Salt formed by berberine and scutellarin, preparation method and application thereof

Abstract

The present invention relates to a salt formed by berberine and scutellarin, and its preparation method and application. The structure of the berberine scutellarin salt is:

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a salt formed by berberine and scutellarin, and a preparation method and application thereof. Background Art

[0002] Berberine (molecular formula: C 20 H 18 NO4, molecular weight: 336.37), is a quaternary ammonium alkaloid isolated from the traditional Chinese medicine Coptis chinensis and is the main active ingredient in its antibacterial properties. Its main clinical application is as an over-the-counter drug for the prevention and treatment of dysentery. Berberine has a broad antibacterial spectrum and has antibacterial effects on a variety of gram-positive and gram-negative bacteria in vitro, such as Shigella dysenteriae, Mycobacterium tuberculosis, Pneumococcus, Salmonella typhi, and Corynebacterium diphtheriae. It has the strongest effect on Shigella dysenteriae and has almost no drug resistance or side effects. In recent years, with the continuous deepening of research, its application scope has seen new developments:

[0003] (1) Modern pharmacological research has confirmed that berberine also has significant effects in regulating blood sugar and lipid metabolism, anti-inflammation, lowering blood uric acid, anti-rheumatoid arthritis, inhibiting tumor cell proliferation, and anti-virus.

[0004] (2) A number of basic and clinical evidences have confirmed that berberine participates in glucose metabolism through various mechanisms, such as retinol binding protein-4 (RBP-4) and glucose transporter-4 (GLUT-4) mechanisms; increasing the expression of hepatocyte nuclear factor-4a (HNF-4a) and glucokinase activity; reversing the phosphorylation of insulin receptor (IRS) Ser307; and upregulating the expression of IRS.

[0005] (3) The lipid-lowering function of berberine, the molecular mechanism involves upregulating the low-density lipoprotein receptor (LDLR) mRNA level, inhibiting the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene, increasing the expression of hepatic apolipoprotein (Apo) E mRNA, activating the AMPK pathway, sterol regulatory element binding protein (SREBP)-C and CCAAT enhancer binding protein (C / EBP)-α, proteasome proliferator-activated receptor (PPAR)-γ, and reducing the transcription level of the subtilisin convertase (PCSK) 9 gene. 1,2 .

[0006] (4) In the treatment of cardiovascular diseases, berberine is also a promising candidate with multiple targets. The cardiovascular pharmacological effects of berberine mainly include anti-arrhythmia, anti-heart failure, vasodilation and blood pressure reduction, lipid metabolism regulation and anti-atherosclerosis, anti-platelet and anti-thrombotic, anti-cerebral ischemia, protection of vascular endothelium, anti-vascular inflammation and inhibition of tumor angiogenesis. The target cells include myocardial cells, vascular endothelial cells, vascular smooth muscle cells, platelets and blood mononuclear macrophages. The specific targets are mainly Ca 2+ Channel, K + channels, M receptors, adenosine receptors, cholinesterase and angiotensin-converting enzyme (ACE), etc. 3 .

[0007] Berberine is a yellow needle-shaped crystal with an extremely bitter taste. It is poorly absorbed orally, with an oral bioavailability of less than 5%. Improve biological Improving the utilization rate and taste are the keys to increase its clinical applicability.

[0008]

[0009] Scutellarin (molecular formula: C 21 H 18 O 12 , molecular weight: 462.363) is a flavonoid compound extracted and separated from the Asteraceae plant Erigeron breviscapus, which has a strong protective function for cardiovascular and cerebrovascular diseases (especially ischemic stroke and coronary heart disease). As a drug for the treatment of cardiovascular and cerebrovascular diseases, baicalin is widely used in clinical treatment, especially in combination with other traditional chemical drugs, which not only significantly enhances the therapeutic effect, but also reduces adverse reactions to a certain extent. Its mechanism includes that baicalin can significantly reduce the levels of inflammatory factors and chemokines in the process of cardiovascular and cerebrovascular damage, inhibit the occurrence of inflammation, and thus play a protective role. The anti-inflammatory effect of baicalin also plays a role in other diseases such as tumors. In addition, baicalin also has antioxidant function, which can increase the activity and content of antioxidant substances such as superoxide dismutase, and play an auxiliary role in the prevention and treatment of many diseases. Recent studies have shown that baicalin can also lower blood lipids and treat diabetes. In addition, baicalin can also inhibit colorectal cancer, bladder cancer, lung cancer, liver cancer, breast cancer, melanoma and other cancers; as well as neuroprotection and ischemia-reperfusion protection. But due to wild Baicalin is almost insoluble in water, has a short half-life, and has low oral bioavailability, which greatly limits its clinical application. 4 .

[0010]

[0011] Although berberine and baicalin have many similar pharmacological activities, and studies have shown that the combination of the two drugs has a synergistic effect, their low bioavailability and significant differences in release and absorption characteristics limit their clinical use. Therefore, finding a method to improve the bioavailability of berberine and baicalin and exert their synergistic effect is of great clinical significance.

[0012] Salt formation (the preparation of acidic and basic drugs into salts) can improve the drug's physical and chemical properties, such as solubility, dissolution rate, stability, and bioavailability, and has become a new approach to drug development. Berberine-scutellarin salts can also effectively reduce the bitterness of berberine and increase patient compliance.

[0013] Based on this, the present invention is proposed.

