Crystalline material of epivitratoside

Abstract

This invention discloses a crystalline form of cimicifuga glycoside, its preparation method, and its uses. This crystalline form has good stability, higher solubility and bioavailability, and advantages in gut microbiota.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, it relates to crystalline substances resembling cimicifugain, their preparation methods, compositions, and uses. Background Technology

[0002] The chemical name of the verbascoside is (E)-(2R,3R,4R,5R,6R)-6-(3,4-dihydroxyphenylethoxy)-5-hydroxy-2-(hydroxymethyl)-4-(((2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3-yl 3-(3,4-dihydroxyphenyl)acrylate, with the molecular formula C 29 H 36 O 15 The structural formula is as follows:

[0003]

[0004] Citrulline-like glycosides are glycoside compounds, also known as verbascoside or ergosterol. Physicochemically, they appear as off-white or pale yellow crystalline powders, slightly soluble in methanol or ethanol, soluble in dimethyl sulfoxide, and insoluble in water. They possess antitumor, anti-anxiety, and antidepressant activities, can improve memory impairment, and are also effective in treating inflammation. [1-9] .

[0005] Because of its poor water solubility, cimicifugain has poor gastric absorption and limited oral efficacy, thus restricting its role in the body. Therefore, improving the water solubility of cimicifugain to better exert its therapeutic effects is of great significance. Summary of the Invention

[0006] One or more embodiments of this application provide a crystalline form of cimicifuga glycoside, having crystal type III, wherein the powder X-ray diffraction pattern of crystal type III exhibits diffuse diffraction peaks, using CuK α The radiation is expressed in 2θ angles, and the peak value of the diffuse diffraction is located at 19.2±0.3°.

[0007] In one or more embodiments, the peak values ​​of the diffuse diffraction peaks are located at 7.9±0.3° and 19.2±0.3°.

[0008] In one or more embodiments, the powder X-ray diffraction pattern of the crystal type III is as follows: Figure 1 As shown.

[0009] In one or more embodiments, when analyzed using attenuated total reflectance Fourier transform infrared spectroscopy, the infrared spectrum of the crystal type III is at 3348 cm⁻¹. -1 ±2cm-1 2933cm -1 ±2cm -1 1687cm -1 ±2cm -1 1628cm -1 ±2cm -1 1600cm -1 ±2cm -1 1516cm -1 ±2cm -1 1444cm -1 ±2cm -1 1370cm -1 ±2cm -1 1257cm -1 ±2cm -1 1155cm -1 ±2cm -1 1112cm -1 ±2cm -1 1089cm -1 ±2cm -1 1011cm -1 ±2cm -1 978cm -1 ±2cm -1 912cm -1 ±2cm -1 877cm -1 ±2cm -1 852cm -1 ±2cm -1 808cm -1 ±2cm -1 784cm -1 ±2cm -1 718cm -1 ±2cm -1 685cm -1 ±2cm -1 589cm -1 ±2cm -1 563cm -1 ±2cm -1 451cm -1 ±2cm -1 There are characteristic peaks in the infrared spectrum at that location.

[0010] In one or more embodiments, the infrared spectrum of the crystal type III is as follows: Figure 2 As shown.

[0011] In one or more embodiments, the differential scanning calorimetry spectrum of the crystal type III has an endothermic peak at 126.68℃±10℃.

[0012] In one or more embodiments, the heating rate is 10°C / min in the range of 30–200°C.

[0013] In one or more embodiments, when thermogravimetric analysis is used, it is observed that when the heating rate is 5°C per minute, there is a weight loss step at 300±5°C in the thermogravimetric spectrum, with a weight loss of 55.1%.

[0014] One or more embodiments of this application provide a method for preparing the crystalline form of the cimicifuga glycoside of this application, which includes a suspension stirring method, an (organic solvent) liquid addition grinding method, a mechanical grinding method, a mechanochemical ball milling method, and a high-temperature crystallization method.

[0015] In one or more embodiments, the suspension stirring method involves weighing ephedrine, adding it to a single solvent or a mixed solvent, stirring at 100-400 r / min for 48-72 hours at a temperature of 20°C-45°C, and drying the product to obtain the crystalline form of ephedrine.

[0016] In one or more embodiments, the solvent is selected from one or more of methanol, ethanol, n-propanol, isopropanol, acetonitrile, acetone, dioxane, tetrahydrofuran, n-hexane, and cyclohexane.

[0017] In one or more embodiments, the volume ratio between any two solvents is 1:10 to 10:1.

[0018] In one or more embodiments, the liquid addition grinding method involves adding 5-100 mL of solvent per gram of cimicifuga glycoside, grinding for 0.1-10 hours, drying after grinding at a temperature of 25-60°C for 2-10 hours.

[0019] In one or more embodiments, the solvent is selected from one or more of methanol, ethanol, n-propanol, isopropanol, acetonitrile, acetone, dioxane, and tetrahydrofuran.

[0020] In one or more embodiments, the volume ratio between any two solvents is 1:10 to 10:1, for example 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1.

[0021] In one or more embodiments, the mechanical grinding method involves weighing ephedrine, placing it in a mortar, grinding it clockwise for 0.5 to 3 hours, and then drying the resulting product.

