astragalus have significant immunomodulatory effects, and the immunomodulatory effect of red astragalus is stronger than that of astragalus, suggesting that the immunomodulatory effect of red astragalus is stronger than that of astragalus, and the clinical efficacy of the former in immunomodulation may have certain advantages over the latter. It is speculated that the pharmacological effect of red astragalus in tonifying the middle and replenishing qi may be stronger than that of astragalus. The results of the comparative analysis of the antioxidant effects of red astragalus and astragalus show that the antioxidant effect of the water extract of red astragalus is slightly stronger than that of the water extract of astragalus, but there is no significant difference in the antioxidant effect of the tonic compound containing red astragalus and astragalus, suggesting that the two can be used interchangeably in clinical terms of antioxidant effect.

[0010] The problem that this proposal needs to solve: How to develop a substance with better performance than astragalus polysaccharide as an active ingredient in drugs for the treatment of allergic rhinitis. Summary of the invention

[0011] The purpose of the present invention is to provide the use of red stilbene polysaccharide as an active ingredient of a drug for treating allergic rhinitis, using red stilbene polysaccharide to inhibit the levels of histamine and IgE in serum, improve nasal mucosal tissue damage, alleviate mast cell and eosinophil infiltration and goblet cell hyperplasia in the nasal mucosa, reduce the content of IL-4, IL-5, and IL-17A in nasal lavage fluid, weaken nasal mucosal cell apoptosis, and increase the expression of PINK1, Parkin, and LC3II proteins in nasal mucosal tissue, thereby playing a role in treating allergic rhinitis;

[0012] In addition, a reagent is disclosed.

[0013] Unless otherwise specified in the present invention: nM represents nanomole / liter, μM represents micromole / liter, mM represents millimole / liter, and M represents mole / liter;

[0014] Red Hepatitis Polysaccharide as an active ingredient in drugs for the prevention and treatment of allergic rhinitis.

[0015] In addition, a reagent is also disclosed, which comprises the above-mentioned stilbene polysaccharide.

[0016] Preferably, the agent is in the form of tablets, capsules, powders, pills, granules, sustained-release controlled agents, sprays, and injection preparations.

[0017] Preferably, the agent is formulated as a nasal spray.

[0018] In practical applications, the above reagents can be made into a form that acts more directly on the nasal cavity. Therefore, more preferably, the reagent is in the form of a nasal spray reagent. Specifically, the types of the nasal spray reagent include but are not limited to aerosols, sprays, and nasal drops.

[0019] More preferably, other polysaccharides may be added without affecting the content of red Astragalus polysaccharide, including but not limited to wolfberry polysaccharide, proteoglycan, lentinan, astragalus polysaccharide, ginseng polysaccharide, and nettle polysaccharide.

[0020] Preferably, the reagent contains 1-99 wt % of stilbene polysaccharide.

[0021] The beneficial effects of the present invention are:

[0022] The present invention uses Astragalus polysaccharide as an active ingredient of a drug for treating allergic rhinitis, utilizes Astragalus polysaccharide to inhibit the levels of histamine and IgE in serum, improves nasal mucosal tissue damage, alleviates mast cell and eosinophil infiltration and goblet cell hyperplasia in the nasal mucosa, reduces the contents of IL-4, IL-5 and IL-17A in nasal lavage fluid, weakens nasal mucosal cell apoptosis, and increases the expression of PINK1, Parkin and LC3II proteins in nasal mucosal tissue, thereby playing a role in treating allergic rhinitis. At the same time, we surprisingly found that when Astragalus polysaccharide is used as an active ingredient of a drug for treating allergic rhinitis, its therapeutic effect on rhinitis is much higher than that of Astragalus polysaccharide, achieving a therapeutic effect far beyond expectations. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Allergic rhinitis construction and drug administration process;

[0024] Figure 2 : HE staining of nasal mucosa tissues of mice in each group (×400);

[0025] Figure 3 : Representative images of Giemsa staining, toluidine blue staining, and PAS staining of nasal mucosal tissues of mice in each group;

[0026] Among them, (A) Giemsa staining image (×400), black arrows indicate eosinophils;

[0027] (B) is the eosinophil count;

[0028] (C) Toluidine blue staining image (×400), red arrows indicate mast cells;