[0014] [References]

[0015] 1. Research progress on the mechanism of berberine regulating glucose and lipid metabolism. Liu Xiaoyan, Liu Jian, Gao Yu, Chinese Journal of Gerontology. 2016, 36(8): 4117-4119 2. Berberine Clinical Research progress. Wang Xiaohong, Medical Frontier. 2013, 27 :380-381

[0016] 3. A Brief Review of Berberine's Cardiovascular Pharmacology. Wang Ruiguo, Fang Taihui, Proceedings of the Fourth National Symposium on Traditional Chinese Medicine Immunology. September 30, 2007

[0017] 4.Wang L,Ma Q.Clinical benefits and pharmacology of scutellarin:Acomprehensive review.Pharmacol Ther.2018Oct;190:105-127.doi:10.1016 / j.pharmthera.2018.05.006.Epub 2018May 6.PMID:29742480.

[0018] 5. Hu Qianlian, Zhang Jieyu, Mei Gege, Li Zhujun, Li Xinyi, Tian Ya, Fang Zhenfeng. Determination of total flavonoids from Scutellaria barbata by different enrichment methods by HPLC[J]. Journal of Central South Pharmacy, 2021, 19(04): 714-718. Summary of the Invention

[0019] The present invention first relates to a berberine scutellarin salt, the structure of which is:

[0020]

[0021] Its NMR spectrum characteristics are:

[0022] 1H NMR (400MHz, DMSO-d6) δ12.60(s,1H),9.90(s,1H),8.85(s,1H),8.08–7.99(m,1H),7.90(d,J=9.4Hz, 1H),7.76(dd,J=8.8,2.3Hz,2H),7.71(d,J=2.7Hz,1H),7.00(s,1H),6.86(d,J=8.5Hz,2H),6.83(s,1H ),6.64(d,J=2.6Hz,1H),6.13(s,2H),5.09(s,1H),5.01–4.94(m,1H),4.91(t,J=6.5Hz,2H),4.05(s, 3H), 3.94 (s, 3H), 3.74–3.61 (m, 1H), 3.37 (s, 3H), 3.28 (dd, J = 16.0, 7.4Hz, 2H), 3.18 (d, J = 6.4Hz, 2H).

[0023] In the berberine and baicalin salts, the dissolution behavior of the two drugs is consistent; preferably, the dissolution rate of berberine is greater than 65% in 6 hours, and the dissolution rate of baicalin is greater than 29% in 6 hours; the dissolution rate is determined by the method recorded in the "Chinese Pharmacopoeia 2020".

[0024] The X-ray diffraction pattern of the berberine scutellarin salt crystals has no characteristic diffraction peak of berberine.

[0025] The X-ray crystal data of the berberine scutellarin salt crystals are:

[0026]

[0027] The infrared spectral properties of the berberine scutellarin salt after tableting with KBr are:

[0028] (1) 3600-3000cm -1 There are no characteristic absorption peaks of berberine and scutellarin;

[0029] (2) Compared with the physical mixture, at 1720.7 cm -1 The C=O characteristic peak at the position disappears;

[0030] (3) There are characteristic peaks of infrared spectrum at 2947, 2897, 1665, 1604, 1570, 1507, 1363, 1276, 1232, 1180, 1065, 1035, 973, 936, 911, 835, 804, 590, 557, 514, and 428 cm-1.

[0031] The allowable deviation of the infrared spectrum characteristic peak is ±2cm -1 .

[0032] The berberine and scutellarin salt is determined by differential scanning calorimetry (DSC) analysis and has no characteristic melting peaks of berberine and scutellarin, wherein the allowable deviation of the characteristic peak of differential scanning calorimetry analysis is ±2 cel.

[0033] The present invention also relates to a method for preparing the berberine scutellarin salt, which comprises the following steps:

[0034] (1) Dissolve berberine hydrochloride or dihydroberberine in 10 to 300 times the volume of water by heating;

[0035] (2) dispersing scutellarin in water and an alcohol solvent, adding 0.05 to 0.1 equivalents of a base, and dissolving it in an oil bath at 30 to 50° C., wherein the base is selected from potassium hydroxide, sodium hydroxide, potassium carbonate, and sodium carbonate; the alcohol solvent is selected from methanol, ethanol, and isopropanol; and the ratio of water to alcohol is 1:1 to 10:1;

[0036] (3) At room temperature, slowly add berberine hydrochloride or dihydroberberine aqueous solution to the above-mentioned scutellarin alkali solution while it is hot, and stir until an amber colloidal solid precipitates;

[0037] (4) Stir at room temperature for 1 to 2 hours, let stand for 1 to 3 hours, filter out the solid, and dry to obtain a salt composed of berberine and scutellarin.

[0038] The berberine is berberine or a pharmaceutically acceptable salt thereof; the structural formula of berberine is:

[0039]

[0040] The pharmaceutically acceptable salt is a salt formed by berberine and an inorganic acid or an organic acid, wherein the inorganic acid includes but is not limited to hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid or nitric acid; the organic acid includes but is not limited to oxalic acid, fumaric acid, maleic acid, succinic acid, citric acid, tartaric acid, methanesulfonic acid, phthalic acid or p-toluenesulfonic acid;

[0041] Preferably, the berberine is berberine hydrochloride.