[0022] In one or more embodiments, the mechanochemical ball milling method involves placing citric acid-like substances into a ball mill jar for grinding, drying the resulting product, using a ball-to-material ratio of 1:1 to 10:1 (e.g., 6:1 to 10:1), a ball milling speed of 20 r / min to 400 r / min, and a grinding time of 1 to 72 h.

[0023] In one or more embodiments, the high-temperature crystallization method involves placing cimicifugoside in a glass desiccator and crystallizing it at 140°C for 20 to 60 minutes.

[0024] One or more embodiments of this application provide a solid mixture comprising the crystalline form of the cimicifuga glycoside of this application, wherein the amount of the crystalline form is 1-99.9% by weight, based on the weight of the solid mixture.

[0025] In one or more embodiments, the amount of the crystalline substance is 10-99.9% by weight, 50-99.9% by weight, or 85-99.9% by weight, for example 10% by weight, 20% by weight, 30% by weight, 40% by weight, 50% by weight, 60% by weight, 70% by weight, 80% by weight, or 90% by weight.

[0026] One or more embodiments of this application provide pharmaceutical compositions comprising a therapeutically effective amount of the crystalline form of the present application's cimicifuga glycoside or a solid mixture of the present application, and a pharmaceutically acceptable carrier.

[0027] In one or more embodiments, the daily dose of the pharmaceutical composition is 10 to 1000 mg, for example 100 mg, 500 mg, or 900 mg.

[0028] In one or more embodiments, the pharmaceutical composition is prepared as a tablet, capsule, pill, injectable formulation, powder for injection, granule, powder, micro-pellet, drop, suppository, film, patch, aerosol, or spray.

[0029] In one or more embodiments, the pharmaceutical composition is prepared as a sustained-release formulation or a controlled-release formulation.

[0030] One or more embodiments of this application provide the use of the crystalline form of the phycocyanin-like substance of this application, the solid mixture of this application, or the pharmaceutical composition of this application in the preparation of a medicament for the prevention and / or treatment of inflammation, anxiety, depression, tumors, and neurodegenerative diseases.

[0031] One or more embodiments of this application provide the use of the crystalline form of the cimicifuga glycoside of this application, the solid mixture of this application, or the pharmaceutical composition of this application in the preparation of a medicament for immunomodulation.

[0032] In one or more embodiments, the amount of organic solvent added in the (organic solvent) addition grinding method is 5 to 100 mL of organic solvent per gram of cimicifuga glycoside; the grinding time is 0.1 to 10 hours (e.g., 5 hours); the drying temperature is 25-60°C (e.g., 40°C); the drying time is 2 to 10 hours (e.g., 6 hours); the pot filling rate is 10% to 50% (e.g., 30%); and the reciprocating speed is 20 to 70 m / min (e.g., 45 m / min).

[0033] In one or more embodiments, the present application has a crystal form of Cimicifuga glycoside (Cimicifuga glycoside crystal form III) which has good solubility.

[0034] In one or more embodiments, the use of a crystalline form of cimicifugoside having crystal type III as an active pharmaceutical ingredient is provided in the preparation of medicaments for the prevention and treatment of inflammation, anxiety, depression, tumors, neurodegenerative diseases, and immunomodulation.

[0035] Morphological characteristics of Cimicifugoside III-type substances

[0036] The morphogenetic glycoside III crystal of this application is a crystal form of morphogenetic glycoside.

[0037] The type III cimicifugoside crystal of this application, when analyzed by powder X-ray diffraction under CuKα radiation conditions, exhibits a diffuse powder X-ray diffraction pattern. Figure 1 ).

[0038] The Cimicifugain Crystal Type III of this application, when analyzed using attenuated total reflectance Fourier transform infrared spectroscopy, showed the following values ​​at 3348, 2933, 1687, 1628, 1600, 1516, 1444, 1370, 1257, 1155, 1112, 1089, 1011, 978, 912, 877, 852, 808, 784, 718, 685, 589, 563, and 451 cm⁻¹. -1 There is an infrared spectral characteristic peak at this location, with an allowable deviation of ±2cm for the infrared spectral characteristic peak. -1 ( Figure 2 ).

[0039] When the cimicifugoside III crystal of this application was analyzed using differential scanning calorimetry, its DSC spectrum showed an exothermic peak at 126.68℃±10℃ within the temperature range of 30~200℃ at a heating rate of 10℃ / min. Figure 3 ).

[0040] The cimicifugain-like crystal type III of this invention, when analyzed by thermogravimetric analysis, exhibits a weight loss step at 300±5℃ in the TG spectrum when the heating rate is 5℃ per minute. Figure 4 ).

[0041] Preparation method of Cimicifugoside type III substance

[0042] The preparation method of the Cimicifuga glycoside type III substance in this application:

[0043] The liquid addition grinding method was adopted, with the addition of organic solvent, and the grinding time was 0.1 hours to 10 hours at room temperature, and the drying time was 2 to 10 hours.

[0044] The mechanochemical ball milling method uses a ball-to-material ratio of 1:1 to 10:1, preferably 6:1 to 10:1; a ball milling speed of 20 r / min to 400 r / min; and a grinding time of 1 to 72 h.

[0045] The organic solvent can be selected from any one or more of methanol, ethanol, acetonitrile, acetone, tetrahydrofuran, dioxane, n-hexane, and cyclohexane, and mixed solvents prepared by different proportions.