[0029] (D) is a mast cell statistical graph;

[0030] (E) PAS staining image (×400), blue arrows indicate goblet cells;

[0031] (F) is a goblet cell statistical diagram;

[0032] Figure 4 : TUNEL staining of nasal mucosal tissues of mice in each group (×400);

[0033] Figure 5 : Immunoblotting of PINK1, Parkin and LC3-II protein expressions in the nasal mucosal tissues of each group of mice A: Comparative Example 1; B: Comparative Example 2; C: Comparative Example 3; D: Example 1; E: Comparative Example 4. DETAILED DESCRIPTION

[0034] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that, if no specific conditions are specified in the embodiments, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0035] The experimental model was constructed as follows Figure 1 As shown:

[0036] Male BALB / c mice around 6 weeks old (provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) were housed under standard laboratory conditions (temperature: 23±2°C, relative humidity: 55±10%, light-dark cycle: 12h light / 12h dark) and adaptively fed for one week with free access to food and water.

[0037] One hundred mice were randomly divided into control group, model group, red astragalus polysaccharide group, astragalus polysaccharide group and loratadine group, 20 mice in each group.

[0038] Basic sensitization stage: Except for the control group, each mouse in the other groups was injected with an OVA-aluminum hydroxide mixture (25 μg of OVA and 1 mg of aluminum hydroxide dissolved in 300 μl of normal saline) by intraperitoneal injection on the 0th, 7th and 14th day from the beginning of modeling, while the mice in the control group were injected with a single intraperitoneal injection of the same volume of normal saline at the same time point.

[0039] Intranasal provocation phase: From the 21st to the 27th day of modeling, the other four groups of mice except the control group received OVA suspension (200 μg of OVA dissolved in 20 μl of normal saline) once a day, with 20 μl dripped into each nostril, while the control group mice received the same volume of normal saline.

[0040] Drug administration: From the 15th day to the 27th day after modeling, mice in the red sage polysaccharide group were given 200 mg / kg of red sage polysaccharide by gavage (administered 2 hours before OVA sensitization each time from the 21st to the 27th day), once a day;

[0041] Mice in the APS group were given 200 mg / kg APS by gavage (administered 2 h before OVA sensitization on days 21 to 27), once a day;

[0042] The mice in the loratadine group were given 2 mg / kg loratadine by gavage (administered 2 h before OVA sensitization each time from day 21 to day 27), once a day;

[0043] The control group and the model group were given the same volume of normal saline by gavage.

[0044] Sample collection and preparation:

[0045] Nasal lavage solution: intraperitoneal injection of 1% sodium pentobarbital (50 mg kg -1 ) for anesthesia and disinfected with 75% ethanol. 1 ml of normal saline was injected through the trachea, and the nasal lavage fluid was collected and centrifuged at 4°C, 12000 rpm / min for 15 min. The supernatant was collected, divided and frozen in a -80°C refrigerator.

[0046] Serum collection: After the mice were anesthetized, blood was collected through the eyeball. After standing for 2 hours, the blood was centrifuged at 4000 rpm / min for 10 minutes, and the upper serum was aspirated, aliquoted and frozen at -80°C.

[0047] Example 1

[0048] This is the Hedysari polysaccharide group in the above experimental model.

[0049] Comparative Example 1

[0050] It is the control group in the above experimental model.

[0051] Comparative Example 2

[0052] It is the model group in the above experimental model.

[0053] Comparative Example 3

[0054] This is the astragalus polysaccharide group in the above experimental model.

[0055] Comparative Example 4

[0056] This is the loratadine group in the above experimental model.

[0057] Statistical analysis

[0058] SPSS 21.0 software was used to perform statistics and analysis on the data. The measurement data were expressed as mean ± standard deviation (x ± s), and SigmaPlot software was used to make statistical graphs. When the data obeyed the normal distribution and the variance was equal, the comparison between multiple groups was performed by one-way analysis of variance (ANOVA) and least significant difference (LSD); if the variance was unequal or did not obey the normal distribution, the Wilcoxon rank sum test was used to compare the differences between groups. If P < 0.05, the difference was considered statistically significant.