[0042] The structure of scutellarin is:

[0043]

[0044] More preferably,

[0045] In step (1), the berberine hydrochloride is berberine hydrochloride chloride; the ratio of berberine hydrochloride chloride to water is 1:80-90 (w / v); heating at 80°C to dissolve;

[0046] In step (2), the water and alcohol solvent are ethanol / water = 2:1; the amount ratio of scutellarin to solvent is 1:30-40 (w / v); after dissolving in a 40°C oil bath, the mixture is allowed to stand until it reaches room temperature.

[0047] The present invention also relates to a medicine or pharmaceutical composition comprising the berberine scutellarin salt, which comprises a therapeutically effective amount of the berberine scutellarin salt and a pharmaceutically acceptable excipient.

[0048] The dosage form of the pharmaceutical composition can be a liquid dosage form, a solid dosage form, or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o / w types, w / o types, and multiple emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.

[0049] The above dosage forms can be made into ordinary preparations, sustained-release preparations, controlled-release preparations, targeted preparations and various microparticle delivery systems, such as liposome preparations.

[0050] The pharmaceutical composition of the present invention can be prepared according to methods known in the art. Berberine scutellarin salt can be combined with one or more pharmaceutically acceptable solid or liquid excipients to form any dosage form suitable for human or animal use. Berberine scutellarin salt or the pharmaceutical composition of the present invention containing the same can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0051] For example, in order to prepare berberine scutellarin salt into tablets, various excipients known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and solubilizers. The diluent may be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent may be water, ethanol, isopropyl alcohol, etc.; the binder may be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrant may be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; the lubricant and solubilizing agent may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0052] The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.

[0053] To prepare capsules, the active ingredient, berberine scutellarin, can be mixed with a diluent and a solubilizing agent, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient, berberine scutellarin, can be first mixed with a diluent, a binder, and a disintegrant to form granules or pellets, which can then be placed into hard or soft capsules. The diluents, binders, wetting agents, disintegrants, and solubilizing agents used to prepare the berberine scutellarin tablets of the present invention can also be used to prepare capsules of the compound of the present invention.

[0054] Furthermore, if necessary, colorants, preservatives, perfumes, flavorings or other additives may be added to the pharmaceutical preparations.

[0055] The dosage of the pharmaceutical composition of the present invention 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, the dosage form, etc. Generally speaking, for administration to human subjects, the dosage of berberine is 1-30 mg / kg / day, preferably 2-25 mg / kg / day, and more preferably 5-20 mg / kg / day, calculated as berberine; the dosage of scutellarin is 1-40 mg / kg / day, preferably 2-35 mg / kg / day, and more preferably 5-30 mg / kg / day, calculated as scutellarin.

[0056] In addition, the above dosage can be administered in one dosage unit or divided into several dosage units, depending on the clinical experience of the physician and the dosage regimen including the use of other treatment methods. Furthermore, the salts of the present invention can be used with other drugs, such as vitamin E, obeticholic acid, bicyclol, polyene phosphatidylcholine, diamine glycyrrhizinate, reduced glutathione, S-adenosylmethionine, ursodeoxycholic acid, etc., as long as they do not impair the effect of berberine-scutellarin salt. Furthermore, when the berberine-scutellarin salt of the present invention is used in combination with other drugs, its dosage should be adjusted according to the actual situation.

[0057] The present invention also relates to the use of the berberine scutellarin salt in the preparation of medicines for preventing or treating metabolic diseases and related diseases.

[0058] The metabolic diseases include dyslipidemia, obesity, and fatty liver disease;

[0059] The lipid metabolism disorder includes high low-density lipoprotein-cholesterol, high total cholesterol, high triglycerides or low high-density lipoprotein-cholesterol;

[0060] Obesity refers to weight gain and increased body fat percentage;

[0061] The fatty liver disease is characterized by increased triglyceride (TG) in liver tissue, increased liver index, increased liver fat rate, increased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and increased liver inflammatory factors.

[0062] The metabolism-related cardiovascular and cerebrovascular diseases include hypercoagulability, atherosclerosis, hypertension, and ischemic stroke.

[0063] The beneficial effects of the present invention include:

[0064] The salt of the present invention is a novel compound distinct from the physical mixture of berberine and baicalin, exhibiting distinct nuclear magnetic resonance (NMR) spectra, powder X-ray diffraction (XRD) spectra, differential scanning calorimetry (DSC) spectra, and infrared spectra. The salt significantly improves the dissolution rate of berberine and baicalin, achieving simultaneous release of the two drugs and enhancing their synergistic pharmacological effects in preventing or treating metabolic diseases. Furthermore, the salt effectively reduces the bitterness of berberine and improves patient compliance.

[0065] In a golden hamster model of hyperlipidemia, hyperglycemia, obesity, hypercoagulopathy, and fatty liver disease induced by a high-sugar, high-fat diet, the salts of the present invention can lower blood sugar and lipid levels, improve liver function, significantly reduce body weight, and reduce platelet aggregation. In an ApoE(- / -) mouse model of atherosclerosis induced by a high-fat diet, the salts of the present invention can reduce arterial plaque formation in the model animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 , NMR spectrum of berberine scutellariae salt.

[0067] Figure 2 , NMR spectrum of the physical mixture of berberine and baicalin.

[0068] Figure 3 , infrared spectrum of berberine scutellariae salt.

[0069] Figure 4 , infrared spectrum of the physical mixture of berberine and baicalin.

[0070] Figure 5 , DSC diagram of berberine and baicalin salt.

[0071] Figure 6 , DSC graph of the physical mixture of berberine and baicalin.

