B cell inhibitor and application thereof in preparation of medicine for treating xerophthalmia

Abstract

The invention relates to a B cell inhibitor and application thereof in preparation of drugs for treating xerophthalmia. The present invention relates to a B cell activity inhibitor selected from the group consisting of formononetin; formononin; astragaloside IV; total flavonoids of an astragalus extract; the components are astragaloside IV and formononetin; astragaloside IV and formononin; the composition is prepared from astragaloside I, astragaloside IV, formononin and formononetin. Compared with the side effect of cyclosporine in the prior art (which is within the renal toxicity of 1 / 3 of a patient who takes the medicine), the astragalus extract general flavone has the effect of inhibiting the activity of B cells so as to inhibit humoral immunity. On the basis of the research on the disease mechanism of pSS, the specific astragalus membranaceus extract general flavone which can be used as a medicine for administration and a medicine for instillation is developed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural product extraction, in particular to a B cell inhibitor and its application in the preparation of a medicament for treating dry eye symptoms, and further relates to an Astragalus extract, in particular Formononetin, Onoin, Astragaloside IV, total flavonoids and specific compositions thereof for inhibiting B cell activity or treating autoimmune diseases. BACKGROUND

[0002] Sjogren's syndrome is a chronic inflammatory autoimmune disease characterized by reduced secretion of lacrimal and salivary glands. The occurrence of the disease is related to infection, endocrine, genetics and other factors, but the exact cause has not been clear. The main pathological manifestations of primary Sjogren's syndrome (pSS) are lymphocyte involvement in exocrine glands (parotid gland, lacrimal gland) and decreased secretion of glands, leading to dry mouth and dry eyes. The current drug for pSS-related dry eye is cyclosporine A eye drops. Sjogren's syndrome can cause patients to have keratoconjunctival dryness (dry eye), and even cause keratitis or conjunctivitis in the patient's eye.

[0003] Activation of the complement system, dysfunction of exocrine glands (such as disorders of neuroendocrine regulation), cytokine imbalance, immune system abnormalities, and genetic factors and environmental triggers all become inducements for Sjogren's syndrome, among which, immune system regulation becomes one of the intervention means that can alleviate Sjogren's syndrome.

[0004] In recent years, many drug combination methods have been given for dry eye in clinical practice, for example:

[0005] Sodium hyaluronate eye drops or artificial tears are used to relieve symptoms, and pilocarpine is used to promote gland secretion;

[0006] Qi and blood circulation of traditional Chinese medicine, hydroxychloroquine sulfate tablets, and prednisone tablets are used to nourish yin and dryness (Sheng Zhenghe et al., Clinical observation on the synergistic effect of warming yang traditional Chinese medicine in the treatment of Sjogren's syndrome and dry eye, Practical Medicine Journal, 2017.33);

[0007] Befu Shu eye drops, sodium hyaluronate eye drops [Zhao Zhi, Shi Chunhe, Efficacy observation of Befu Shu (recombinant bFGF) eye drops in the treatment of dry eye, Journal of Jiangsu University, 2004, 014].

[0008] As can be seen from the above-mentioned prior art, the components of the eye drops for inhibiting conjunctivitis or keratitis are mostly compound drug components, which need to interact with other components of similar eye drop structures that have a moisturizing effect on the eye to ensure that the eye drops will not be rejected by the eye when used.

[0009] Further, for pSS-related dry eye symptoms, cyclosporine A eye drops are mainly used clinically. From the perspective of drug adverse reactions, the routine use of cyclosporine has other serious adverse reactions in addition to common and mild adverse reactions. About 1 / 3 of the drug users will have nephrotoxicity, such as Figure 1 Neurological complications are also common in drug users.

[0010] Considering that natural products generally have better biocompatibility and are less resistant to long-term use of Sjogren's syndrome, there is currently a hot research on eye drop ingredients composed of natural product ingredients that are specifically targeted at keratitis and conjunctivitis and can provide a moist ocular epithelium with a structure similar to tears.

[0011] For example: Patent application with publication number CN117615773A discloses the use of Cistanche tubulosa extract to prepare a medicine for relieving dry eye symptoms. The symptoms include ocular burning, itching, stinging, excessive tearing, foreign body sensation, pain, redness, photophobia, and blurred vision. The medicine is an oral medicine and cannot directly act on the eye, and there is no clear treatment effect on conjunctivitis and keratitis.

[0012] Or as disclosed in patent application with publication number CN106581446A, the use of Dendrobium extract in the preparation of a medicine for treating dry eye. Dendrobium extract can prolong tear film break-up time and repair corneal damage, thereby achieving the purpose of treating and preventing dry eye.

[0013] From the above disclosure of the application of natural extracts, it can be seen that natural extracts can be used for the treatment of dry eye. Although these extracts have shown certain potential in the treatment of dry eye, they mostly rely on specific production processes and have not been deeply explored in terms of effective ingredients and their optimal proportions, nor have they been systematically optimized in combination with clinical mechanisms. This lack of clear identification of effective ingredients limits further process improvement and technological development, making it difficult to achieve precise formula optimization based on the mechanism of action. For dry eye or its complications caused by Sjogren's syndrome, the present application proposes a medicine containing total flavonoids of Astragalus extract, aiming to provide a more effective and targeted treatment regimen through clear component analysis and scientific proportioning.

[0014] B lymphocytes (B cells) are the core components of the adaptive immune system, responsible for mediating humoral immune responses. B cells recognize antigens through their surface-expressed B cell receptors (BCRs), are activated by the synergistic action of antigen-presenting cells and T cells, differentiate into plasma cells and secrete specific antibodies, while forming memory B cells to maintain long-term immune memory. This process involves complex regulation of signaling pathways, including the interaction of BCR signals, costimulatory molecules (such as CD40 / CD40L, BAFF / BAFF-R), and cytokines (such as IL-4, IL-6). However, abnormal activation or dysfunction of B cells can lead to various pathological states, such as autoimmune diseases, B cell malignancies, and transplant rejection.

[0015] In autoimmune diseases, the pathogenic mechanisms of B cells include: 1) secreting autoantibodies to attack host tissues (such as the role of anti-dsDNA antibodies in systemic lupus erythematosus); 2) activating autoreactive T cells through antigen presentation and cytokine release; 3) forming immune complexes to initiate inflammatory responses. For example, a large number of B cells infiltrate the synovial membrane of rheumatoid arthritis (RA) patients, and the rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) they secrete are directly involved in joint destruction. In B cell malignancies (such as chronic lymphocytic leukemia, non-Hodgkin's lymphoma), uncontrolled proliferation and apoptosis resistance of malignant clones are the core of disease progression.