[0046] The physcinoside-type III substance of this application is mixed with other chemical substances in any non-zero proportion and by conventional methods.

[0047] Pharmaceutical formulations containing physcinoside type III substances, dosage characteristics and pharmaceutical uses

[0048] The pharmaceutical composition of this application comprises a type III substance of citronellol crystals and a pharmaceutically acceptable carrier.

[0049] The pharmaceutical composition of this application comprises a mixture of solids of a type III phytoestrogen-like substance and a pharmaceutically acceptable carrier.

[0050] The pharmaceutical composition of this application contains a daily dose of 10 to 1000 mg of a substance resembling cimicifugain crystal type III.

[0051] The dosage form of the pharmaceutical composition of this application can be various tablets, capsules, pills, injectable preparations, sustained-release preparations or controlled-release preparations.

[0052] The Cimicifugoside III-type substance described in this application has applications in the prevention and treatment of inflammation, anti-anxiety, anti-depression, anti-tumor, neurodegenerative diseases, and immune regulation.

[0053] The pharmaceutical composition of this application, which uses citric acid crystal type III as the active ingredient, can be prepared according to methods known in the art.

[0054] The phytoesphate-like crystal type III substance of this application can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants to formulate any dosage form suitable for human or animal use.

[0055] The content of the phytoestrogen type III substance and the mixed solid containing phytoestrogen type III substance in the pharmaceutical composition of this application is 10% to 90% by weight.

[0056] The Cimicifugoside III-type substance of this application can be administered in unit dose form, and the route of administration can be enteric or non-enteric, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0057] The dosage form of this application may be a solid dosage form, which may be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, microcapsules, droplets, suppositories, films, patches, aerosols, sprays, etc.

[0058] The physcinoside type III substance and the mixed solid substance containing physcinoside type III substance of this application can be formulated into ordinary formulations, as well as sustained-release formulations, controlled-release formulations, targeted formulations and various microparticle delivery systems.

[0059] In order to prepare the physcinoside type III substance of this application into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0060] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0061] To formulate the drug delivery unit into capsules, the active ingredient, cimicifugoside type III, can be mixed with a diluent and a flow aid, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient, cimicifugoside type III, can be first formulated into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. Various diluents, binders, wetting agents, disintegrants, and flow aids used to prepare tablets of cimicifugoside type III can also be used to prepare capsules of the cimicifugoside type III of this invention.

[0062] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0063] To achieve the intended therapeutic purpose and enhance the therapeutic effect, the drug of the present invention can be administered using any known method of drug administration.

[0064] The dosage of the pharmaceutical composition of the physcinoside type III substance of this application can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration, and the dosage form. The above dosage can be administered as a single unit or divided into several units, depending on the physician's clinical experience and the dosing regimen, including the use of other treatment methods.

[0065] The ephedrine-like crystal type III substance or composition of this application can be taken alone or in combination with other therapeutic or symptomatic drugs. When the ephedrine-like crystal type III substance of this invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0066] Beneficial technical effects of the present invention:

[0067] The Cimicifuga glycoside type III substance of this application has advantages in terms of safety, solubility, stability, bioavailability, absorption, and gut microbiota.

[0068] The cimicifugoside III-type substance of this application does not contain any crystallization solvent and has good advantages in terms of safety in drug formulation;

[0069] The physcinoside-type III substance described in this application is stable under conditions of high temperature (60℃), high humidity (25℃, relative humidity 90%±5%), and light exposure (4500 lx±500 lx), exhibiting certain advantages in drug stability. Figure 5 ).

[0070] The type III substance of citric acid glycosides in this application is superior to type I citric acid glycosides in terms of equilibrium solubility. Figure 6 );

[0071] The biological absorption of the drug and its composition containing cimicifugoside III as an active ingredient after oral administration, as described in this application, involves rapid and substantial release through the gastrointestinal tract or bloodstream. Compared to cimicifugoside type I crystals, cimicifugoside type III exhibits a higher in vivo exposure level and a shorter time to peak concentration (T0). max Later, the maximum blood drug concentration C max It has a higher content of active ingredients and higher bioavailability than type I crystals, thus playing a superior role in disease prevention and treatment. Figure 7 ).

[0072] In one or more embodiments, the dosage of the active ingredient in the pharmaceutical composition of the physcinoside crystal type III substance of this application is affected by many factors, such as: different uses for prevention and treatment resulting in different daily dosages; different nature and severity of the disease resulting in different daily dosages; differences in patient gender, age, body surface area, route of administration, frequency of administration, and treatment purpose resulting in different daily dosages; in addition, differences in absorption and blood drug concentration between crystal samples also result in a suitable daily dosage range of 0.002-20 mg / kg body weight, for example, 0.01-10 mg / kg body weight, for example, when using the physcinoside crystal type III substance. When using it, different total dosage schemes of the active ingredient of the physcinoside crystal type III substance should be formulated according to the actual needs of prevention and treatment, and it can be administered in multiple or single doses. Attached Figure Description

[0073] Figure 1 The powder X-ray diffraction pattern of the Cimicifuga glycoside type III substance.

[0074] Figure 2 The infrared absorption spectrum of the Cimicifugoside III crystal is shown.