[0072] Figure 7 , powder X-ray diffraction pattern of berberine scutellariae salt.

[0073] Figure 8 , powder X-ray diffraction pattern of the physical mixture of berberine and baicalin.

[0074] Figure 9 The cumulative dissolution curves of berberine and baicalin in the physical mixture of berberine and baicalin and berberine baicalin salt in water. There are four dissolution curves in the figure, namely berberine in the physical mixture (physical mixture-berberine), baicalin in the physical mixture (physical mixture-baicalin), berberine (salt-berberine) in berberine baicalin salt, and baicalin (salt-baicalin) in berberine baicalin salt.

[0075] Figure 10 , Comparison of liver triglyceride content in experimental animals of different drug groups in the golden hamster hyperglycemia, hyperlipidemia, obesity, hypercoagulability and fatty liver disease model induced by a high-sugar and high-fat diet.

[0076] Figure 11 , Comparison of plasma blood glucose levels in experimental animals in different drug groups in the golden hamster hyperglycemia, hyperlipidemia, obesity, hypercoagulability and fatty liver disease model induced by a high-sugar and high-fat diet.

[0077] Figure 12 , Comparison of total cholesterol levels in the plasma of experimental animals in different drug groups in the golden hamster hyperglycemia, hyperlipidemia, obesity, hypercoagulability and fatty liver disease model induced by a high-sugar and high-fat diet.

[0078] Figure 13, Comparison of low-density lipoprotein-cholesterol levels in the plasma of experimental animals in the golden hamster hyperglycemia, hyperlipidemia, obesity, hypercoagulability and fatty liver disease models induced by a high-sugar and high-fat diet.

[0079] Figure 14 , Comparison of triglyceride levels in the plasma of experimental animals in the golden hamster hyperglycemia, hyperlipidemia, obesity, hypercoagulability and fatty liver disease models induced by a high-sugar and high-fat diet.

[0080] Figure 15 , Comparison of high-density lipoprotein-cholesterol levels in the plasma of experimental animals in the golden hamster hyperglycemia, hyperlipidemia, obesity, hypercoagulability and fatty liver disease models induced by a high-sugar and high-fat diet.

[0081] Figure 16 , Comparison of plasma alanine aminotransferase levels in experimental animals in the golden hamster hyperglycemia, hyperlipidemia, obesity, hypercoagulability and fatty liver disease models induced by a high-sugar and high-fat diet.

[0082] Figure 17 , Comparison of plasma aspartate aminotransferase levels in experimental animals in the golden hamster hyperglycemia, hyperlipidemia, obesity, hypercoagulability and fatty liver disease models induced by a high-sugar and high-fat diet.

[0083] Figure 18 , Oil red staining images of liver tissue of experimental animals in the golden hamster hyperglycemia, hyperlipidemia, obesity, hypercoagulability and fatty liver disease model induced by a high-sugar and high-fat diet.

[0084] Figure 19 , Comparison of body weight of experimental animals in the golden hamster hyperglycemia, hyperlipidemia, obesity, hypercoagulability and fatty liver disease models induced by a high-sugar and high-fat diet.

[0085] Figure 20 Platelet aggregation rate (%) in golden hamster models of hyperglycemia, hyperlipidemia, obesity, hypercoagulability, and fatty liver disease induced by a high-sugar and high-fat diet

[0086] Figure 21 , Quantitative results of gross oil red staining of the aorta of experimental animals in different dosing groups in the ApoE(- / -) mouse model induced by a high-fat diet.

[0087] Figure 22 , HE staining images of the aorta of experimental animals in different drug groups in the ApoE(- / -) mouse model induced by a high-fat diet.

[0088] Figure 23 , the crystal structure diagram of berberine scutellariae salt (ignoring the solvent part).

[0089] Figure 24, the unit cell stacking diagram of berberine scutellariae salt. DETAILED DESCRIPTION

[0090] Materials and equipment

[0091] Berberine hydrochloride was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., chemically pure, with a purity of ≥98%;

[0092] Berberine was purchased from Shanghai MacLean Biochemical Technology Co., Ltd., chemically pure, with a purity of ≥97%;

[0093] Scutellarin was purchased from Wuhan Qiongge Biotechnology Co., Ltd., chemically pure, with a purity of ≥98%;

[0094] KBr was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., chromatographically pure, with a purity of 99%;

[0095] C57 mice, Syrian golden hamsters, and ApoE(- / -) mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.;

[0096] High-sugar and high-fat diet feed was purchased from Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd.

[0097] Example 1. Preparation of berberine scutellarin salt

[0098] (1) Add 3.6 g of berberine chloride (chloride of hydrochloride) to 300 mL of water and heat at 80°C to dissolve;

[0099] (2) 4.5 g of scutellarin was added to a mixed solution of 150 mL of water and ethanol (ethanol / water = 2:1), and 10 mL of sodium hydroxide aqueous solution containing 0.4 g of sodium hydroxide was added (0.4 g of sodium hydroxide was dissolved in 10 mL of water, and then all 10 mL of sodium hydroxide solution was added). The mixture was placed in a 40°C oil bath, stirred for half an hour, and allowed to stand until it reached room temperature.

[0100] (3) The berberine chloride aqueous solution was slowly added dropwise to the scutellarin solution under stirring. After the addition was complete, the mixture was stirred at room temperature for 1 hour, allowed to stand for 2 hours, filtered, dried at room temperature for 2 days, and dried at 50°C for 5 hours to obtain 7.94 g of berberine scutellarin salt.

