CN110003230B

CN110003230B - Clerodane diterpenoid compound, pharmaceutical composition and application thereof

Info

Publication number: CN110003230B
Application number: CN201910321049.XA
Authority: CN
Inventors: 赵勤实; 王贺瑶; 范敏; 王婷; 吴兴德; 黄俊上; 彭丽艳
Original assignee: Kunming Institute of Botany of CAS; Shanghai Institute of Materia Medica of CAS
Current assignee: Kunming Institute of Botany of CAS; Shanghai Institute of Materia Medica of CAS
Priority date: 2019-04-21
Filing date: 2019-04-21
Publication date: 2021-10-01
Anticipated expiration: 2039-04-21
Also published as: CN110003230A

Abstract

The invention discloses a clerodane diterpenoid compound shown as structural formulas (1) and (2), a pharmaceutical composition taking the clerodane diterpenoid compound as an active ingredient, a preparation method of the pharmaceutical composition, and application of the pharmaceutical composition in preparation of medicines and health-care foods for treating or preventing type II diabetes and hyperlipidemia. The pharmacological activity test proves that: the compounds 1 and 2 of the invention can obviously reduce the blood sugar of type II diabetes C57BL/KsJdb/db mice, improve the sugar tolerance and insulin tolerance of db/db mice and obviously reduce the content of serum triglyceride.

Description

Clerodane diterpenoid compound, pharmaceutical composition and application thereof

The technical field is as follows:

the invention belongs to the technical field of medicines, and particularly relates to a

clerodane diterpenoid compound

1 or 2, a pharmaceutical composition taking the

clerodane diterpenoid compound

1 or 2 as an active ingredient, a preparation method of the pharmaceutical composition, and application of the pharmaceutical composition in preparation of medicines and health-care foods for treating or preventing type II diabetes and hyperlipidemia.

Background art:

the metabolic syndrome is a syndrome of glucose metabolism and lipid metabolism disorder characterized by dyslipidemia, abnormal blood sugar, obesity, fatty liver, hypertension, hypercoagulability, type II diabetes, atherosclerosis, non-alcoholic fatty liver disease, and the like. With economic development and changes in people's lifestyle, the incidence of diabetes and cardiovascular diseases caused by metabolic syndrome is rapidly increasing. Diabetes is a chronic endocrine and metabolic disease, wherein the type II diabetes accounts for 90%, and the number of diabetes patients in China is more than 2000 ten thousand, and the diabetes patients have the characteristics of multiple occurrence and youthfulness, thereby seriously threatening the health of the people. The main symptoms of type II diabetics are hyperglycemia, hyperlipidemia, insulin resistance, etc., which are also the main causes of cataract, infection, fatty liver, cardiovascular disease, diabetic foot, even death, etc. Diabetes has severely affected people's quality of life, causing significant medical costs. Therefore, in addition to control by exercise and diet, the search for drugs for treating diabetes remains a hot spot of current pharmaceutical research.

Active natural products separated and found in plant resources are widely applied to the treatment of various diseases for years. Medicinal plants and natural product monomers have been successfully applied to diabetes control in many countries, and have also become one of the important sources of safe and effective hypoglycemic drugs.

The salvia plant is an important medicinal plant, especially the salvia is widely applied in various places of China all the time, and has the effects of promoting blood circulation to remove blood stasis, activating collaterals to remove obstruction of qi, nourishing the heart to calm the nerves, removing toxicity to cool blood, reducing swelling and relieving pain and the like. Pharmacological experiments prove that the danshenquinone compounds have the effects of diminishing inflammation, expanding the crown, resisting platelet aggregation and the like, and in recent years, new pharmacological activities such as antitumor activity, free radical elimination and the like are discovered successively (Wu, Y.B.; Ni, Z.Y.; Shi, Q.W.; Dong, M.; Kiyota, H.; Gu, Y.C.; Cong, B.; chem.Rev.; 2012,112 (11); 5967). Chia (Salvia hispanica) is an annual herbaceous plant of the genus Chia of the family labiatae, known as Chia (Chia), native to the south of mexico and the north of guatemala. Seeds of chia are also known as chia seeds and have a long history of eating and medicinal use (Joseph, c.p., gene. resource. crop. ev.2004,51(7), 773). Besides being directly eaten, the edible fungus is also used for producing food such as biscuits, bread, yoghourt and the like. And chia seed can be used as nutrition enhancer and food additive. The chia seeds are approved as new food raw materials in 2014 in China. Research shows that chia seeds are rich in various proteins, mineral elements, vitamins, omega-3 series polyunsaturated fatty acids, antioxidants and other components, have the effects of maintaining normal blood lipid level, resisting tumors and oxidation, improving diabetes and cardiovascular diseases and the like, and are widely applied to the aspects of medicines, foods, cosmetics and the like (Poudyal, H., Panchal, S.K., Waanders, J., Ward, L., Brown, L., J.Nutr.Bichem.2012,23(2),153) (Taga, M.S., Miller, E.E., Pratt, D.E., am.Oil chem.Soc.1984,61(5), 928). Although chia seeds have a variety of biological activities and are widely studied, the chemical composition of aerial parts of plants is rarely reported. The