[0016] Targeted therapy against B cells has become an important strategy for the above diseases. The first generation of B cell inhibitors is represented by anti-CD20 monoclonal antibodies (such as rituximab), which clear peripheral B cells through complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The subsequent development of obinutuzumab (Obinutuzumab) further enhances the ADCC effect through glycosylation modification. In addition, inhibitors targeting B cell survival factors (such as BAFF / BLyS inhibitor belimumab) can block B cell maturation and survival signals and show efficacy in the treatment of systemic lupus erythematosus. In recent years, small molecule drugs such as Bruton's tyrosine kinase (BTK) inhibitors (ibrutinib, acalabrutinib) selectively inhibit B cell activation by interfering with the BCR downstream signaling pathway, and have made breakthroughs in B cell lymphoma and autoimmune diseases.

[0017] However, the existing B cell inhibitors still have limitations: 1) some patients develop resistance to CD20-targeted therapy, which may be related to down-regulation of CD20 expression or variation of the targeted epitope; 2) long-term B cell depletion can increase the risk of infection; 3) treatment targeting a single target point can not completely inhibit the multi-pathway activation mechanism of B cells. Therefore, the development of new B cell inhibitors needs to consider target specificity, action durability and safety, such as simultaneously targeting CD20 and CD3 by bispecific antibodies to enhance T cell-mediated killing, or designing reversible BTK inhibitors to reduce off-target effects.

[0018] In addition, on the one hand, there are differences in the understanding of those skilled in the art; on the other hand, a large number of literatures and patents have been studied by the applicant when making the present application, but due to the limitation of space, all the details and contents are not listed in detail, which does not mean that the present application does not have these prior art characteristics, on the contrary, the present application has all the characteristics of the prior art, and the applicant reserves the right to add relevant prior art in the background art. SUMMARY

[0019] In view of the problems of the prior art, the present application relates to the use of total flavonoids of Astragalus extract in the preparation of a medicament for treating dry eye symptoms of Sjogren's syndrome.

[0020] According to a preferred embodiment, the preparation method of total flavonoids of Astragalus extract comprises the following steps: after Astragalus is crushed, it is mixed with an alcohol compound; water bath reflux; ultrasonic extraction after cooling to room temperature; microwave extraction in a microwave reactor, solid-liquid separation; column chromatography to separate the liquid product of solid-liquid separation; and vacuum recovery of the solid product to obtain total flavonoids of Astragalus extract.

[0021] According to a preferred embodiment, the preparation method of total flavonoids of Astragalus extract comprises the following steps: after Astragalus is crushed, it is mixed with an alcohol compound; water bath reflux; ultrasonic extraction after cooling to room temperature; microwave extraction in a microwave reactor, solid-liquid separation; column chromatography to separate the liquid product of solid-liquid separation; and vacuum recovery of the solid product to obtain total flavonoids of Astragalus extract.

[0022] According to a preferred embodiment, the method for preparing the total flavonoids of Astragalus extract comprises the following steps: crushing Astragalus in a pulverizer and passing it through a 60-mesh sieve, adding 50-100% ethanol solution, 10 ml of 50-100% ethanol solution for every 5 g of Astragalus; refluxing the mixture in a constant-temperature water bath at 80°C for 0.5-4 h; performing ultrasonic extraction at a constant power after cooling to room temperature; transferring into a microwave reactor for microwave extraction, and performing suction filtration after cooling to room temperature; mixing 2 g of AB-8 macroporous resin with 15 ml of the above-mentioned suction-filtered product until the AB-8 macroporous resin reaches the maximum adsorption capacity; filling the AB-8 macroporous resin that has reached the maximum adsorption capacity into a chromatographic column according to the wet method, and eluting with ethanol solution at a flow rate of 2-4 Bv / h to obtain the separated product; and recovering the solid product under reduced pressure to obtain the total flavonoids of Astragalus extract. Preferably, the flow rate is set to 3 Bv / h. Preferably, the reflux time is set to 1 h. Preferably, the reflux time is set to 2 h. Preferably, the reflux time is set to 3 h. Preferably, the reflux time is set to 4 h. Preferably, the reflux time is set to 0.5 h. Preferably, the ethanol solution is set to 50%. Preferably, the ethanol solution is set to 100%. Preferably, the ethanol solution is set to 75%. Preferably, the ultrasonic temperature is set to 50°C, the ultrasonic time is set to 30 min, and the ultrasonic power is set to 400 W. Preferably, the microwave power is set to 200 W, the microwave extraction time is set to 3 h, and the extraction times is set to 3 times.

[0023] According to a preferred embodiment, the method for preparing the polysaccharides of Astragalus extract comprises the following steps: mixing crushed Astragalus with sterile water; water bath refluxing; ultrasonic extraction after cooling to room temperature; microwave extraction in a microwave reactor, solid-liquid separation; column chromatography to separate the liquid product of the solid-liquid separation; and recovering the solid product under reduced pressure to obtain the polysaccharides of Astragalus extract.

[0024] According to a preferred embodiment, the method for preparing the polysaccharides of Astragalus extract comprises the following steps: mixing crushed Astragalus with sterile water; constant-temperature water bath refluxing; ultrasonic extraction after cooling to room temperature; microwave extraction in a microwave reactor, suction filtration; and then column chromatography to separate the suction-filtered product in AB-8 macroporous resin; and recovering the solid product under reduced pressure to obtain the polysaccharides of Astragalus extract.

[0025] According to a preferred embodiment, the preparation method of the Astragalus extract polysaccharide comprises the following steps: crushing Astragalus in a pulverizer and passing through a 60-mesh sieve, adding sterile water, 10 ml of sterile water per 5 g of Astragalus; refluxing the mixture under the condition of a constant temperature water bath at 80℃ for 0.5-4 h; after cooling to room temperature, performing ultrasonic extraction at a constant power; transferring into a microwave reactor for microwave extraction, and after cooling to room temperature, performing suction filtration; mixing 2 g of AB-8 macroporous resin with 15 ml of the above-mentioned suction-filtered product until the AB-8 macroporous resin reaches the maximum adsorption capacity; filling the AB-8 macroporous resin reaching the maximum adsorption capacity into a chromatographic column in a wet column packing mode, and using sterile water for elution at a flow rate of 2-4 Bv / h to obtain a separated product; and recovering the solid product under reduced pressure to obtain the Astragalus extract polysaccharide. Preferably, the flow rate is set to 3 Bv / h. Preferably, the refluxing time is set to 1 h. Preferably, the refluxing time is set to 2 h. Preferably, the refluxing time is set to 3 h. Preferably, the refluxing time is set to 4 h. Preferably, the refluxing time is set to 0.5 h. Preferably, the ultrasonic temperature is set to 50℃, the ultrasonic time is set to 30 min, and the ultrasonic power is set to 400 W. Preferably, the microwave power is set to 200 W, the microwave extraction time is set to 3 h, and the extraction times is set to 3 times.

