A kaempferol conjugated derivative and its use in the preparation of a medicament for treating diarrhea

Abstract

Hyperthyroid diarrhea refers to hyperthyroid diarrhea, which is mostly intractable diarrhea, due to excessive thyroid hormone causing increased intestinal peristalsis excitability and diarrhea when the thyroid function is increased, which is caused by endocrine metabolic disorder leading to gastrointestinal function disorder. The present application studies and observes the treatment effect of kaempferol coupling derivative JC-1 on diarrhea of hyperthyroid model mice, and provides a new drug treatment option for diarrhea type colon dysfunction.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of small organic molecule drugs, in particular to a kaempferol conjugated derivative, its extraction method and its use for treating diarrhea. BACKGROUND

[0002] In order to seek new drugs for clinically intractable diseases, many Chinese medicine ingredients have begun to enter the research field (“Luteolin as an anti-inflammatory and neuroprotective agent”, Brain Res Bull., Vol. 119, pp. 1-11, 201510; “Luteolin suppresses colonic smooth muscle motility via inhibiting L-type calcium channel currents in mice”, Meng Yang et al., Gen Physiol Biophys, Vol. 39, pp. 49-58, 202001; “Inhibitory effect and mechanism of resveratrol on colonic motility in mice”, Yang Meng et al., Journal of Shanghai Jiaotong University (Medical Science), Vol. 40, pp. 165-172, 202002).

[0003] Flavonoids are polyphenolic compounds that are ubiquitous in nature. In recent years, studies have shown that these compounds have a variety of biological activities and can effectively improve the pathological state of diseases. Kaempferol, also known as kaempferol, kaempferol flavonol, etc., has a molecular structure formula of C15H10O6 and a relative molecular mass of 286.236. Kaempferol and its derivatives are flavonoids widely used in the medical field and are widely found in natural plants. They are one of the main active ingredients of many Chinese herbal medicines (“Flavonoids as the main active ingredient of single herb and compound Chinese medicine and their pharmacological effects”, Guo Meng et al., Journal of Shenyang Medical College, Vol. 20, pp. 558-561, 564, 2018). They have a wide range of pharmacological effects, including anti-tumor, antioxidant, anti-inflammatory, anti-anxiety, analgesic, and anti-allergic effects.

[0004] Colonic transit dysfunction is a common clinical symptom. Systemic diseases accompanied by colonic transit dysfunction, such as diabetic slow transit constipation, hyperthyroidism accompanied by diarrhea, etc. ("Clinical characteristics analysis and treatment of 60 cases of hyperthyroidism diarrhea", Hui Huanqing, Clinical Medicine, Vol. 6, pp. 73-74, 2013), have complex pathogenesis and poor treatment effect. Hyperthyroid diarrhea refers to hyperthyroid diarrhea, which is often intractable diarrhea. It is due to excessive thyroid hormone causing increased intestinal motility excitability and diarrhea when the thyroid function is hyperthyroid. It is caused by endocrine metabolic disorders leading to gastrointestinal dysfunction. Patients with hyperthyroid diarrhea all show paste-like stool, unsmooth defecation, anal burning sensation, and defecation 5-15 times a day. Patients are extremely emaciated, some showing cachexia, and umbilical abdominal burning pain. Recent studies have shown that the pathogenesis is related to the ENS / SIP syncytial axis ("Colonic Transit Disorder Mediated by Downregulation of Interstitial Cells of Cajal / Anoctamin-1 in Dextran Sodium Sulfate-induced Colitis", Lu C et al., Neurogastroenterology & Motility, Vol. 30, pp. 316-331, 201904; "Diabetes-induced colonic slow transit mediated by the up-regulation of PDGFRα+cells / SK3 in streptozotocin-induced diabetic mice. eurogastroenterol", Song NN et al., Neurogastroenterology & Motility, Vol. 30, pp. e e13326, 201803), so it is urgent to develop new drugs based on new theories.