compounds

1 and 2 are clerodane diterpenoid compounds, wherein the compound 1 is a new compound, and the activity effects of the 2 compounds are not reported.

The invention content is as follows:

the invention aims to: provides 2 clerodane diterpenoid compounds, a pharmaceutical composition taking the clerodane diterpenoid compounds as active ingredients, a preparation method thereof, and application of the compounds and the pharmaceutical composition in preparing medicaments and health-care foods for treating or preventing type II diabetes and hyperlipidemia. Pharmacological activity tests prove that the

compounds

1 and 2 can obviously reduce the blood sugar of type II diabetes C57BL/KsJdb/db mice, improve the sugar tolerance and the insulin tolerance of the db/db mice and obviously reduce the content of serum triglyceride.

In order to achieve the above purpose of the present invention, the present invention provides the following technical solutions:

a clerodane-type diterpene compound 1 represented by the following structural formula (1),

the application of the

clerodane diterpenoid compound

1 or 2 shown in the following structural formula in the preparation of the drugs for treating or preventing the II type diabetes and the hyperlipidaemia,

application of the

clerodane diterpenoid compound

1 or 2 in preparing health-care food for treating or preventing type II diabetes and hyperlipidemia.

A pharmaceutical composition comprising a clerodane-

type diterpene compound

1 or 2 and a pharmaceutically acceptable carrier.

The preparation method of the

clerodane diterpenoid compound

1 or 2 comprises the following process steps: drying and pulverizing aerial parts of Salvia splendens (Salvia hispanica), extracting with acetone for three times, mixing extractive solutions, concentrating under reduced pressure to obtain total extract, subjecting the extract to silica gel column chromatography (petroleum ether/acetone, 9:1, 8:2, 7:3, and 6:4), detecting by TLC, and mixing according to main spot to obtain 5 components. The 5 th fraction was separated by reversed phase medium pressure liquid chromatography (MPLC MCI), and combined into 5 subfractions (Fr.5.1-F.5.5) by gradient elution with an ethanol water system (70:30, 75:25, 80:20, 85:15, 90:10 and 95: 5). The component Fr.5.1 is placed for a long time to precipitate a large amount of crystals to obtain the compound 1. The 4 th component is chromatographed by silica gel column (petroleum ether/chloroform/ethyl acetate, 4:4:1) to obtain compound 2.

The application of the

clerodane diterpenoid compounds

1 and 2 in preparing medicaments and health-care foods for treating or preventing type II diabetes and hyperlipoidemia is realized by remarkably reducing fasting blood glucose of a mouse to be administrated, improving the glucose tolerance and the insulin tolerance of a db/db mouse and reducing the content of serum triglyceride.

The pharmaceutically acceptable carrier as mentioned above means a pharmaceutical carrier which is conventional in the pharmaceutical field, for example, water, glucose, lactose, gum arabic and the like and other carriers suitable for use in preparing formulations in the form of solid, semisolid, liquid or aerosol. The composition may additionally contain stabilizers, thickeners, and/or coloring agents and fragrances.

The composition prepared from the clerodane diterpenoid compound and the pharmaceutically acceptable carrier thereof can be orally or non-orally administered, the administration amount is different according to different medicines, and 1-100mg is suitable for adults.

For oral administration, the compound is first mixed with conventional pharmaceutical adjuvants such as excipient, disintegrant, binder, lubricant, antioxidant, coating agent, colorant, aromatic agent, surfactant, etc., and made into granules, capsules, tablets, etc.: for parenteral administration, the administration may be in the form of injection, infusion solution, suppository, or the like. In preparing the above formulation, conventional formulation techniques may be used.