[0059] Evaluation of rhinitis symptoms in mice:

[0060] 2 hours after the last administration, the nasal cavity of mice was locally stimulated with OVA, and then the mice were placed in a quiet environment and observed and recorded for 10 minutes for sneezing, scratching the nose, and runny nose. The results are shown in Table 1.

[0061] Table 1: Record of sneezing and nose scratching in mice

[0062]

[0063] Result analysis:

[0064] The number of sneezes and nose scratches in mice in Comparative Example 2 increased significantly, which was statistically significant compared with Comparative Example 1 (P<0.05); compared with Comparative Example 2, Hesperidium polysaccharide and Astragalus polysaccharide can significantly reduce the number of sneezes and nose scratches in mice with allergic rhinitis (P<0.05), and the number of nose scratches and sneezes in mice in Example 1 were significantly lower than those in Comparative Example 3, indicating that Hesperidium polysaccharide has a more obvious inhibitory effect on the number of sneezing and nose scratching in mice with allergic rhinitis than Astragalus polysaccharide and the effect is very significant, but compared with Comparative Example 4, the inhibitory effect of Hesperidium polysaccharide on the number of sneezing and nose scratching in mice with allergic rhinitis is still lower than that of loratadine.

[0065] Enzyme-linked immunosorbent assay (ELISA):

[0066] The collected serum and nasal lavage fluid were used to measure the levels of IgE and histamine in serum and the levels of cytokines such as IL-4, IL-5 and IL-17A in nasal lavage fluid according to the instructions of the ELISA kit. The test results are shown in Tables 2 and 3.

[0067] Table 2: Statistical table of OVA-sIgE and histamine levels in the serum of mice in each group

[0068]

[0069] By comparing Comparative Example 2 with Comparative Example 1, as allergic rhinitis occurs in mice, the levels of OVA-sIgE and histamine in the serum increase significantly. By comparing Example 1 and Comparative Example 3, it can be found that the inhibitory effect of the Hedysari polysaccharide in Example 1 on the growth of OVA-sIgE and histamine levels is more obvious and the effect is very significant. At the same time, compared with Comparative Example 4, there is a certain gap, but the gap is not large.

[0070] Table 3: Statistical table of cytokine IL-4, IL-5 and IL-17A content in nasal lavage fluid of mice in each group

[0071]

[0072] By comparing Comparative Example 2 with Comparative Example 1, with the occurrence of allergic rhinitis in mice, the levels of cytokines IL-4, IL-5 and IL-17A in the nasal lavage fluid increased significantly. By comparing Example 1 and Comparative Example 3, it can be found that Example 1 has a more significant inhibitory effect on the growth of the levels of cytokines IL-4, IL-5 and IL-17A. At the same time, compared with Comparative Example 4, there is a certain gap, but the gap is not large.

[0073] Pathological examination of nasal mucosa tissue:

[0074] The nasal tissue was fixed in tissue fixative at room temperature for 48 h, and then decalcified in 20% EDTA decalcification solution for 14 days. The decalcified samples were embedded in paraffin. Serial sections (thickness of 5 μm) were cut, and then hematoxylin and eosin (HE), Giemsa, toluidine blue, periodic acid-Schiff (PAS) and TUNEL staining were used to observe the morphology of the nasal mucosa, eosinophils, mast cells, goblet cells and apoptotic cells according to conventional methods. After imaging by the digital slice scanning system, the average value of 5 sections was randomly counted to detect the number of eosinophils and mast cells. The calculation of goblet cell hyperplasia was performed using the following formula: relative proportion of goblet cells (%) = number of goblet cells in the field of view / number of epithelial cells in the field of view × 100%. The calculation of cell apoptosis was performed as follows: apoptosis index (%) = number of TUNEL-positive (red) cells / number of DAPI-labeled (blue) cells × 100%.

[0075] Immunoblotting:

[0076] The isolated nasal mucosa tissue was cut and ground, and then lysed with a lysis buffer. The protein concentration was determined according to the BCA kit, and the protein was separated by SDS-PAGE method through the conventional immunoblotting operation process, and then transferred to a PVDF membrane. After blocking, diluted PINK1 (1:1000), Parkin (1:1000), and GAPDH (1:3000) were added and incubated at 4°C overnight, and then incubated with horseradish peroxidase-labeled goat anti-rabbit IgG (1:2000) at room temperature for 1 hour. ECL luminescent solution was used for development under a chemiluminometer, and finally the gray value was analyzed by Image J software.