[0075] Figure 3 Differential scanning calorimetry (DSC) spectrum of Cimicifugoside III crystals.

[0076] Figure 4 Thermogravimetric spectrum of Cimicifugoside III type III substance.

[0077] Figure 5 The spectrum is a stability study image of Cimicifugoside III-type substances.

[0078] Figure 6 This is an equilibrium solubility diagram of Cimicifugoside III-type substances.

[0079] Figure 7 The blood concentration and time curves of the Cimicifugoside III-type substance are shown.

[0080] Figure 8 Venn diagram of type III cimicifugoside crystals.

[0081] Figure 9 Dilution curves for intestinal flora analysis of Cimicifugoside III-type substances.

[0082] Figure 10 A graph showing the significant differences in gut microbiota analysis of Cimicifugoside III-type substances. Detailed Implementation

[0083] To better illustrate the technical solution of the present invention, the following embodiments are provided, but the present invention is not limited thereto.

[0084] Example 1

[0085] Preparation method of Cimicifugoside type III substance 1: Suspension stirring method

[0086] Weigh an appropriate amount of cimicifugoside and place it in a clean container. Add 0.5–20 mL of organic solvent and stir at 100–400 r / min for 48–72 h at 20℃–45℃. Filter the resulting suspension and dry it under vacuum, or allow it to air dry or evaporate. Perform powder X-ray diffraction analysis on the suspension. Its diffraction pattern is similar to… Figure 1 The consistency indicates that the obtained sample is a type III substance similar to cimicifugoside crystals.

[0087] Table 1 Specific examples of preparation method 1

[0088]

[0089] Preparation method of Cimicifugoside III-type substance: Mechanical grinding method

[0090] Weigh an appropriate amount of cimicifugoside and place it in a clean mortar. Grind it clockwise for 0.5 to 3 hours, then dry the resulting product. Perform powder X-ray diffraction analysis on it. Its diffraction pattern is similar to... Figure 1 The consistency indicates that the obtained sample is a type III substance similar to cimicifugoside crystals.

[0091] Table 2 Specific examples of preparation method 2

[0092]

[0093] Preparation method of Cimicifugoside III-type substance: Mechanochemical ball milling method

[0094] Mechanochemical ball milling was employed. An appropriate amount of cimicifugoside was weighed and placed in a clean ball mill jar, and ground according to a specific ball-to-material ratio and rotation speed. After grinding for a certain time, the resulting product was dried. Powder X-ray diffraction analysis was performed on the ground sample, and its diffraction pattern was consistent with... Figure 1 The consistency indicates that the obtained sample is a type III substance similar to cimicifugoside crystals.

[0095] Table 3 Specific examples of preparation method 3

[0096]

[0097] Preparation method of Cimicifugoside III-type substance: High-temperature crystallization method

[0098] Weigh an appropriate amount of Cimicifugain I-type crystals and place them in a clean glass desiccator. Perform high-temperature crystallization at 140℃ for 20-60 minutes, and then perform powder X-ray diffraction analysis. The diffraction pattern is similar to... Figure 1 The consistency indicates that the obtained sample is a type III substance similar to cimicifugoside crystals.

[0099] Example 2: Stability of Type III cimicifugoside crystals

[0100] High-temperature test: A sample of cimicifugoside type III crystals was placed in an open, clean petri dish and stored at 60°C for 10 days. Samples were taken on days 0, 5, and 10. Powder X-ray diffraction analysis of the samples obtained at the above sampling points showed that cimicifugoside type III crystals were stable under the high-temperature influence factor test.

[0101] High humidity test: The sample of Cimicifugain III crystals was placed in an open, clean petri dish and stored at 25°C and 90% ± 5% relative humidity for 10 days. Samples were taken on days 0, 5, and 10. Powder X-ray diffraction analysis of the samples obtained at the above sampling points showed that Cimicifugain III crystals were stable under high humidity conditions.

[0102] Illumination test: The sample of cimicifugoside III crystals was placed in an open, clean petri dish and then placed in an illumination box equipped with a fluorescent lamp at an illuminance of 4500 lx ± 500 lx for 10 days. Samples were taken on days 0, 5, and 10. The samples obtained from these sampling points were subjected to powder X-ray diffraction analysis. Figure 5 The results showed that the Cimicifuga glycoside type III substance was stable under light conditions.

[0103] Stability studies of cimicifugoside type III have demonstrated that cimicifugoside type III remains stable for 10 days under high temperature and light conditions, exhibiting excellent drug stability.

[0104] Example 3: Solubility of Type III cimicifugoside crystals

[0105] The solubility characteristics of ephedrine type III and ephedrine type I crystals were investigated in hydrochloric acid buffer (pH 1.2), acetate buffer (pH 4.5), phosphate buffer (pH 6.8), and pure water (pH 7.0). Using the equilibrium solubility concentration of ephedrine as the evaluation index and ephedrine type I crystals as the reference, the content of ephedrine type III crystals was determined by high-performance liquid chromatography (HPLC) at a wavelength of 365 nm. The dissolution concentration was calculated using the external standard method. Specific data are shown in Table 4. Figure 6 As shown.

[0106] Table 4. Equilibrium solubility data of Cimicifugain Type III and Cimicifugain Type I crystals.