Description of the drawings:

FIG. 1 is a schematic structural view of a clerodane-type diterpene compound of the present invention;

FIG. 2 is a schematic diagram of the single crystal X-ray diffraction structure of Compound 1 of the present invention;

FIG. 3 is a graph showing the change in body weight of mice in each group:

FIG. 4 is a graph showing the results of blood glucose measurements for each group of mice:

FIG. 50 is a schematic view of the GTT curve and the area under the curve:

FIG. 6 schematic diagram of the ITT curve and the area under the curve:

FIG. 7 is a diagram showing the measurement results of serum-related indicators of various groups of mice.

The specific implementation mode is as follows:

the following description will further explain the substance of the present invention by using the embodiments of the present invention with reference to the accompanying drawings, but the present invention is not limited thereto. Modifications of the invention which are in accordance with the spirit of the invention are within the scope of the invention.

Example 1:

the preparation method and the structure identification of the

clerodane diterpenoid compounds

1 and 2 are as follows:

the preparation method comprises the following steps: drying and pulverizing aerial parts of Salvia splendens (Salvia hispanica), extracting with acetone for three times, mixing extractive solutions, concentrating under reduced pressure to obtain total extract, subjecting the extract to silica gel column chromatography (petroleum ether/acetone, 9:1, 8:2, 7:3, and 6:4), detecting by TLC, and mixing according to main spot to obtain 5 components. The 5 th fraction was separated by reversed phase medium pressure liquid chromatography (MPLC MCI), and combined into 5 subfractions (Fr.5.1-F.5.5) by gradient elution with an ethanol water system (70:30, 75:25, 80:20, 85:15, 90:10 and 95: 5). The component Fr.5.1 is placed for a long time to precipitate a large amount of crystals to obtain the compound 1. The 4 th component is chromatographed by silica gel column (petroleum ether/chloroform/ethyl acetate, 4:4:1) to obtain compound 2.

And (3) structural identification: the molecular structural formulas (1) and (2) of the compound of the invention correspond to the compounds 1 and 2:

compound 1 (salvihispin): colorless crystals;

UV(MeOH)λ_max(logε)206(4.36),279(3.50)nm；IR(KBr)ν_max 3432,2927,1754,1309,1045,732and 603cm^-1；¹H NMR and ¹³C NMR data see Table 2.12；ESIMS(positive)m/z 375[M+Na]⁺；HRESIMS(positive)m/z377.0791[M+K]⁺(calcd for C₂₀H₁₈O₅K,377.0786).

compound 2 (salvificaricin): a white powder; c₂₀H₂₀O₅；¹H NMR(600MHz,acetone-d6):δ_H6.01(1H,dd,J＝9.3,2.0,H-1),6.31(1H,m,H-2),6.82(1H,d,J＝5.5,H-3),2.04(1H,m,H-6a),1.33(1H,d,J＝14.0,H-6b),4.27(1H,d,J＝3.7,H-7),1.95(1H,m,H-8),2.80(1H,d,J＝5.6,H-10),2.80(1H,m H-11a),1.97(1H,m,H-11b),5.28(1H,t,J＝7.8,H-12),6.37(1H,brs,H-14),7.49(1H,overlap,H-15),7.49(1H,overlap,H-16),1.33(3H,d,J＝7.0,Me-17),4.04(1H,d,J＝8.0,H-19a),4.93(1H,d,J＝8.0,H-19b),5.31(1H,s,H-20)；¹³CNMR(150MHz,acetone-d6):δ_C134.5(d, C-1),124.2(d, C-2),127.8(d, C-3),129.9(s, C-4),59.0(s, C-5),40.0(t, C-6),84.9(d, C-7),40.1(d, C-8),39.0(s, C-9),49.5(d, C-10),39.0(t, C-11),76.1(d, C-12),127.7(s, C-13),109.6(d, C-14),144.5(d, C-15),139.8(d, C-16),15.1(q, C-17),169.6(s, C-18),81.1(t, C-19),110.6(d, C-20). Prepared by mixing with salvifiicin (Savona, G.; Raffa, D.; Bruno, M.; Rodriguez, B.phytochemistry 1983,22,784.) (Eguren, L.; Fayos, J.; metals, A.; Savona, G.; Rodriguez, B.Phytochemistry 1984,23,466)¹H and¹³c nuclear magnetic data comparison shows that the same compound is identified.

TABLE 1 Hydrogen and carbon spectra data for Compound 1

^a Measured at ^a600MHz,^b150MHz.