[0026] According to a preferred embodiment, the Astragalus total extract comprises Astragalus extract polysaccharide and Astragalus extract total flavones. Preferably, the Astragalus total extract comprises Astragalus extract polysaccharide and Astragalus extract total flavones obtained by using the preparation method involved in the present application.

[0027] Preferably, the dry eye symptom caused by Sjogren's syndrome is a dry eye symptom caused by primary Sjogren's syndrome.

[0028] Another aspect of the present application relates to the use of Astragalus extract total flavones in the preparation of a medicament for treating conjunctivitis caused by Sjogren's syndrome.

[0029] Another aspect of the present application relates to the use of Astragalus extract total flavones in the preparation of a medicament for treating keratitis caused by Sjogren's syndrome.

[0030] The present application proves, through the research results of a mouse model of Sjogren's syndrome, that Astragalus extract total flavones have the pharmacodynamic effect of improving the dry eye symptom, conjunctivitis, and keratitis of Sjogren's syndrome. The preparation method of the dry eye symptom of Sjogren's syndrome provided by the present application is simple, the raw materials are easy to obtain, has no cytotoxicity, and has good antioxidant and autoimmune B cell activation enhancement effects in pSS, and is suitable for promotion. Figure 5 B) of the present application.

[0031] According to a preferred embodiment, the step of column chromatography comprises: eluting the product in AB-8 macroporous resin with 30% ethanol solution at a flow rate of 3Bv / h; eluting the product in AB-8 macroporous resin with 50% ethanol solution at a flow rate of 3Bv / h; eluting the product in AB-8 macroporous resin with 70% ethanol solution at a flow rate of 3Bv / h; eluting the product in AB-8 macroporous resin with 90% ethanol solution at a flow rate of 3Bv / h; eluting the product in AB-8 macroporous resin with 100% ethanol solution at a flow rate of 3Bv / h; and mixing the above products to obtain the product to be recovered under reduced pressure. Preferably, the AB-8 macroporous resin mixed with 20ml of the product is continuously eluted with gradient concentrations of 30%, 50%, 70%, 90%, and 100% ethanol solution (volume fraction) at a flow rate of 3Bv / h, and the elution time of each concentration of ethanol is set to 2h, and after elution, the product is recovered to dryness under reduced pressure to obtain the total flavonoids of astragalus extract.

[0032] Another aspect of the present application relates to a pharmaceutical composition for treating dry eye symptoms caused by Sjogren's syndrome. Preferably, the pharmaceutical composition comprises total flavonoids of astragalus extract. Preferably, the pharmaceutical composition comprises astragalus total extract.

[0033] According to a preferred embodiment, the pharmaceutical composition comprising total flavonoids of astragalus extract comprises an oral dosage form.

[0034] According to a preferred embodiment, the pharmaceutical composition comprising polysaccharides of astragalus extract comprises an oral dosage form.

[0035] According to a preferred embodiment, the pharmaceutical composition comprising total flavonoids of astragalus extract comprises an oral dosage form.

[0036] According to a preferred embodiment, the effective concentration of total flavonoids of astragalus extract is 50mg / kg under gavage or oral conditions.

[0037] According to a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, such as an antioxidant, a diluent, a preservative, a disintegrant, a binder, a wetting agent, an emulsifying agent, a sweetener, a coloring agent, a flavoring agent, a buffer, a solubilizer, a stabilizer, or a coating agent.

[0038] According to a preferred embodiment, the diluent can be starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.

[0039] According to a preferred embodiment, the binder can be water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, sugar paste, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone, etc.

[0040] According to a preferred embodiment, the disintegrant can be dry starch, alginate, agar powder, fucoidin, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium dodecyl sulfonate, methyl cellulose, ethyl cellulose, etc.

[0041] Another aspect of the present application relates to the use of the Astragalus extract in the preparation of a medicament for inhibiting the secretion of antibodies. Preferably, the Astragalus extract comprises one or more of the following combinations: a. Astragalus extract total flavones; b. Astragalus extract polysaccharides.

[0042] According to a preferred embodiment, the autoantibody comprises Anti-SSA.

[0043] Another aspect of the present application relates to the use of the Astragalus extract in the preparation of a medicament for regulating diseases caused by B cell overreaction. Preferably, the Astragalus extract comprises one or more of the following combinations: a. Astragalus extract total flavones; b. Astragalus extract polysaccharides.

[0044] Another aspect of the present application relates to the use of the Astragalus extract total flavones in the preparation of a medicament for inhibiting humoral autoimmunity.

[0045] Another aspect of the present application relates to the use of the Astragalus extract total flavones in the preparation of a medical device or medical facility for treating dry eye symptoms caused by Sjogren's syndrome.

[0046] Another aspect of the present application relates to a preparation method of the Astragalus extract total flavones for dry eye. The preparation method comprises the following steps: mixing Astragalus powder with ethanol; constant temperature water bath reflux; cooling to room temperature and then ultrasonic extraction; transferring into a microwave reactor for microwave extraction, and then suction filtration; and then transferring into AB-8 macroporous resin for column chromatography separation of the suction filtered product; and then recovering the solid product under reduced pressure to obtain the Astragalus extract total flavones.

[0047] Another aspect of the present application relates to a saliva stimulant. The saliva stimulant comprises the Astragalus extract total flavones. Preferably, the saliva stimulant comprises an oral dosage form.

[0048] The present application also relates to a B cell activity inhibitor comprising one or more of formononetin, calycosin, and Astragalus saponin IV. Preferably, the B cell activity inhibitor is composed of formononetin, calycosin, and Astragalus saponin IV in an equimolar concentration ratio.

[0049] The present invention also relates to the use of a B-cell activity inhibitor in the preparation of a medicament for treating dry eye symptoms caused by Sjögren's syndrome, wherein the B-cell activity inhibitor is composed of equimolar amounts of styracin, styracin, and astragaloside IV.

[0050] The present invention also relates to an eye drop comprising gallnut extract, styracin, styracin glycoside and astragaloside IV.

[0051] The beneficial effects of this technical solution are:

[0052] Compared to cyclosporine, which acts on dendritic cells, macrophages, and neutrophils in existing technologies, considering the side effects of cyclosporine (nephrotoxicity and neuritis observed in approximately one-third of users; nephrotoxicity results are available in...),... Figure 1 (A schematic diagram of cells involved in cyclosporine-induced glomerular filtration dysfunction). This invention, starting from the mechanism, clarifies that the target of total flavonoids from Astragalus extract is B cells, specifically inhibiting B cell activity to suppress humoral autoimmunity (see the results of Example 3; antibody OD450 decreased from 0.7 to 0.4, and a lower antibody OD450 value indicates a lower antibody quantity: total flavonoids from Astragalus extract can inhibit antibody secretion). Given that Anti-SSA autoantibodies are closely involved in the excessive response process of B cells, it can be confirmed that total flavonoids from Astragalus extract have immunomodulatory effects on the excessive response of B cells.