[0005] Spiraea belongs to Rosaceae and contains more than 90 shrubs growing in temperate regions of the Northern Hemisphere and East Asia. Many Spiraea species are used as effective drugs for treating inflammation, malaria and diarrhea. One of the Spiraea species is Spiraea brahuica, which is distributed in Asia and grows in large quantities in the Ziarat Valley of Balochistan, Pakistan. It has been reported that Spiraea brahuica contains a large amount of flavonoids, and the extraction of corresponding flavonoids from Spiraea brahuica as a drug for treating diarrhea has research prospects. SUMMARY

[0006] In order to solve the problem of poor treatment effect of hyperthyroid diarrhea, the present application takes the chrysanthemum as the extraction raw material, and obtains a novel kaempferol coupling derivative JC-1 through extraction, and the structural formula is:

[0007]

[0008] And on the basis of the theory of ENS / SIP syncytial axis, the effect and mechanism of JC-1 on colon transmission are studied, and the treatment effect of JC-1 on diarrhea of hyperthyroid model mice is studied, so as to provide a theoretical basis for clinical drug research and development and provide a new idea for gastrointestinal drugs.

[0009] The technical scheme of the present application is as follows:

[0010] A kaempferol coupling derivative JC-1 and its pharmaceutically acceptable salt, characterized in that the structural formula of the compound JC-1 is:

[0011]

[0012] In the second aspect, the present application also relates to an extraction method of the kaempferol coupling derivative JC-1, characterized in that the extraction method comprises the following steps:

[0013] S1. Dry chrysanthemum is placed in an organic solvent for multiple extractions, and the multiple extraction liquids are combined and concentrated at room temperature to obtain a residue;

[0014] S2. The residue is dissolved in water and extracted with multiple organic solvents in sequence; and the obtained extraction liquid is separated by VLC chromatography to obtain components A-J;

[0015] S3. Component E is separated by CC chromatography to obtain components E.1-E.5;

[0016] S4. Component E.4 is separated again by CC chromatography to obtain a binary mixture, and then purified by TLC to obtain compound JC-1.

[0017] In step S1, the organic solvent is ethanol, the use amount of chrysanthemum and organic solvent is 1:1-1.4g / mL, the number of multiple extractions is 3-5 times, and the extraction temperature is 25-40 DEG C.

[0018] In step S2, the use amount of the residue and water is 1:2-10g / mL, the organic solvents used for extraction are hexane and chloroform in sequence, and the use amount of the residue and the organic solvent for extraction is 10:1-10g / mL.

[0019] Further, in step S2, the extractant for the VLC chromatographic separation is chloroform extractant, after loading, first elution is performed with hexane, hexane / chloroform (1:1, V:V), chloroform in sequence, the elution volume is three times of the loading volume, then gradient elution is performed with chloroform and methanol, the elution volume is five times of the loading volume.

[0020] In step S3, component E is first eluted with chloroform, the elution volume is three times of the loading volume, then gradient elution is performed with chloroform and methanol, the elution volume is five times of the loading volume.

[0021] In step S4, component E.4 is first gradient eluted with chloroform and methanol, the elution volume is five times of the loading volume, to obtain a binary mixture, then gradient elution is performed with chloroform and methanol to obtain compound JC-1.

[0022] In a third aspect, the present application also relates to a pharmaceutical composition comprising a therapeutically effective amount of JC-1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.

[0023] The JC-1 compound or a pharmaceutically acceptable salt thereof of the present application can be mixed with a pharmaceutically acceptable carrier, diluent or excipient to prepare a pharmaceutical preparation suitable for oral or parenteral administration. The administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal and oral routes. The preparation can be administered by any route, for example, by infusion or bolus, by the route of absorption through the epithelial or skin mucosa (for example, oral mucosa or rectum, etc.). The administration can be systemic or local. Examples of oral administration preparations include solid or liquid dosage forms, in particular, tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions, etc. The preparation can be prepared by methods known in the art, and contains carriers, diluents or excipients commonly used in the field of pharmaceutical preparations.