[0101] Comparative Example Preparation of Berberine-Scutellarin Mixture

[0102] 2.2 g of berberine and 2.8 g of baicalin were put into a mortar, and the mixture was repeatedly ground and mixed to obtain 4.95 g of a berberine-baicalin mixture.

[0103] Example 2, berberine scutellarin salt nuclear magnetic resonance 1 H NMR determination

[0104] The berberine scutellarin salt and the berberine-scutellarin mixture prepared in Example 1 were subjected to H-NMR analysis. Figure 1 (berberine scutellarin salt), 2 (berberine-scutellarin physical mixture) as samples 1 H NMR spectrum.

[0105] As can be seen from the figure, berberine scutellarin salt and berberine-scutellarin mixture have completely different NMR spectra.

[0106] Berberine scutellarin characteristic peaks ( Figure 1 )for:

[0107] 1 H NMR (400MHz, DMSO-d6) δ12.60(s,1H),9.90(s,1H),8.85(s,1H),8.08–7.99(m,1H),7.90(d,J=9.4Hz, 1H),7.76(dd,J=8.8,2.3Hz,2H),7.71(d,J=2.7Hz,1H),7.00(s,1H),6.86(d,J=8.5Hz,2H),6.83(s,1H ),6.64(d,J=2.6Hz,1H),6.13(s,2H),5.09(s,1H),5.01–4.94(m,1H),4.91(t,J=6.5Hz,2H),4.05(s, 3H),3.94(s,3H),3.74–3.61(m,1H),3.37(s,3H),3.28(dd,J=16.0,7.4Hz,2H),3.18(d,J=6.4Hz,2H).

[0108] Characteristic peaks of berberine-scutellarin mixture ( Figure 2 )for:

[0109] 1H NMR(400MHz,DMSO-d6)δ12.70(s,1H),10.46(s,1H),9.86(s,1H),8.90(s,1H),8.60(s,1H),8.1 6(d,J=9.0Hz,1H),7.96(d,J=9.1Hz,1H),7.90(d,J=8.6Hz,2H),7.76(s,1H),7.06(s,1H),6.98– 6.91(m,3H),6.78(s,1H),6.16(s,2H),5.56(s,1H),5.43(s,1H),5.21(d,J=7.3Hz,1H),4.92(t ,J=6.3Hz,2H),4.08(s,3H),4.06(s,1H),4.04(s,3H),3.48–3.38(m,5H),3.19(t,J=6.3Hz,2H).

[0110] Example 2: Infrared Spectroscopy Determination of Berberine and Scutellarin

[0111] Take appropriate amount of each sample prepared in Example 1 and mix with KBr and press into pellets. The scanning range is 4000-400 cm -1 . Figure 3 、 4 Berberine and scutellarin salts ( Figure 3 ), berberine-scutellarin physical mixture ( Figure 4 )’s infrared spectrum.

[0112] As can be seen from the figure, in the infrared spectrum of berberine scutellarin salt, 3374.6cm -1 The free vibration peak of OH at 1720.7cm -1 The characteristic peak of carbonyl group disappeared.

[0113] Example 3, DSC determination of berberine scutellarin salt

[0114] 2-5 mg of the sample prepared in Example 1 was accurately weighed and placed in an aluminum crucible. An empty crucible of the same type was used as a reference. The protective atmosphere was 99% pure nitrogen at a flow rate of 60 ml / min. The sample was scanned at a rate of 10°C / min over a range of 25-280°C.

[0115] Berberine scutellarin salt ( Figure 5 ), berberine-scutellarin physical mixture ( Figure 6 ), it can be seen from the DSC spectrum of berberine and baicalin salt that there are endothermic peaks at 137.2°C and 186.0°C, respectively, and the physical mixture of berberine and baicalin has endothermic peaks at 79.1°C, 160.6°C and 194.1°C.

[0116] Example 4: X-ray powder diffraction determination of berberine scutellarin salt

[0117] An appropriate amount of each sample prepared in Example 1 was placed in an aluminum sample holder to prepare an analytical sample, and then subjected to X-ray powder diffraction. Measurement conditions: CuKa target as X-ray source; voltage: 40 kV, current: 35 mA, scanning range: 3° < 2θ < 40°. The physical mixture of berberine and scutellarin ( Figure 8 ), berberine scutellarin salt ( Figure 7 ) is shown in the attached X-ray powder diffraction pattern. It can be seen that the physical mixture of berberine and baicalin has multiple sharp diffraction peaks, while the number of diffraction peaks of berberine baicalin salt is significantly reduced.

[0118] The berberine scutellarin salt crystals prepared in Example 1 were further subjected to structural analysis. The crystal analysis parameters are shown below. The crystal structure diagram (ignoring the solvent portion) is shown below. Figure 23 As shown, the unit cell stacking diagram is as follows Figure 24 shown.

[0119] Berberine scutellarin salt crystal data and structure:

[0120]

[0121]

[0122] Example 5: Cumulative dissolution curve determination

[0123] Chromatographic conditions for berberine determination:

[0124] Chromatographic column: Kromasil C18 column (150 mm × 4.6 mm, 5 μm);

[0125] Mobile phase: acetonitrile-0.05 mol / L sodium dihydrogen phosphate (adjusted to pH = 3 with phosphoric acid) (28:72);

[0126] Flow rate: 1.0 ml / min; column temperature: room temperature (25°C); detection wavelength: 345 nm; injection volume: 10 μL.