[0053] This invention reveals that the application of total flavonoids from Astragalus extract aligns with the latest understanding of the pathogenesis of pSS, and further demonstrates through relevant animal experiments that total flavonoids from Astragalus extract have significant effects on humoral stimulation. Figure 5 A shows the salivary flow rate, which is the most important indicator for measuring saliva secretion and a key indicator of changes in body fluids. Through gavage experiments in mice, it was found that, due to the properties of natural products, gavage administration of total flavonoids from Astragalus extract produced a good therapeutic effect on the eyes of mice (see the results of Example 3; compared to before treatment, the average reduction rate of corneal inflammation area after treatment was 90.78%), while also exhibiting extremely low side effects, especially without any damage to glomerular filtration function. No significant side effects have been observed so far.

[0054] Based on the investigation of the disease mechanism of pSS, this invention has developed a targeted total flavonoid extract of Astragalus membranaceus that can be taken orally. Data from Example 2 shows that the total flavonoid extract of Astragalus membranaceus can act on BAFF factor, which is crucial for the survival and proliferation of autoimmune B cells, thereby achieving a humoral-promoting effect in patients with Sjögren's syndrome. This invention, by exploring the mechanism of action of the total flavonoid extract of Astragalus membranaceus, further clarifies the stability and direction of action of the total flavonoid extract of Astragalus membranaceus in clinical applications, providing clearer and more specific guidance for medication use in clinical treatment.

[0055] Meanwhile, patent application CN105918726A discloses a Chinese yam beverage with thirst-quenching and saliva-generating effects, which contains astragalus polysaccharides. However, the saliva flow rate index given in Example 2 of this application ( Figure 5 A) reveals that Astragalus extract polysaccharides have a negative effect on promoting salivation, while the component with a positive effect on promoting salivation is total flavonoids from Astragalus extract. This also demonstrates the current discrepancies in research understanding regarding salivation promotion in humans, and this application clarifies these discrepancies through relevant experiments. For patients with Sjögren's syndrome, this application provides a more targeted natural product component for promoting salivation (total flavonoids from Astragalus extract) based on experimental results related to salivary flow rate, and excludes factors that affect salivation (Astragalus extract polysaccharides), enabling patients with Sjögren's syndrome to use a more effective component in the treatment of salivary glands or salivation secretion.

[0056] This invention relates, in one aspect, to a B-cell activity inhibitor selected from the group consisting of:

[0057] Brassioides flavonoids;

[0058] Mangzhihua glycoside;

[0059] Astragaloside IV;

[0060] Total flavonoids from Astragalus membranaceus extract;

[0061] Astragaloside IV and cymenein;

[0062] Astragaloside IV and gentianoside;

[0063] Astragaloside IV, gentianoside and gentianoside flavonoids.

[0064] According to a preferred embodiment, the ratio of astragaloside IV to basidioflavone is 1:1 to 2. Preferably, the ratio of astragaloside IV to basidioflavone is 1:1.

[0065] According to a preferred embodiment, the ratio of astragaloside IV to gentianoside is 1:1 to 2. Preferably, the ratio of astragaloside IV to gentianoside is 1:1.

[0066] According to a preferred embodiment, the ratio of Astragaloside IV, Ononin and Formononetin is 1:1:1.

[0067] According to a preferred embodiment, the B cell activity inhibitor is used at a concentration of 8-32 μM.

[0068] Another aspect of the present application relates to a method for cultivating mature B cells, comprising cultivating the mature B cells with a culture solution comprising the following components:

[0069] Formononetin;

[0070] Ononin;

[0071] Astragaloside IV;

[0072] Total flavonoids of Astragalus extract;

[0073] Astragaloside IV and Formononetin;

[0074] Astragaloside IV and Ononin;

[0075] Astragaloside IV, Ononin and Formononetin.

[0076] Another aspect of the present application relates to a method for inhibiting the development of B cells into plasma cells,

[0077] Another aspect of the present application relates to a method for preparing an eye drop, comprising

[0078] Step one: mixing the extract of Galla Chinensis and Formononetin;

[0079] Step two: adding Astragaloside IV and Ononin.

[0080] According to a preferred embodiment, the method for preparing further comprises pretreatment of the extract of Galla Chinensis, comprising:

[0081] mechanically stirring with 60% ethanol at a solid-liquid ratio of 1:15 for 30 min;

[0082] ultrasonically treating with 70% ethanol at a solid-liquid ratio of 1:12 for 20-30 min;

[0083] ultrasonically treating with 80% ethanol at a solid-liquid ratio of 1:10 for 20-30 min;

[0084] microwaving with 90% ethanol at a solid-liquid ratio of 1:8 for 20 min,

[0085] After mixing the four extracts, concentrate, adjust pH and filter.

[0086] According to a preferred embodiment, the extract of Galla Chinensis and Formononetin are mixed and then left to stand for 35-45 min.

[0087] Another aspect of the application relates to the use of an inhibitor of B cell activity for the preparation of a medicament for the treatment of dry eye symptoms caused by Sjogren's syndrome. The inhibitor of B cell activity is selected from the group consisting of:

[0088] Formononetin;

[0089] Baptiside;

[0090] Astragaloside IV;

[0091] Total flavonoids of Astragalus extract;

[0092] Astragaloside IV and Formononetin;

[0093] Astragaloside IV and Baptiside;

[0094] Astragaloside IV, Baptiside and Formononetin.

[0095] According to a preferred embodiment, the inhibitor of B cell activity inhibits differentiation of B cells.

[0096] According to a preferred embodiment, inhibiting differentiation of B cells comprises inhibiting differentiation of B cells into plasma cells.

[0097] Another aspect of the application relates to a pharmaceutical composition for the treatment of dry eye symptoms caused by Sjogren's syndrome, selected from the group consisting of:

[0098] Formononetin;

[0099] Baptiside;

[0100] Astragaloside IV;

[0101] Total flavonoids of Astragalus extract;

[0102] Astragaloside IV and Formononetin;

[0103] Astragaloside IV and Baptiside;

[0104] Astragaloside IV, Baptiside and Formononetin.