[0024] In a fourth aspect, the present application also relates to the use of a JC-1 compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the JC-1 compound or the salt thereof, in the preparation of a medicament for treating diarrhea.

[0025] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features specifically described below (for example, in the examples) can be combined with each other to form a new or preferred technical scheme. Due to the limited space, they will not be repeated here.

[0026] The present application has the following beneficial effects relative to the prior art:

[0027] The application adopts muscle strip tension experiment, observes the effect of kaempferol coupling derivative JC-1 on spontaneous contraction of normal mouse colon smooth muscle; preliminarily explores the mechanism of the effect of kaempferol coupling derivative JC-1 on colon transmission; explores the ion channel mechanism of the effect of kaempferol coupling derivative, adopts single cell membrane patch clamp technology, observes the influence of kaempferol coupling derivative on L-type calcium channel, ANO1 and SK3 channel current of mouse colon smooth muscle on fresh isolated cells, and finally provides a new theoretical basis for development and application of kaempferol coupling derivative JC-1.

[0028] The application researches and observes the treatment effect of kaempferol coupling derivative JC-1 on diarrhea of hyperthyroidism model mice, and provides a new drug treatment option for diarrhea type colon dysfunction. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The curve graph of kaempferol coupling derivative JC-1 colon smooth muscle contraction wave

[0030] Figure 2 The curve graph of BayK8644 blocking kaempferol coupling derivative JC-1 inhibition of contraction wave

[0031] Figure 3 The Iba inhibition curve graph of kaempferol coupling derivative JC-1 DETAILED DESCRIPTION

[0032] Example 1. Extraction and separation of kaempferol coupling derivative JC-1

[0033] The meadowsweet used for extraction was provided by the Wuhan Botanical Garden, Chinese Academy of Sciences. Air-dried meadowsweet (5 kg) was extracted with ethanol (3 × 12 L, 10 days each time) at room temperature. After extraction, the combined extracts were concentrated at room temperature. The residue obtained after concentration was dissolved in 800 mL of water, first extracted with 50 mL of hexane, and then extracted with 70 mL of chloroform. The chloroform extract was separated by VLC, eluted sequentially with three times the loading volume of hexane, hexane / chloroform (1:1, V:V), and chloroform, followed by a gradient elution with five times the loading volume of chloroform and methanol to obtain fraction AJ. The eluent fraction with a chloroform:methanol ratio of 90:10 to 95:5 (fraction E) was separated by CC, first eluted with three times the loading volume of chloroform, and then eluted with five times the loading volume of chloroform and methanol in a gradient elution to obtain fractions E.1-E.5. The eluent fraction of chloroform:methanol = 90:10 to 95:5 (component E.4) was separated again by CC chromatography. Gradient elution was performed using five times the sample volume of chloroform and methanol. The eluent fraction of chloroform:methanol = 90:10 was obtained to obtain a binary mixture. The obtained binary mixture was separated by TLC (developing solvent was chloroform:methanol = 85:15) to obtain compound JC-1.

[0034] Example 2. Effects and mechanisms of kaempferol-coupled derivative JC-1 on colonic transit in normal mice.

[0035] (1) Determining the effect of kaempferol conjugate JC-1 on colonic transit in mice

[0036] The spontaneous contraction of colonic smooth muscle in normal mice before and after administration of kaempferol-coupled derivative JC-1 was recorded using a muscle strip tension experiment. Figure 1 As shown, the concentration-dependent inhibition of the kaempferol coupling derivative JC-1 significantly suppressed the contraction wave (P<0.05). 10M, 20M and 30 μM of the kaempferol coupling derivative JC-1 inhibited the colonic contraction wave by 7-19%, 20-61% and 60-87%, respectively.