[0127] Chromatographic conditions for determination of scutellarin

[0128] Chromatographic column: GALAK EF-C18 M column;

[0129] Mobile phase: acetonitrile (A)-0.1% formic acid aqueous solution (B), gradient elution (0-5 min, 20% A; 5-20 min, 20%-28% A; 20-30 min, 28-30% A; 30-35 min, 30%-35% A; 35-50 min, 35%-60% A; 50-51 min, 60%-90% A; 51-60 min, 90% A);

[0130] Flow rate: 0.8 mL / min; column temperature: 30°C; detection wavelength: 335 nm; injection volume: 20 μL.

[0131] Each sample was taken according to the paddle method provisions in the appendix of the 2020 edition of the Chinese Pharmacopoeia, with a speed of 100±1r / min, a water bath temperature of 37°C, a dissolution medium of water, and a dosage equivalent to 15 mg of berberine and 20 mg of scutellarin. 2 ml of samples were taken at 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 18, and 24 hours, respectively, and filtered through a 0.45 um filter membrane. At the same time, 2 ml of dissolution medium at the same temperature was added, and 1 ml of the filtrate was taken. After dilution with the mobile phase, the cumulative dissolution amount was determined by the above HPLC method.

[0132] Figure 9 Cumulative dissolution curves of berberine and scutellarin in each sample.

[0133] Depend on Figure 9 It can be seen that 90% of berberine in the physical mixture was dissolved in 15 minutes, while only 4% of scutellarin was dissolved in 24 hours.

[0134] Berberine scutellarin salt slowed down the dissolution rate of berberine, dissolving 90% in 4 hours. At the same time, the solubility of scutellarin was significantly increased, with the cumulative dissolution increasing to 40% in 4 hours. The dissolution behavior of the two drugs was consistent.

[0135] It can be seen that the dissolution behavior of berberine scutellarin salt and the physical mixture of the two drugs is significantly different, which is conducive to the synergistic effect of the two drugs.

[0136] Example 6: Berberine-scutellarin can effectively treat metabolic-related diseases

[0137] In this example, Syrian golden hamsters were fed a high-sugar, high-fat diet to establish a model of hyperlipidemia, hyperglycemia, obesity, hypercoagulability, and fatty liver disease, and the therapeutic effect of berberine scutellarin on metabolic-related diseases was then investigated.

[0138] 1. Experimental Design

[0139] Syrian golden hamsters (8 weeks old) were randomly divided into 4 groups, with 6 hamsters in each group:

[0140] control group (normal diet);

[0141] model group (high sugar, high fat diet);

[0142] Berberine and baicalin physical mixture (high-fat diet + berberine and baicalin physical mixture);

[0143] Berberine scutellarin group (high-fat diet + berberine scutellarin).

[0144] The animals in each group were given the drug by gavage, and the hamsters in the control group and the model group were given 10 mL / kg / d of distilled water.

[0145] The dosages of the berberine and baicalin physical mixture group and the berberine baicalin salt group were 100 mg / kg / d (berberine) and 135 mg / kg / d (baicalin), respectively, administered once a day for 8 consecutive weeks.

[0146] After the administration, the animals were anesthetized and killed by intraperitoneal injection of 1 ml of 20% chloral hydrate, and the eyeballs were removed for blood collection and liver removal. Blood biochemical indicators were measured using an automatic biochemical analyzer.

[0147] The experimental results show that berberine scutellarin salt has the effects of lowering triglycerides in the liver, lowering blood lipids, lowering blood sugar, reducing weight gain and anti-platelet aggregation, and its effects are better than the physical mixture of berberine and scutellarin.

[0148] Therefore, berberine scutellariae salt can effectively prevent and treat metabolic diseases such as hyperlipidemia, hyperglycemia, obesity, platelet aggregation, and fatty liver disease.

[0149] 2. Effects of berberine and scutellarin on triglyceride content in the liver of experimental animals

[0150] The results of liver triglyceride (liver TG) content determination are shown in Figure 10 and Table 1.

[0151] Table 1

[0152]

[0153] The results showed that triglycerides in the livers of the model animals were significantly higher than those in the blank control group. Berberine-scutellarin salt effectively reduced the increase in liver triglyceride levels caused by a high-sugar, high-fat diet, and the degree of reduction was more significant than that of the berberine-scutellarin physical mixture.

[0154] 3. Effects of berberine and scutellarin on blood glucose and blood lipids in experimental animals

[0155] The results of blood glucose levels are shown in Figure 11 and Table 2

[0156] Table 2

[0157]

[0158] The results of plasma cholesterol (CHO) content are shown in Figure 12 and Table 3.

[0159] Table 3

[0160]

[0161] Plasma low-density lipoprotein cholesterol (LDL-c) levels are shown in Figure 13 and Table 4.

[0162] Table 4

[0163]

[0164] The results of plasma triglyceride (TG) content are shown in Figure 14 and Table 5.

[0165] Table 5

[0166]

[0167] Plasma high-density lipoprotein-cholesterol (HDL-c) levels are shown in 15 and Table 6.