[0107]

[0108] The experimental data show that the maximum concentration of the ephedrine-like glycoside type III material in the four buffer media is significantly improved compared with that of ephedrine-like glycoside type I crystal. Specifically, the ephedrine-like glycoside type III material has higher solubility, which can improve the speed and extent of ephedrine-like glycoside bioabsorption.

[0109] Example 4: In vivo bioabsorption of Cimicifugoside Type III substances

[0110] Standard solution: Accurately weigh 1.00 mg of citric acid, dissolve and mix thoroughly in 1 mL of acetonitrile to prepare a 1.00 mg / mL citric acid stock solution. Store at -20℃. Dilute with mobile phase solution to prepare standard solutions of different concentrations immediately before use. Internal standard solution: Accurately weigh 1.00 mg of genistein, dissolve and mix thoroughly in 1 mL of acetonitrile to prepare a 1.00 mg / mL genistein stock solution. Store at -20℃. Dilute with mobile phase solution to prepare a 500 ng / mL internal standard working solution immediately before use.

[0111] Eighteen male SD rats were randomly divided into three groups and fed under standard conditions with free access to water. After fasting for 12 hours, two groups of SD rats were administered 100 mg / kg of cimicifugain type I and type III crystals via gavage. One group of SD rats was administered 10 mg / kg of cimicifugain type I crystals intravenously. In the solid gavage group, approximately 0.3 mL of blood was collected from the posterior ocular venous plexus before administration and at 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 10 h after administration and placed into heparinized anticoagulant tubes. In the intravenous injection group, approximately 0.3 mL of blood was collected from the posterior ocular venous plexus before administration and at 2 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 10 h after administration and placed into heparinized anticoagulant tubes. The tubes were centrifuged at 4°C and 5000 rpm for 10 min, and the supernatant plasma was separated and stored at -40°C for later analysis.

[0112] Accurately pipette 100 μL of heparin-anticoagulated plasma into a 1.5 mL centrifuge tube, add 10 μL of genistein working solution, then add 1 mL of ethyl acetate, shake thoroughly for 5 min, centrifuge at 13400 rpm for 10 min, collect the supernatant in the centrifuge tube, and dry under nitrogen at room temperature. Reconstitute with 100 μL of reconstitution solution (acetonitrile:water = 90:10, v:v), shake for 5 min, centrifuge at 13400 rpm for 10 min, and inject the supernatant into an inner tube for analysis. Prepare a series of plasma samples with concentrations of 0, 2.5, 5, 10, 25, 50, 75, 100, 250, and 500 ng / mL using different concentrations of citric acid-like glycoside standard solution. Process the samples according to plasma processing methods and perform analysis under the corresponding LC-MS / MS conditions. Calculate pharmacokinetic parameters using a non-compartmental model with DAS 3.0 software.

[0113] Detection conditions: Column: ACQUITY BEH C 18 (1.7 μm, 2.1 × 50 mm); Column temperature: 40℃; Flow rate: 0.3 mL / min; Injection volume: 5 μL

[0114] Table 5. Gradient elution program for mobile phase

[0115]

[0116] Mass spectrometry conditions: Multiple reaction monitoring (MRM) negative ion monitoring mode; curtain gas: 40.0 Psi; collision gas: 9 Psi; spray voltage: -4500.0 V; nebulization temperature: 550℃; nebulizing gas: 40 Psi; auxiliary gas: 40 Psi. The quantitative ion pairs and parameter settings for the analytes are shown in Table 6.

[0117] Table 6. Quantitative ion pairs and parameter settings for the analytes

[0118]

[0119] A series of plasma samples with concentrations of 0, 2.5, 5, 10, 25, 50, 75, 100, 250, and 500 ng / mL were prepared using a phytoescin standard solution. After processing, the plasma samples were analyzed under the corresponding LC-MS / MS conditions. The standard curve regression equation was obtained as y = 0.001x - 0.00360.1132, r 2 =0.9994, indicating that cimicifugain showed good linearity in the range of 2.5-500 ng / mL, with a limit of quantification of 2.5 ng / mL. Pharmacokinetic parameters were calculated using a non-compartmental model with DAS (Data Analysis System 3.0) software.

[0120] Table 7. Blood concentrations of 100 mg / kg of citric acid-like drug in rats after oral administration (n=6)

[0121]

[0122] Table 8. Blood concentrations of 10 mg / kg of cimicifugain-like drug in rats after intravenous injection (n=6)

[0123]

[0124] Table 9. Main pharmacokinetic parameters of phytoesin-like compounds in rats (oral administration of 100 mg / kg / intravenous injection of 10 mg / kg, n=6)

[0125]

[0126] Based on oral and intravenous AUC (0-∞) Bioavailability was calculated, and the bioavailability of emodin type III crystals was 1.23%, while that of emodin type I crystals was 0.81%. The pharmacokinetic processes of emodin type I and emodin type III crystals differed in rats after solid gavage administration. A comparison of the two sets of data... Figure 7 Compared to type I cimicifugoside crystals, type III cimicifugoside crystals have higher in vivo exposure levels and a peak time T. max Later, the maximum blood drug concentration C max It is 1.78 times more potent than Cimicifugoside Type I crystals, and its bioavailability is higher.