[0105] Advantages of the technical solution of the present application:

[0106] According to the experimental results disclosed in Example 2, compared with the results of the control group, calycosin-7-O-β-D-glucoside and calycosin not only failed to inhibit the transformation of B cells into CD138+plasma cells, but also significantly increased the number of CD138+plasma cells, which suggested that calycosin-7-O-β-D-glucoside and calycosin had the ability to promote the activation and differentiation of B cells into plasma cells, and were suitable for symptoms requiring enhanced B cell immune response. On the contrary, astragalus extract total flavones, astragaloside IV, ononin, formononetin, composition 1, composition 2, and composition 3 significantly reduced the number of CD138+plasma cells transformed from B cells, and composition 3 showed a significant inhibitory effect. The above results indicated that astragalus extract total flavones, astragaloside IV, ononin, formononetin, composition 1 (astragaloside IV and formononetin), composition 2 (astragaloside IV and ononin), and composition 3 (astragaloside IV, ononin, and formononetin) were of great significance for treating diseases related to B cell overactivation, such as autoimmune diseases. In particular, Examples 3 and 4 took astragalus extract total flavones as an example to explore the therapeutic effect of autoimmune diseases under the influence of B cells by astragalus extract components, such as Sjogren's syndrome (especially dry eye caused by Sjogren's syndrome). Based on this, the present application proposes a B cell activity inhibitor containing astragalus extract (total flavones, astragaloside IV, ononin, formononetin, composition 1, composition 2, and composition 3) and its application in the preparation of a medicament for treating dry eye symptoms caused by Sjogren's syndrome. BRIEF DESCRIPTION OF DRAWINGS

[0107] Figure 1 Cell smears for cyclosporine-induced glomerular filtration function damage;

[0108] Figure 2 Mechanism diagram for cyclosporine action;

[0109] Figure 3 Mechanism diagram for CsA immunosuppression;

[0110] Figure 4 Cell smears for pSS patient labial gland biopsy tissues;

[0111] Figure 5 Result diagram of salivary flow rate (A) and Anti-SSA autoantibody (B) of Sjogren's syndrome mice under the action of astragalus extract total flavones involved in the present application;

[0112] Figure 6 Result diagram of Anti-SSA autoantibody concentration of ESS model mice injected with different concentrations of antigens;

[0113] Figure 7A cell smear of lacrimal gland lymphocyte infiltration of a display model mouse involved in the present application;

[0114] Figure 8 A schematic diagram of fluorescein sodium staining of an ocular surface of a model mouse involved in the present application;

[0115] Figure 9 A schematic diagram of fluorescein staining score of an ocular surface (cornea) of a model mouse involved in the present application;

[0116] Fig. 10 is a mass spectrum of each extract of total flavones of Astragalus extract involved in the present application;

[0117] Figure 11 A schematic diagram of the number of plasma cells, wherein the number of plasma cells is proportional to the degree of B cell differentiation, and it can be understood that the fewer the number of plasma cells, the better the inhibitory effect of the components in the extract on B cell differentiation under stimulation. DETAILED DESCRIPTION

[0118] The following will be described in detail with reference to the accompanying drawings.

[0119] The following examples are only used to further explain the beneficial effects of total flavones of Astragalus extract in the present application, and should not limit the protection scope of the present application. For ordinary skilled persons in the art, the specific meanings of the terms in the present application can be understood according to the specific circumstances. The experimental procedures described in the following examples are all conventional procedures unless otherwise specified. The reagents used in the following examples can be obtained through commercial channels unless otherwise specified. The terms "comprising" and "having" and any variations thereof in the present application are intended to cover non-exclusive inclusion. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but can optionally further include steps not listed, or can optionally further include other steps inherent to these processes, methods, products or equipment.

[0120] Example 1

[0121] (I) This example relates to the extraction method of total flavones of Astragalus extract.

[0122] After the Astragalus is crushed in a crusher and passed through a 60-mesh sieve, 5 g is added to 10 ml of 75% ethanol solution, the bottle opening is closed, and it is refluxed in a 80℃ constant temperature water bath for 1 h, and then the weight loss is made up with 10 ml of 75% ethanol. After cooling to room temperature, ultrasonic extraction is carried out at constant power, the ultrasonic temperature is 50℃, the ultrasonic time is 30 min, and the ultrasonic power is 400 W. Then it is moved into a microwave reactor for microwave extraction, the microwave power is 200 W, the microwave extraction time is 3 h, and the extraction times is 3 times. After cooling to room temperature, suction filtration is carried out.

[0123] Take 2g AB-8 macroporous resin and 15ml of the above extract on a vortex mixer, mix well and oscillate on a shaker, set as follows: T=40℃, speed=120r / min. Every half hour, determine the absorbance of the extract by aluminum chloride colorimetry once, calculate the mass concentration, and determine the maximum adsorption capacity of AB-8 resin.

[0124] When the maximum adsorption capacity is reached, AB-8 macroporous resin is filled into a chromatography column according to the wet method, and the above 20ml product is continuously eluted according to 3Bv / h flow rate with 30%, 50%, 70%, 90%, and 100% ethanol solution, and the elution time of each concentration of ethanol is set to 2h. After elution, it is recovered to dryness under reduced pressure to obtain total flavonoids of astragalus extract.

[0125] (II) This example relates to the extraction method of polysaccharides in astragalus extract.

[0126] After the astragalus is crushed in a crusher and passed through a 60-mesh sieve, 5g is added to 10ml of sterile water solution, the bottle opening is closed, and it is refluxed in a 80℃ constant temperature water bath for 1h, then the weight loss is made up with 10ml of sterile water. After cooling to room temperature, ultrasonic extraction is performed at constant power, the ultrasonic temperature is 50℃, the ultrasonic time is 30min, and the ultrasonic power is 400W. Then it is moved into a microwave reactor for microwave extraction, the microwave power is 200W, the microwave extraction time is 3h, and the extraction times is 3. After cooling to room temperature, it is filtered.

[0127] Take 2g AB-8 macroporous resin and 15ml of the above extract on a vortex mixer, mix well and oscillate on a shaker, set as follows: T=40℃, speed=120r / min. Every half hour, determine the absorbance of the extract by aluminum chloride colorimetry once, calculate the mass concentration, and determine the maximum adsorption capacity of AB-8 resin.

[0128] When the maximum adsorption capacity is reached, AB-8 macroporous resin is filled into a chromatography column according to the wet method, and the above 20ml product is continuously eluted according to 3Bv / h flow rate with 30%, 50%, 70%, 90%, and 100% ethanol solution, and the elution time of each concentration of ethanol is set to 2h. After elution, it is recovered to dryness under reduced pressure to obtain total flavonoids of astragalus extract.

[0129] (III) The total extract of astragalus is obtained by mixing the total flavonoids and polysaccharides of astragalus extract extracted at a time.