[0168] Table 6

[0169]

[0170] The results showed that after the high-sugar and high-fat diet model was established, the levels of glucose, cholesterol, low-density lipoprotein-cholesterol and triglycerides in the animal plasma increased significantly. The physical mixture of berberine and baicalin had the effect of lowering blood lipids, and berberine and baicalin salt could also effectively reduce the high-sugar and high-fat diet. High-fat diet causes increased blood sugar and blood lipids, and the efficacy is better than berberine- The physical mixture of scutellarin is more significant .

[0171] 4. Changes of berberine and baicalin on liver function in experimental animals

[0172] Figure 16 Table 7 shows the comparison of alanine aminotransferase (ALT) levels in Syrian golden hamsters in each group after the intervention.

[0173] Table 7

[0174]

[0175] As shown in the figure, compared with the blank control group, the ALT level in the model group was significantly increased. Berberine Baicalin salt significantly reduces ALT levels caused by high-sugar and high-fat diets, and its efficacy is better than that of berberine-scutellarin physical mixture. More obvious .

[0176] Figure 17 Table 8 shows the comparison of aspartate aminotransferase (AST) levels in Syrian golden hamsters in each group after the intervention.

[0177] Table 8

[0178]

[0179] As shown in the figure, compared with the blank control group, the AST level in the model group was significantly increased. Berberine Baicalin salt significantly reduces AST levels caused by high-sugar and high-fat diets, and its efficacy is better than that of berberine-scutellarin physical mixture. More obvious .

[0180] Figure 18 The results of oil red staining of liver tissue sections of experimental animals.

[0181] As shown in the figure, compared with the blank control group, the lipid deposition in the liver tissue of the model group increased significantly; the liver oil of the hamsters in the berberine scutellarin salt group increased significantly. The red staining was significantly reduced and the liver lipid deposition was reduced. The results showed that drug intervention could inhibit the liver The deposition of neutral fats such as triglycerides in the liver can improve liver fatty degeneration, and the efficacy of improving liver fatty degeneration should be Significantly stronger than the berberine and scutellarin physical mixture group .

[0182] 5. Effects of berberine and baicalin on the body weight of experimental animals

[0183] The body weight of the animals was measured once a week during the administration period. The results are shown in Table 9. Figure 19 .

[0184] Table 9

[0185]

[0186] Compared with the blank control group, the body weight of the animals in the model group increased significantly. Berberine scutellarin can reduce the body weight caused by high sugar and high fat diet The drug effect was significantly stronger than that of the berberine and baicalin physical mixture group. .

[0187] 6. Effects of berberine and baicalin on platelet aggregation in experimental animals

[0188] Platelet aggregation results are shown in Figure 20 and Table 10

[0189] Table 10

[0190]

[0191] The results showed that the platelet aggregation rate of the high-sugar and high-fat diet model animals was significantly higher than that of the control group, and berberine scutellarin salt could reduce the platelet aggregation rate of the high-sugar and high-fat diet model animals. Platelet aggregation caused by high-fat diet, and the efficacy is significantly stronger than that of berberine and baicalin physical mixture Compound .

[0192] Example 8. Berberine and scutellarin have a significant taste-masking effect on berberine

[0193] 1. Preparation of each solution

[0194] Reference solution: 2.2365g KCl + 0.045g tartaric acid in 1000mL purified water;

[0195] Internal solution: Add 248.2 g KCl to 900 mL distilled water and stir until completely dissolved. Make up to 1000 mL, add 10 mg AgCl, and stir magnetically for at least 8 h.

[0196] Cleaning solution: Negative solution: 500mL water + 300mL ethanol + 8.3mL HCL, dilute to 1000mL

[0197] Objective solution: 7.46g KCl + 500mL water + 300mL ethanol + 0.56g KOH, dilute to 1000mL

[0198] 2. The effect of berberine and baicalin salt on masking the bitterness of berberine

[0199] Berberine, scutellarin, a berberine-scutellarin physical mixture, and berberine-scutellarin salt were prepared into solutions containing berberine at a concentration of 0.01 mg / ml, and the bitterness value was measured using an electronic tongue. The results are shown in Table 11. As can be seen from the results, scutellarin did not respond to the sensor, while the berberine-scutellarin physical mixture had a bitterness response consistent with berberine. Berberine-scutellarin salt reduced the bitterness response of berberine, indicating that berberine-scutellarin salt has a certain taste-masking effect.

[0200] Table 11 Taste quality based on electronic tongue

[0201]

[0202] It can be seen that berberine scutellarin salt has a good taste masking effect on the alkaline bitterness B-bitterness of berberine and the alkaline salt bitterness H-bitterness.