[0127] Example 5: In vivo gut microbiota dominance of Cimicifugoside Type III substances

[0128] To clarify the differences in the effects of Cimicifugoside I and Cimicifugoside III on gut microbiota, 16S rDNA sequencing technology was used to compare the differences in gut microbiota in the cecal contents of SD rats, and correlation analysis was performed on different microbiota in each group.

[0129] After sequencing, the raw data (RawData) is spliced ​​using overlap, and quality control and chimera filtering are performed to obtain high-quality CleanData. Through steps such as "repeat removal," representative sequences with single-base precision are obtained. The core of DADA2 is noise reduction, followed by the construction of an OUT-like table to obtain the final ASV feature table and feature sequences, which are then further used for diversity analysis.

[0130] ASV distribution Venn plot: Venn plot ( Figure 8The Venn diagram visually presents the number of ASVs shared by and unique to each group. Each circle in the Venn diagram represents a group, the number of overlapping circles represents the number of ASVs shared by the two groups, and the non-overlapping parts represent the number of ASVs unique to each group.

[0131] Alpha diversity: Alpha diversity refers to the diversity within a specific environment or ecosystem, primarily used to reflect species richness and evenness, as well as sequencing depth. Alpha diversity is mainly reflected indices such as Chao1, Observed Species, Goods Coverage, Shannon, Simpson, and Pielou-E to indicate richness and evenness. (Dilution curve) Figure 9 , Figure 10 By simulating the resampling process, the trend of species change is observed, and the species richness in the environment is estimated. Dilution curves are plotted to statistically analyze the richness of ASVs (autoimmune species). Comparing the dilution curves of different samples visually demonstrates the differences in species diversity between samples. Dilution curves directly reflect the appropriateness of the sequencing data volume and indirectly reflect the species richness in the samples. When the curve flattens, it indicates that the sequencing data volume is gradually appropriate; more data will only generate a small number of new species (ASVs). Among them: Chaol and observed_species (… Figure 9 A and C) mainly estimate the number of species contained in the community; Goods_coverage( Figure 9 The "B" refers to microbial coverage; the higher the value, the lower the probability that new species in the sample were not detected. This index actually reflects whether the sequencing results represent the true situation of the sample; the Simpson score ( Figure 9 The Simpson value (D) ranges from 0 to 1. When there is only one species in the community, the Simpson value is at its minimum of 0, which also intuitively represents the minimum diversity. When there are an infinite number of species (highest richness) and the number of each species is the same (highest evenness), the Simpson value is at its maximum of 1. The Pielou-E index (…) Figure 9 The Shannon index (E), or Shannon's evenness, only reflects uniformity; the higher the value, the more uniform the uniformity. Figure 9 The F) comes from information entropy. The larger the Shannon index, the greater the uncertainty. The greater the uncertainty, the more unknown factors there are in this community, which means high diversity.

[0132] Figure 9Dilution curves were plotted according to the groups, with the horizontal axis representing the number of randomly selected sequences and the vertical axis representing the magnitude of each index for each sample when the same number of sequences were selected, to show the effects of Cimicifugain Type III and Cimicifugain Type I crystals on the richness, evenness and diversity of the intestinal flora of SD rats. Figure 10 The p-values ​​in the top left corner are the p-values ​​obtained by using the rank-sum test for all groups in the figure. The significance of the differences between pairs of groups is indicated by * for significant differences, ** for extremely significant differences, and ns for no significant differences.

[0133] Alpha diversity results indicate that, according to the Chaol and observed_species results, the type III group of emodin crystals contains the largest number of species; the Simpson index results show that the type III group has a higher species diversity; the Pielou-E index results show that the type III group has high evenness; and the Shannon index results show that the type III group has high diversity. These results suggest that after oral administration of the drug to SD rats, the type III group of emodin crystals exhibits greater diversity in gut microbiota species compared to the type I group of emodin crystals.

[0134] Example 6: Preparation of Tablet Formulation

[0135] Preparation method: Pure cimicifugoside type III substance was used as the active pharmaceutical ingredient (API) of the combination drug, and several excipients were used as tablet excipients. Tablets containing 10–500 mg of drug per tablet were prepared according to a certain ratio. Table 10 shows the tablet formulation ratios.

[0136] Table 10 Formulations for Preparing Tablets of Type III Cimicin Crystals

[0137]

[0138] The method for preparing tablet formulations using pure cimicifugoside type III substances as raw materials is as follows: mix several excipients with the raw material evenly and compress directly into tablets; or mix the excipients and dry granulate, then mix evenly with the raw material and compress into tablets.

[0139] Example 7: Preparation method of combination drug formulation 1 (tablet)

[0140] Preparation method: Pure cimicifugoside III crystals were used as raw materials, and several excipients were used as tablet excipients. The tablets were prepared in a certain proportion with a drug content of 10-500 mg per tablet. Table 11 shows the tablet formulation ratio.

[0141] Table 11 Formulations for preparing tablets containing type III cimicifugoside crystals

[0142]

[0143] The method for preparing tablet formulations using pure cimicifugoside type III substances as raw materials is as follows: several excipients are mixed evenly with the raw materials, an appropriate amount of 1% sodium hydroxymethyl cellulose solution is added to form a soft material, which is then granulated by sieving, the wet granules are dried, sieved and sized, magnesium stearate and talc are added and mixed evenly, and then tableted to obtain the final product.