[0130] (IV) Mass spectrometric analysis of astragalus extract

[0131] Calycosin-7-O-β-Glucoside, Calycosin, Formononetin, Onoin, Astragaloside IV, and Total Flav were obtained from Astragalus extract, respectively. The mass spectrometry results are shown in Figure 10.1 and 10.2

[0132] Example 2

[0133] The effects of Calycosin-7-O-β-Glucoside, Calycosin, Formononetin, Onoin, Astragaloside IV (hereinafter can be abbreviated as Astra IV), Total Flav, and various compositions on B cell activity in vitro were detected by CD138+plasma cell detection method.

[0134] To simulate the effects of Astragalus extract on B cells, this example relates to the experimental results of B cell incubation.

[0135] (I) Experimental design

[0136] Control group (DMSO), single compound experiment group (Calycosin-7-O-β-Glucoside, Calycosin, Formononetin, Onoin, Astragaloside IV), and composition experiment group (Mixture 1, Mixture 2, Mixture 3, Total Flav).

[0137] The Mixture 1 group (Composition 1) contains Formononetin and Astra IV mixed in equal proportions (molar concentration ratio 1:1). The Mixture 2 group (Composition 2) contains Onoin and Astra IV mixed in equal proportions (molar concentration ratio 1:1). The Mixture 3 group (Composition 3) contains Formononetin, Onoin, and Astra IV mixed in equal proportions (molar concentration ratio 1:1:1).

[0138] (II) Experimental cell culture

[0139] ​Peripheral blood samples were obtained from healthy volunteers or animal models, and B cells were isolated using density gradient centrifugation or magnetic bead separation. B cells were cultured in FBS-containing medium (product: Sigma-Aldrich RPMI1640) and placed in a sterile incubator at 37°C.

[0140] (III) Group Culture and Cell Stimulation

[0141] B cells were grouped according to the experimental design, with each group having the same number of cells.

[0142] Add the same molar concentration of Calycosin-7-O-β-Glucoside, Calycosin, Formononetin, Onoin, Astragaloside IV, Mixture 1, Mixture 2, Mixture 3, and Total Flav to each culture dish (i.e., the concentration of Calycosin-7-O-β-Glucoside, Calycosin, Formononetin, Onoin, Astragaloside IV, Mixture 1, Mixture 2, Mixture 3, and Total Flav after adding these components to the culture dish is 20 μM). Add an equal amount of DMSO to the control group.

[0143] Stimulants (1 μg / mL anti-CD40 antibody, 5 μg / mL anti-IgM antibody and 50 ng / mL IL-21) were added to cells in all groups to promote the differentiation of B cells into plasma cells;

[0144] Continue incubation for 3 days in a sterile incubator at 37°C.

[0145] (iv) Cell Markers

[0146] Collect the cells and wash them once with PBS;

[0147] CD138 fluorescent antibody was used for cell surface labeling;

[0148] Cells were resuspended after labeling;

[0149] Flow cytometry was used to detect the labeled cells in order to collect and analyze the number of CD138+ plasma cells.

[0150] (V) Data Analysis

[0151] like Figure 11 As shown, compared to the control group (CD138+ plasma cell count was 0.786 x 10⁻⁶), the number of plasma cells was significantly lower. 6 The number of plasma cells was 0.867 x 10⁻⁶ (Calycosin(CD138+)).6 ) and Calycosin-7-O-β-Glucoside (CD138+ plasma cell number was 0.907 x 10 6 ) significantly increased the number of CD138+ plasma cells; Formononetin (CD138+ plasma cell number was 0.470 x 10 6 ), Onoin (CD138+ plasma cell number was 0.399 x 10 6 ), Astragaloside IV (CD138+ plasma cell number was 0.325 x 10 6 ) significantly decreased the number of CD138+ plasma cells; in the composition, Total Flav (CD138+ plasma cell number was 0.462 x 10 6 ), Mixture 1 (CD138+ plasma cell number was 0.256 x 10 6 ), Mixture 2 (CD138+ plasma cell number was 0.261 x 10 6 ), Mixture 3 (CD138+ plasma cell number was 0.115 x 10 6 ) significantly decreased the number of CD138+ plasma cells.

[0152] B cells are a type of white blood cell in the immune system that are activated when they encounter a specific antigen and differentiate into plasma cells. Plasma cells are a subtype of B cells and their main function is to produce and secrete large amounts of antibodies that can recognize and neutralize invading pathogens. CD138 is a protein that is highly expressed on the surface of plasma cells and plays a role in cell adhesion and signal transduction, making it a highly specific marker for plasma cells.

[0153] According to the above results, Formononetin, Onoin, Astragaloside IV, Total Flav, Mixture 1, Mixture 2, and Mixture 3 can inhibit the functional activity of B cells, especially the functional activity of B cells in secreting CD138 upon stimulation. In addition, the number of CD138+ plasma cells in the group with the addition of Mixture 3 was the lowest, indicating that the combination of Mixture 3 is the best for inhibiting the functional activity of B cells.

[0154] Example 3

[0155] This example provides research results for verifying the mechanism of action of BAFF factor, which is essential for the survival and proliferation of autoimmune B cells in pSS.

[0156] According to relevant literatures, cyclosporine acts on dendritic cells (DC), macrophages (Mφ) and neutrophils, as shown in Figure 2 , by inhibiting phosphatase calcineurin, preventing the activation of downstream nuclear factor of activated T cell (NFAT), and inhibiting the expression of downstream genes.

[0157] Specifically, as shown in Figure 3 , in DC and Mφ, CsA prevents the release of cytokines, which are essential for the proliferation and survival of early T cells, especially IL-2, through the above-mentioned pathway. As shown in Figure 3 , in neutrophils, CsA causes a decrease in neutrophil function by inhibiting the production of intracellular reactive oxygen species (ROS) and neutrophil extracellular traps (NETs).

[0158] According to the above discussion, the main effect of CsA immunosuppression is on the intracellular enzyme phosphatase calcineurin, which inhibits the proliferation and activation of early T cells.

[0159] With the publication of recent research results on the role of B lymphocytes in the exocrine glands, the relevant results show that B lymphocytes have an important influence on the course of pSS. For example: The literature published in The Journal of Rheumatology shows that the main infiltrating cells in the lip biopsy tissue of pSS patients are CD4 + T cells, as well as CD20 + B cells, as shown in Figure 4 ( Figure 4 A and Figure 4 B). This literature discloses the presence of ectopic germinal centers, hyperimmunoglobulinemia and the secretion of multiple autoantibodies in the target organs of patients, as well as the increase of Tfh cells in the peripheral blood and salivary glands, Tfh cells migrate to the B cell follicle area and promote the differentiation of germinal center B cells and plasma cells. The above-mentioned experimental results prove that the overactivation of B cells (including cell activation and proliferation, as well as differentiation into plasma cells) plays an important role in the disease process of pSS.