[0077] Test results:

[0078] 1. HPS alleviated nasal mucosal damage in OVA-induced allergic mice;

[0079] refer to Figure 2The nasal mucosal epithelial cells of the mice in Comparative Example 1 were arranged neatly without structural damage; the nasal mucosal epithelial cells of the mice in Comparative Example 2 were arranged disorderly, with obvious epithelial destruction, accompanied by interstitial edema, and obvious infiltration of inflammatory cells; Example 1, Comparative Example 3, and Comparative Example 4 weakened the nasal mucosal damage of mice with allergic rhinitis, and the difference between Example 1, Comparative Example 3, and Comparative Example 4 was not obvious.

[0080] 2. HPS alleviated the infiltration of eosinophils and mast cells and goblet cell hyperplasia in the nasal mucosa of OVA-induced allergic mice;

[0081] refer to Figure 3 Compared with Comparative Example 1, the number of eosinophils and mast cells in the nasal mucosal tissue of mice in Comparative Example 2 was significantly increased (P<0.05); compared with Comparative Example 2, the number of eosinophils and mast cells in the nasal mucosal tissue of mice in Example 1, Comparative Example 3 and Comparative Example 4 was significantly reduced (P<0.05), and the number of eosinophils and mast cells in the nasal mucosal tissue of mice in Example 1 was significantly lower than that in Comparative Example 3, but there was no significant difference between Comparative Example 4;

[0082] Compared with Comparative Example 1, the relative proportion of goblet cells in the nasal mucosal tissue of mice in Comparative Example 2 was significantly increased (P<0.05); compared with Comparative Example 2, the relative proportion of goblet cells in the nasal mucosal tissue of mice in Example 1, Comparative Example 3 and Comparative Example 4 was significantly decreased (P<0.05), and the relative proportion of goblet cells in the nasal mucosal tissue of mice in Example 1 was significantly lower than that in Comparative Example 3, but there was no significant difference between it and Comparative Example 4.

[0083] 3. HPS alleviated OVA-induced apoptosis of nasal mucosal cells in allergic mice;

[0084] refer to Figure 4 Compared with Comparative Example 1, the number of apoptotic cells in the nasal mucosal tissue of mice in Comparative Example 2 increased significantly, which was statistically significant (P<0.05); compared with Comparative Example 2, Comparative Example 3, and Comparative Example 4, the number of apoptotic cells in the nasal mucosal tissue of mice in Example 1 decreased (P<0.05), and was significantly lower than that in Comparative Example 2 and Comparative Example 3.

[0085] 4. HPS enhanced the protein expression of PINK1, Parkin, and LC3-II in the nasal mucosa tissue of allergic mice;

[0086] refer to Figure 5Compared with Comparative Example 1, the protein expression levels of PINK1, Parkin and LC3-II in the nasal mucosal tissue of mice in Comparative Example 2 increased, which was statistically significant (P<0.05); here, because mitochondrial autophagy is a protective mechanism initiated by the body in response to the stimulation of allergic rhinitis, and the protein expression levels of PINK1, Parkin and LC3-II accelerate the acceleration of mitochondrial autophagy, it can be seen that the protein levels of PINK1, Parkin and LC3-II in the nasal mucosal tissue of mice in Example 1, Comparative Example 3 and Comparative Example 4 are higher than those in Comparative Example 2, and the difference is significant (P<0.01), indicating that the mitochondrial autophagy phenomenon of Example 1, Comparative Example 3 and Comparative Example 4 is stronger than that of Comparative Example 2. At the same time, the promoting effect of Example 1 on the expression of PINK1, Parkin and LC3-II is significantly stronger than that of Comparative Example 3 and Comparative Example 4.

Claims

1. The application of Hedysari polysaccharide as the only active ingredient in drugs for the prevention and treatment of allergic rhinitis.

2. The use according to claim 1, characterized in that: The medicine is in the form of tablets, capsules, powders, pills, granules, sprays, and injection preparations.

3. The use according to claim 2, characterized in that: The spray is a nasal spray preparation.

4. The use according to claim 2, characterized in that: The medicine contains 1-99wt% of spatholobi polysaccharide.

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