[0144] Preparation method 2 for combination drug formulations (tablets)

[0145] Preparation method: Using citric acid glycoside type III substances and several excipients as excipients for preparing combination drug tablets, tablet samples containing 5-500 mg of citric acid glycoside type III substances per tablet were prepared according to a certain ratio. Table 12 shows the tablet formulation ratios:

[0146] Table 12 Formulations for the Preparation of Type III Lutein Crystal Drug Tablets

[0147]

[0148] The method for preparing tablet formulations using citric acid type III substances as raw materials is as follows: several excipients are mixed evenly with the raw material, an appropriate amount of 1% sodium hydroxymethyl cellulose solution is added to form a soft material, which is then granulated by sieving, the wet granules are dried, sieved and sized, magnesium stearate and talc are added and mixed evenly, and then tableted to obtain the final product.

[0149] Preparation method 3 of combination drug formulation (capsules)

[0150] Preparation method: Pure cimicifugoside III crystals were used as the raw material for the combination drug, and several excipients were used as excipients for preparing the combination drug capsules. The capsule samples with a drug content of 10-500mg per tablet were prepared according to a certain ratio. Table 13 shows the capsule formulation ratio.

[0151] Table 13 shows the active pharmaceutical ingredient and excipient formulations for capsule preparations containing type III citronellol crystals.

[0152]

[0153] The method for preparing a tablet formulation from the active pharmaceutical ingredient of citric acid crystalline type III is as follows: several excipients are mixed evenly with the active pharmaceutical ingredient, an appropriate amount of 1% sodium carboxymethyl cellulose solution is added, wet granules are formed, dried, sieved and granulated, magnesium stearate is added and mixed evenly, and then inserted into capsules; or, without using the granulation step, the active pharmaceutical ingredient of citric acid crystalline type III is directly mixed evenly with several excipients and excipients, sieved, and directly filled into capsules.

[0154] Example 8: Preparation of Pharmaceutical Composition Formulation

[0155] Dosage Form 1 (Tablet) of Cimicifugoside Type III Substance

[0156] The pharmaceutical composition developed using citric acid glycoside type III as the active pharmaceutical ingredient, with a daily dosage of 0.5-300 mg, can be prepared into ordinary tablets containing 10, 100, 200, 300, and 500 mg of active ingredient, in doses of 1-6 tablets per dose.

[0157] Dosage of Cimicifugoside Type III Substance 2 (capsules)

[0158] The pharmaceutical composition developed using citric acid glycoside type III as the active pharmaceutical ingredient, with a daily dosage of 10-3000 mg, can be prepared into capsules containing 10, 100, 200, 300, and 500 mg of active ingredient per dose, consisting of 1-6 capsules each time.

[0159] References

[0160] [1] Wen Yanli, Huo Shixia, Zhang Wei, et al. Research progress on pharmacological effects and pharmacokinetics of citric acid-like substances [J]. Chinese Journal of Traditional and Folk Medicine, 2015, 24(14): 26-27+29.

[0161] [2] Tan Xue, Gao Li, Ren Jia, et al. Effect of phytoesin-like compound on scopolamine-induced learning and memory impairment in mice [J]. Chinese Journal of New Drugs, 2018, 27(23): 2812-2818.

[0162] [3] Pu Jinghua, Pu Xiaoping, Ma Jian, et al. The effect of phytohemagglutinin on scopolamine-induced memory acquisition impairment [J]. Chinese Journal of Pharmacology, 2001, (06): 625-627.

[0163] [4] Ren Jia, Gao Li, Tan Xue, et al. Effects of citric acid-like glycosides on learning and memory abilities and inflammatory factors in rats with cerebral ischemia-reperfusion injury [J]. China Journal of Traditional Chinese Medicine Information, 2019, 26(04): 47-51.

[0164] [5] Ren Jia, Gao Li, Tan Xue, et al. Effects of citric acid on learning and memory function and oxidative stress in rats with vascular dementia [J]. Chinese Journal of Behavioral Medicine and Brain Science, 2018, 27(9): 777-782.

[0165] [6] Chen Jiayuan, Gao Li, Luo Jianan, et al. Effects of citric acid-like substances on behavioral and hormonal changes in APP / PS1 double transgenic mice [J]. China Medical Herald, 2020, 17(08): 9-12.

[0166] [7] Wang Dongyan, Sheng Shudong, Sun Yun. Experimental study on the effect of cimicifugain on D-galactose-induced aging model mice [C] / / Chinese Pharmaceutical Association, China Association of Traditional Chinese Medicine, Hotan Prefectural Committee, Administrative Office. Proceedings of the 6th Symposium on Cistanche deserticola and Psammophytic Medicinal Plants. School of Medicine, Yangzhou Environmental Resources College; School of Medicine, Yangzhou University; 2011: 1.

[0167] [8] Gao Li, Yan Ming, Huo Shixia. Use of phytoescin-like compounds in the preparation of anti-anxiety drugs: CN201611145131.4[P]. CN106581019B.

[0168] [9] Gao Li, Yan Ming, Huo Shixia. Use of phytoesin in the preparation of antidepressant drugs: CN201610390189.9[P].CN105878258B.