[0160] Based on this, the inventors explore the treatment effect of total flavonoids of astragalus extract on Sjogren's syndrome or Sjogren's syndrome caused by overreaction of B cells by gavage of Sjogren's syndrome modeling mice with total flavonoids of astragalus extract. The total flavonoids of astragalus extract, the polysaccharides of astragalus extract and the total extract of astragalus gavaged to mice are prepared by the extraction method involved in Example 1.

[0161] As shown in Figure 5 As shown in A, the saliva flow rate of the mouse gavaged with the total extract of astragalus is 325 μl / 15 min, and the saliva flow rate of the mouse gavaged with the total flavonoids of astragalus extract is 330 μl / 15 min, which is much higher than the saliva flow rate of the mouse gavaged with the polysaccharides of astragalus extract (240 μl / 15 min) and the saliva flow rate of the control group mouse (255 μl / 15 min). The saliva flow rate of the mouse gavaged with the total flavonoids of astragalus extract is increased by 29% compared with the saliva flow rate of the control group mouse. The saliva flow rate of the mouse gavaged with the total extract of astragalus is increased by 27% compared with the saliva flow rate of the control group mouse. The saliva flow rate of the mouse gavaged with the polysaccharides of astragalus extract is decreased by 5% compared with the saliva flow rate of the control group mouse.

[0162] As shown in Figure 5 As shown in B, the Anti-SSA autoantibody quantity of the mouse gavaged with the total extract of astragalus is 0.3 (OD450), and the Anti-SSA autoantibody quantity of the mouse gavaged with the total flavonoids of astragalus extract is 0.4 (OD450), which is much lower than the Anti-SSA autoantibody quantity of the mouse gavaged with the polysaccharides of astragalus extract (0.6 OD450) and the Anti-SSA autoantibody quantity of the control group mouse (0.7 OD450).

[0163] The above results show that the total flavonoids of astragalus extract can inhibit the secretion of autoantibodies, and the results prove that the total flavonoids of astragalus extract have immune regulation effect on the overreaction of B cells.

[0164] Example 4

[0165] This example relates to the application of total flavonoids of astragalus extract in model mice.

[0166] (I) Construction of model mice

[0167] As shown in Figure 6 As shown in A, the saliva flow rate of the mouse gavaged with the total extract of astragalus is 325 μl / 15 min, and the saliva flow rate of the mouse gavaged with the total flavonoids of astragalus extract is 330 μl / 15 min, which is much higher than the saliva flow rate of the mouse gavaged with the polysaccharides of astragalus extract (240 μl / 15 min) and the saliva flow rate of the control group mouse (255 μl / 15 min). The saliva flow rate of the mouse gavaged with the total flavonoids of astragalus extract is increased by 29% compared with the saliva flow rate of the control group mouse. The saliva flow rate of the mouse gavaged with the total extract of astragalus is increased by 27% compared with the saliva flow rate of the control group mouse. The saliva flow rate of the mouse gavaged with the polysaccharides of astragalus extract is decreased by 5% compared with the saliva flow rate of the control group mouse.

[0168] The lacrimal gland lymphocytes of the model mice were collected. The results show that, as shown in Figure 7 the right graph, the model mouse lacrimal gland lymphocyte infiltration. This indicates that the model mouse lacrimal gland is inflamed.

[0169] (ii) Administration

[0170] Gavage, once a day, 50 mg / kg per mouse.

[0171] (iii) Model mouse ocular surface fluorescein sodium staining

[0172] As shown in Figure 8 , compared with Figure 8 the four model mice with severe corneal point staining before treatment, Figure 8 The corneal fluorescence area of the four model mice after treatment (2 weeks after administration) decreased, with an average reduction rate of 90.78%.

[0173] Fluorescein sodium staining score is mainly used to evaluate dry eye. This scoring method quantifies the degree of ocular surface damage by observing the staining of the cornea. The scoring criteria usually include scoring of different staining areas and degrees in order to grade the corneal damage.

[0174] In the mouse experiment, the ocular surface was divided into 4 quadrants, and each quadrant was scored according to the staining, with the specific scoring criteria being 0 points for no staining, 1 point for scattered and punctate staining, 2 points for clustered punctate staining, and 3 points for sheet-like staining; The total score of the four quadrants is the corneal staining score of the mouse.

[0175] From the scoring results, it can be seen that before treatment, the staining scores of the four mice were 9, 12, 13 and 14, respectively, while after treatment, they decreased to 0, 0, 2 and 4, respectively. From the above scoring results, it can be seen that the ocular inflammation area (or damage area) of the four mice is greatly reduced. Figure 9 This result proves that the gavage of total flavonoids of Astragalus extract has a significant therapeutic effect on keratitis or other ocular surface inflammation.

[0176] According to its cause and the corneal layer involved, keratitis can be divided into various types.

[0177] Epithelial keratitis: Epithelial keratitis mainly affects the outermost epithelial cells of the cornea. Its common types include keratitis caused by herpes simplex virus (HSV) which leads to the destruction of epithelial cells and corneal ulcers, and punctate keratitis which presents as small punctate defects in the epithelial layer caused by viruses, bacteria or trauma. The symptoms of patients with epithelial keratitis include pain, photophobia, tearing and blurred vision.

[0178]

[0179] ​Stromal keratitis: Stromal keratitis affects the middle thick layer of the cornea, common types include bacterial keratitis caused by bacterial infection (e.g. Pseudomonas aeruginosa or Staphylococcus aureus) that can lead to severe stromal destruction and fungal keratitis associated with plant trauma caused by fungal infection (e.g. Aspergillus or Candida). Symptoms of stromal keratitis patients include severe pain, red eye, vision loss and corneal opacity.

[0180] Endothelial keratitis: Endothelial keratitis mainly affects the innermost layer of the cornea endothelial cells, common types include corneal endothelitis caused by, for example, herpes simplex virus, which leads to corneal edema, and uveo-corneal endothelial syndrome, which affects endothelial cells and leads to corneal edema / opacity. Symptoms of endothelial keratitis include blurred vision, corneal edema and iritis.

[0181] Full-thickness keratitis: Full-thickness keratitis affects all layers of the cornea, common types include corneal ulcer caused by severe infection or trauma, which leads to destruction of all layers of the cornea, and keratitis with perforation caused by severe bacterial / fungal / viral infection. Symptoms of full-thickness keratitis include severe pain, vision loss, eye redness and corneal perforation.

[0182] Other special types of keratitis: keratocorneal keratitis affecting the junction of the cornea and the sclera (common in immune diseases such as Sjogren's syndrome); keratocorneal keratitis affecting the peripheral area of the cornea (common in autoimmune diseases such as rheumatoid arthritis).