[0037] (2) Exploring the mechanism of action of kaempferol-coupled derivative JC-1 on colonic transit

[0038] First, we used TTX to block enteric nerve activity, L-NAME to inhibit NO production, Ani9 to block the ANO1 channel of the ICC, TEA to block non-selective K+ channels, glibenclamide to block ATP-dependent potassium channels, and Apamine to block SK3. We found that the inhibitory effect of the kaempferol-coupled derivative JC-1 was unaffected. However, when BayK8644 (an L-type Ca2+ channel agonist) was administered, such as... Figure 2As shown, it can be found that BayK8644 significantly blocked the inhibitory effect of kaempferol conjugate derivative JC-1 (P<0.05). Before treatment with BayK8644, kaempferol conjugate derivative JC-1 inhibited 20-61% of the contraction wave; after treatment with BayK8644, kaempferol conjugate derivative JC-1 inhibited 8-15% of the contraction wave. These results show that kaempferol conjugate derivative JC-1 can inhibit calcium channels of smooth muscle cells.

[0039] (3) Determine the ion channel mechanism of kaempferol conjugate derivative JC-1 on the effect of colon transmission.

[0040] Using single cell patch clamp technique, the effect of kaempferol conjugate derivative JC-1 on L-type calcium channel current of mouse colon smooth muscle was observed. In freshly isolated colon smooth muscle cells, the membrane potential was clamped at -80 mV in the conventional whole cell mode, and a step stimulus with an increase amplitude of 10 mV every 10 s was used to depolarize it from -40 mV to 70 mV in turn, with a time duration of 440 ms, to record barium current (IBa). As shown in the I-V relationship curve, from -20 mV to 50 mV, 20 mol / L kaempferol conjugate derivative JC-1 significantly reduced IBa (P<0.05), and IBa was inhibited by (25-61)% at 0 mV. Figure 3

[0041] Example 3. Therapeutic effect of kaempferol conjugate derivative JC-1 on diarrhea of hyperthyroid model mice

[0042] To determine the therapeutic effect of kaempferol conjugate derivative JC-1 on diarrhea of hyperthyroid model mice, 60 mice were randomly divided into 2 groups: 20 normal control mice and 40 model mice. The normal group was injected subcutaneously with normal saline, and the model group was injected subcutaneously with 350 μg / kg of levothyroxine sodium. Specifically, levothyroxine sodium was prepared into a solution with a concentration of 35 μg / mL using normal saline, and was injected subcutaneously at a dose of 0.1 mL / 10 mg of body weight for a total of 9 days. During this period, the spontaneous activity and stool condition of the mice were observed.

[0043] After 9 days, according to the amount of loose stool, the hyperthyroid mice with diarrhea were selected and randomly divided into an untreated group and a treated group. The normal group and the untreated group continued the previous injection, and the treated group was injected with JC-1 at the same time as the injection of levothyroxine sodium. JC-1 was prepared into a mother liquor with a concentration of 50 mol / L using ethanol, and was diluted with ethanol to 0.2 ml at a dose of 3 μL / 10 g of body weight, then was injected subcutaneously for a total of 6 days. During this period, the changes in stool were observed, and the stool paper was changed every hour for a total of 24 hours.

[0044] ​The diarrhea degree was expressed by the weight of stool, and the eyeball was enucleated to take blood on the 15th day, and the serum T3 and T4 were detected. As shown in Table 1, the kaempferol coupling derivative JC-1 significantly reduced the weight of loose stool of the hyperthyroid model mice (P<0.05).

[0045] Table 1. The detection results of serum T3, T4 and the weight of loose stool of mice

[0046]

[0047] Compared with the normal control group, *P<0.05, compared with the model group, #P<0.05.

[0048] The above results show that the kaempferol coupling derivative JC-1 can significantly inhibit the colon transmission of mice by inhibiting the calcium channel, and can significantly improve the diarrhea symptoms of the hyperthyroid model mice. The present application provides a theoretical basis for clinical drug research and development and gastrointestinal drugs.

Claims

1. Use of a kaempferol coupling derivative JC-1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating diarrhea, wherein the structural formula of the kaempferol coupling derivative JC-1 is:

2. The use as described in claim 1, characterized in that: The diarrhea described is hyperthyroid diarrhea.

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