Claims

1. A crystalline substance resembling cimicifuga glycoside, having a crystal type III, wherein the powder X-ray diffraction pattern of the crystal type III exhibits diffuse diffraction peaks, as determined by CuK... α The radiation is expressed in 2θ angles, and the peak value of the diffuse diffraction is located at 19.2±0.3°; preferably, the peak value of the diffuse diffraction is located at 7.9±0.3° and 19.2±0.3°; more preferably, the powder X-ray diffraction pattern of the crystal type III is shown in Figure 1.

2. The crystalline form of cimicifugain as described in claim 1, wherein when analyzed using attenuated total reflectance Fourier transform infrared spectroscopy, the infrared spectrum of the crystal type III is at 3348 cm⁻¹. -1 ±2cm -1 2933cm -1 ±2cm -1 1687cm -1 ±2cm -1 1628cm -1 ±2cm -1 1600cm -1 ±2cm -1 1516cm -1 ±2cm -1 1444cm -1 ±2cm -1 1370cm -1 ±2cm -1 1257cm -1 ±2cm -1 1155cm -1 ±2cm -1 1112cm -1 ±2cm -1 1089cm -1 ±2cm -1 1011cm -1 ±2cm -1 978cm -1 ±2cm -1 912cm -1 ±2cm -1 877cm -1 ±2cm -1 852cm -1 ±2cm -1 808cm -1 ±2cm -1 784cm -1 ±2cm -1 718cm -1 ±2cm -1 685cm -1 ±2cm -1 589cm -1 ±2cm -1 563cm -1 ±2cm -1 451cm -1 ±2cm -1 There are characteristic infrared spectral peaks at the location; preferably, the infrared spectrum of the crystal type III is shown in Figure 2.

3. The crystalline form of the cimicifuga glycoside according to claim 1, wherein the differential scanning calorimetry spectrum of the crystal type III has an endothermic peak at 126.68℃±10℃; preferably, the heating rate is 10℃ / min in the range of 30~200℃.

4. The crystalline form of the cimicifuga glycoside according to claim 1, when analyzed by thermogravimetric analysis, shows a weight loss step at 300±5℃ in the thermogravimetric spectrum when the heating rate is 5℃ per minute, with a weight loss of 55.1%.

5. The method for preparing the crystalline form of cimicifuga glycoside according to any one of claims 1-4, wherein the method comprises suspension stirring, liquid addition grinding, mechanical grinding, mechanochemical ball milling, or high-temperature crystallization. Preferably, in the suspension stirring method, physcinoside is weighed, added to a single solvent or a mixed solvent, and stirred at 100-400 r / min for 48-72 h at a temperature of 20℃-45℃. The product is then dried to obtain the crystalline form of physcinoside. Preferably, the solvent is selected from one or more of methanol, ethanol, n-propanol, isopropanol, acetonitrile, acetone, dioxane, and tetrahydrofuran. Preferably, the volume ratio between any two solvents is 1:10 to 10:

1. Preferably, in the liquid-addition grinding method, the amount of liquid added is 5-100 mL per gram of cimicifuga glycoside, the grinding time is 0.1-10 hours, and the mixture is dried after grinding at a temperature of 25-60°C for 2-10 hours; preferably, the solvent is selected from one or more of methanol, ethanol, n-propanol, isopropanol, acetonitrile, acetone, dioxane, and tetrahydrofuran; preferably, the volume ratio between any two solvents is 1:10 to 10:

1. Preferably, in the mechanical grinding method, the following steps are taken: weighing ephedrine, placing it in a mortar, grinding in a clockwise direction for 0.5 to 3 hours, and then drying the resulting product; Preferably, the mechanical chemical ball milling method is used, in which citric acid-like substances are placed in a ball mill jar for grinding, the resulting product is dried, the ball-to-material ratio is 1:1 to 10:1, preferably 6:1 to 10:1, the ball milling speed is 20 r / min to 400 r / min, and the grinding time is 1 to 72 h. Preferably, the high-temperature crystallization method involves placing the cimicifugoside in a glass desiccator and crystallizing it at 140°C for 20 to 60 minutes.

6. A solid mixture comprising the crystalline form of cimicifuga glycoside according to any one of claims 1-4, wherein the amount of the crystalline form is 1-99.9% by weight based on the weight of the solid mixture; preferably 10-99.9% by weight, 50-99.9% by weight, or 85-99.9% by weight.

7. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline form of cimicifuga glycoside according to any one of claims 1-4 or the solid mixture according to claim 6, and a pharmaceutically acceptable carrier; preferably, the daily dose of the pharmaceutical composition is 10-1000 mg; preferably, the pharmaceutical composition is prepared as tablets, capsules, pills, injectable formulations, powder for injection, granules, powders, microcapsules, drops, suppositories, films, patches, aerosols, or sprays; preferably, the pharmaceutical composition is prepared as a sustained-release or controlled-release formulation.

8. Use of the crystalline form of cimicifuga glycoside according to any one of claims 1-4, the solid mixture according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for the prevention and / or treatment of inflammation, anxiety, depression, tumors, and neurodegenerative diseases.

9. Use of the crystalline form of cimicifuga glycoside according to any one of claims 1-4, the solid mixture according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for immunomodulation.

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