[0183] The total flavonoids of Astragalus extract provided by the present application can produce a significant therapeutic effect on one or more of epithelial keratitis, stromal keratitis, endothelial keratitis, full-thickness keratitis, keratocorneal keratitis and keratocorneal keratitis, i.e. significantly reducing the area of corneal inflammation, thereby avoiding the impact of keratitis disease on the eyes of Sjogren's syndrome patients, such as reduced vision, limited eye displacement and decreased muscle capacity.

[0184] Compared with the anti-infection treatment (using antibiotics, antiviral drugs or antifungal drugs according to the cause) for keratitis, anti-inflammatory treatment (using non-steroidal anti-inflammatory drugs or glucocorticoids to reduce inflammation), supportive treatment (using artificial tears and corneal protectants to protect the cornea and relieve symptoms) and surgical treatment in the prior art, the natural product Astragalus extract total flavonoids involved in the present application can not only provide targeted treatment (dry eye symptoms of Sjogren's syndrome patients), but also avoid pharmacological conflicts between the use of Western medicines (Sjogren's syndrome patients will take Western medicines for the treatment of other complications).

[0185] Example 5

[0186] The present example relates to an eye drop. The eye drop comprises extract of Rhus coriaria, For, Onoin and Astra IV.

[0187] (I) Preparation of raw materials

[0188] Extract of Rhus coriaria:

[0189] a. Pretreatment of medicinal materials

[0190] Rhus coriaria was soaked in deionized water, then dried in the air, crushed, and the crushed particle size was controlled between 20-40 mesh.

[0191] b. Fractional extraction

[0192] Four-stage extraction was performed under the conditions set forth in the following table. The power of the ultrasonic extraction method used in the second stage extraction was 300 W. The power of the ultrasonic extraction method used in the third stage extraction was 300 W. The power of the microwave extraction method used in the fourth stage extraction was 500 W.

[0193] Table 1

[0194]

[0195] c. Concentration and purification

[0196] The extract of each stage was mixed and concentrated in a rotary evaporator at 50°C, and the ethanol recovery rate was controlled to be more than 80%.

[0197] d. Optimization of color depth

[0198] An appropriate amount of citric acid was added to the eluent to adjust the pH value to 3.5-4.0 to optimize the color depth of the extract of Rhus coriaria (tannin).

[0199] e. Filtration

[0200] Filtration was performed using a microporous filter membrane with a pore size of 0.22 μm to obtain the solution after adjustment of the pH value, and the concentration of the extract of Rhus coriaria was confirmed using a spectrophotometer.

[0201] For, Onoin and Astra IV: For, Onoin and Astra IV were extracted from Astragalus membranaceus according to the preparation method involved in Example 1.

[0202] (II) Preparation of eye drops

[0203] For and the extract of Rhus coriaria were mixed first, and the mixture was placed in the dark for 4±1 hours;

[0204] Then, Onoin and Astra IV were added, and low-speed stirring was performed at a stirring speed of 50±5 rpm.

[0205] The polyphenol compounds present in the extract of Chinese gallnut can effectively capture free radicals due to the presence of multiple phenolic hydroxyl groups, thereby having antioxidant properties, and the hydroxyl groups in the polyphenols can form hydrogen bonds with For molecules and form steric hindrance to increase the stability of For in solution.

[0206] Specifically, the present embodiment obtains the optimal binding time of For and the extract of Chinese gallnut at different concentrations by referring to molecular dynamics simulation and free energy calculation in quantum pharmacology.

[0207] Taking tannic acid as an example, the molecular structures of tannic acid and formononetin are imported into AMBER, the self-binding process of the two is simulated, and the binding kinetic parameters are analyzed based on the representative conformations (minimum binding free energy, corresponding to the most stable binding state) extracted from the simulation trajectories, and finally the standing time of the mixture of tannic acid and formononetin is obtained as shown in Table 2, and the sensory evaluation of the eye drops prepared by mixing 0.1M tannic acid and formononetin at different standing times is confirmed based on the skin patch test as shown in Table 3.

[0208] Table 2

[0209]

[0210] Table 3

[0211]

[0212]

[0213] Therefore, the tannins in the extract of Chinese gallnut can bind with proteins to have an adhesion effect. When the eye drops are used, the tannins in the extract of Chinese gallnut can increase the residence time of the eye drops in the eye and increase the sustained-release effect of other effective components.

[0214] It should be noted that the above specific embodiments are exemplary, and those skilled in the art can think of various solutions under the inspiration of the disclosure of the present application, and these solutions also belong to the disclosed range of the present application and fall within the protection scope of the present application. Those skilled in the art should understand that the specification and drawings of the present application are illustrative and do not constitute a limitation on the claims. The protection scope of the present application is defined by the claims and their equivalents. The specification of the present application contains multiple inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", which all indicate that the corresponding paragraph discloses an independent concept, and the applicant reserves the right to file a divisional application according to each inventive concept. Throughout the text, the features introduced by "preferably" are only optional ways, and should not be understood as necessarily provided, therefore the applicant reserves the right to abandon or delete the relevant preferred features at any time.

Claims

1. Application of total flavonoids from Astragalus extract in the preparation of drugs for inhibiting B cell activity.

2. A pharmaceutical composition for inhibiting B cell activity, characterized in that, The pharmaceutical composition contains total flavonoids from Astragalus extract.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition is an oral dosage form.

4. The application of Astragalus extract in the preparation of drugs that inhibit autoantibody secretion, characterized in that, Astragalus extract contains one or more of the following combinations: a. Total flavonoids from Astragalus extract; b. Astragalus extract polysaccharides.

5. The application according to claim 4, characterized in that, The autoantibody contains Anti-SSA.

6. The use of Astragalus extract in the preparation of drugs for regulating diseases caused by excessive B-cell response, characterized in that, Astragalus extract contains one or more of the following combinations: a. Total flavonoids from Astragalus extract; b. Astragalus extract polysaccharides.

7. A B-cell activity inhibitor, characterized in that, The B-cell activity inhibitor contains one or more of the following: styracin, styracin, and astragaloside IV.

8. A B-cell activity inhibitor, characterized in that, The B-cell activity inhibitor is composed of equimolar amounts of gentianin, gentianoside, and astragaloside IV.

9. The use of B-cell activity inhibitors in the preparation of medicaments for treating dry eye symptoms caused by Sjögren's syndrome, characterized in that, The B-cell activity inhibitor is composed of equimolar amounts of gentianin, gentianoside, and astragaloside IV.

10. An eye drop, characterized in that, The eye drops contain gallnut extract, styracin, styracin, and astragaloside IV.

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