[0203] In summary, berberine scutellarin can effectively prevent and treat hyperglycemia, hyperlipidemia, metabolic fatty liver disease and atherosclerosis. Finally, it should be noted that the above examples are only used to help those skilled in the art understand the essence of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. A berberine scutellarin salt, the structure of which is: Its nuclear magnetic characteristics are: 1H NMR (400MHz, DMSO-d6) δ12.60 (s, 1H), 9.90 (s, 1H), 8.85 (s, 1H), 8.08–7.99 (m, 1H), 7.90 (d, J = 9.4 Hz, 1H), 7.76 (dd, J = 8.8, 2.3 Hz, 2H), 7.71 (d, J = 2.7 Hz, 1H), 7.00 (s, 1H), 6.86 (d, J = 8.5 Hz, 2H), 6.83 (s, 1H ),6.64(d,J=2.6Hz,1H),6.13(s,2H),5.09(s,1H),5.01–4.94(m,1H),4.91(t,J=6.5Hz,2H),4.05(s, 3H),3.94(s,3H),3.74–3.61(m,1H),3.37(s,3H),3.28(dd,J=16.0,7.4Hz,2H),3.18(d,J=6.4Hz,2H); Prepared by the following steps: (1) Dissolve berberine hydrochloride in 10 to 300 times the volume of water; (2) Disperse baicalin in water and an alcohol solvent, add 0.05 to 0.1 equivalents of a base, and dissolve it in an oil bath at 30 to 50°C, wherein the base is selected from potassium hydroxide, sodium hydroxide, potassium carbonate, and sodium carbonate; the alcohol solvent is selected from methanol, ethanol, and isopropanol; and the ratio of water to alcohol is 1:1 to 10:1; (3) At room temperature, slowly add the aqueous solution of berberine hydrochloride to the aqueous solution of the baicalin base solution while it is hot, stir, and an amber colloidal solid precipitates; (4) Stir at room temperature for 1 to 2 hours, let it stand for 1 to 3 hours, filter out the solid, and dry it to obtain a salt composed of berberine and baicalin.

2. The berberine scutellarin salt according to claim 1, characterized in that In the berberine and scutellarin salt, the dissolution behaviors of the two drugs are consistent.

3. The berberine scutellarin salt according to claim 2, characterized in that The dissolution rate of berberine is greater than 65% in 6 hours, and the dissolution rate of scutellarin is greater than 29% in 6 hours.

4. The berberine scutellarin salt according to claim 3, characterized in that The dissolution rate of the berberine scutellarin salt was determined according to the method recorded in the "Chinese Pharmacopoeia 2020".

5. The berberine scutellarin salt according to claim 1, characterized in that (1) The X-ray diffraction pattern of the berberine scutellarin salt crystals does not have the characteristic diffraction peak of berberine; Or the infrared spectral properties of the berberine scutellarin salt described in (2) after being pressed into KBr tablets are: 1) at 3600-3000cm -1 There is no characteristic absorption peak of berberine and scutellarin at 1720.7cm -1 The C=O characteristic peak at 2947, 2897, 1665, 1604, 1570, 1507, 1363, 1276, 1232, 1180, 1065, 1035, 973, 936, 911, 835, 804, 590, 557, 514, 428 cm -1 There are characteristic peaks in the infrared spectrum. Or the berberine scutellarin salt described in (3) is determined by differential scanning calorimetry and has no characteristic melting peaks of berberine and scutellarin.

6. The method for preparing the berberine scutellarin salt according to any one of claims 1 to 5, comprising the following steps: (1) Dissolve berberine hydrochloride in 10 to 300 times the volume of water; (2) dispersing scutellarin in water and an alcohol solvent, adding 0.05 to 0.1 equivalents of a base, and dissolving it in an oil bath at 30 to 50° C., wherein the base is selected from potassium hydroxide, sodium hydroxide, potassium carbonate, and sodium carbonate; the alcohol solvent is selected from methanol, ethanol, and isopropanol; and the ratio of water to alcohol is 1:1 to 10:1; (3) At room temperature, slowly add the aqueous solution of berberine hydrochloride dropwise to the aqueous solution of scutellarin alkali solution while it is still hot, and stir until an amber colloidal solid precipitates; (4) Stir at room temperature for 1 to 2 hours, let stand for 1 to 3 hours, filter out the solid, and dry to obtain a salt composed of berberine and scutellarin.

7. The method according to claim 6, characterized in that In step (1), the berberine hydrochloride is berberine hydrochloride chloride; the ratio of berberine hydrochloride chloride to water is 1:80-90 (w / v); heating at 80°C to dissolve; In step (2), the water and alcohol solvent are ethanol / water = 2:1; the amount ratio of scutellarin to solvent is 1:30-40 (w / v); after dissolving in a 40°C oil bath, the mixture is allowed to stand until it reaches room temperature.

8. A medicament or pharmaceutical composition comprising the berberine scutellarin salt according to any one of claims 1 to 5, comprising a therapeutically effective amount of the berberine scutellarin salt and a pharmaceutically acceptable excipient.

9. The drug or pharmaceutical composition according to claim 8, characterized in that The dosage form of the drug or pharmaceutical composition can be a liquid dosage form, a solid dosage form or a semisolid dosage form.

10. The medicine or pharmaceutical composition according to claim 8, characterized in that The medicine or pharmaceutical composition can be a conventional preparation, a sustained-release preparation, a controlled-release preparation, a targeted preparation or various microparticle preparation systems.

11. Use of the berberine scutellarin salt according to any one of claims 1 to 5, or the medicine or pharmaceutical composition according to any one of claims 8 to 10 in the preparation of a medicine for preventing or treating metabolic diseases and related diseases.

12. The use according to claim 11, characterized in that The metabolic diseases include dyslipidemia, obesity, fatty liver disease, and metabolic-related cardiovascular and cerebrovascular diseases; the dyslipidemia includes high low-density lipoprotein-cholesterol, high total cholesterol, high low-density lipoprotein-cholesterol, high triglycerides or low high-density lipoprotein-cholesterol; the obesity is weight gain and increased body fat rate; the fatty liver disease is increased liver tissue triglycerides (TG), increased liver index, increased liver fat rate, increased serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and increased liver inflammatory factors; the metabolic-related cardiovascular and cerebrovascular diseases include hypercoagulability, atherosclerosis, hypertension, and ischemic stroke.

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