Example 2:

activity of

compounds

1 and 2 in diabetes and metabolic syndrome related diseases:

experimental animals and groups:

the C57BL/KsJdb/db mouse (db/db mouse) is a congenital type II diabetes mouse caused by a receptor gene defect of a receptor (Leptin), mainly shows hyperphagia, obesity and glycolipid metabolic abnormality characteristics such as hyperglycemia, hyperinsulinemia, insulin resistance, hyperlipidemia and the like due to the lack of response to a satiety substance (Leptin), and is very similar to clinical symptoms of human type II diabetes.

The tested C57BL/KsJdb/db mice were male mice of 6 weeks of age, SPF-rated, and 20-24g in weight, and were bred and purchased from Shanghai pharmaceutical research institute of Chinese academy of sciences. The feed is fed according to the standard feeding operation rules of SPF animals, except necessary fasting time, water is freely drunk, and the feed is conventional feed.

Experimental materials:

the

compound

1 and 2 beige crystals are prepared by grinding a mixed solution of PEG400 and water as a solvent (VPEG400: V water: 3:7) into fine powder by using a mortar, adding PEG400 in portions, grinding uniformly, adding proper amount of water in portions, and continuously grinding uniformly. The experimental principles are in accordance with standard operating procedures.

The experimental method comprises the following steps:

1) animal grouping: group 1 was a model control group, group 2 was a metformin-administered group, group 3 was a compound 1-administered group, group 4 was a compound 2-administered group, group 5 was a littermate normal mouse, and the groups of experimental animals and the administered doses were as shown in table 2.

TABLE 2 groups of experimental animals

Group of	Laboratory animal	Number of animals (n)	Test drug	Test dose (mg/kg)
					1	db/db	10	5 per mill CMC-Na (solvent)	0
2	db/db	10	Metformin	200
					3	db/db	10	1	100
4	db/db	10	2	100
					5	C57	10	5 per mill CMC-Na (solvent)	0

2) Administration and detection

And (3) detecting fasting blood glucose: carrying out routine monitoring on db/db mice, and carrying out blood sugar and weight detection again 1 day (P-1) before the administration of the medicine after adaptive feeding for one week; dissolving and diluting the test substance according to the concentration in the table, and performing intragastric administration (PO), wherein the administration day is marked as P0; 14 parts per day: dosing between 00p.m. -16:00p.m. for 34 days, during which body weight is tracked; measuring food consumption every 3 days after administration, measuring fasting blood sugar for 6h every 7 days, and regularly recording the food consumption;

glucose tolerance (OGTT) test: 1 day before the experiment, after fasting for 12 hours to detect the blood sugar of each group of mice, feeding glucose at 0.25g/kg by intragastric administration, measuring the blood sugar of each group at five time points of 15min,30min,60min,90min and 120min respectively by tail veins, drawing an OGTT curve and calculating the area under the curve;

insulin resistance (ITT) test: on the experimental day, mice in each group are fasted, 0.8IU/kg of insulin is injected into the abdominal cavity, blood sugar in each group is measured by tail veins at four time points of 30min,60min,90min and 120min respectively, an ITT curve is drawn, and the area under the curve is calculated;

detecting related indexes such as serum triglyceride and cholesterol level: after the experiment is finished, serum is taken to measure related indexes.

Animal performance, body weight, and food intake during the experiment were routinely recorded.

3) Statistical method

All Statistics were performed using PASW Statistics 18(SPSS 18.0). The blood glucose value or body weight is used as ordinate, and the administration groups are respectively used as abscissa to make statistical chart. And taking time as an abscissa and blood glucose concentration as an ordinate to coordinate OGTT and ITT curves. The serum index is used as the ordinate, and the tested medicine is used as the abscissa to make a statistical chart. Statistical data are presented using mean ± standard deviation, with One-Way analysis of variance (One Way ANOVA), p <0.05 considered significant differences.

The experimental results are as follows:

1) effect of

Compounds

1 and 2 of the invention on body weight in db/db diabetic mice:

db/db mice were weighed 1 day prior to dosing, every 3 days, and the weights of the groups were compared. The body weight change curve of the mice is shown in fig. 3.

The experimental result shows that the weight of the mice in the administration group is not obviously reduced compared with the control group.

2) Effect of

Compounds

1 and 2 of the invention on fasting plasma glucose in db/db diabetic mice:

detecting fasting blood glucose of db/db mice 1 day before administration, detecting fasting blood glucose of 6h every 7 days after continuous administration of

compounds

1 and 2, and regularly recording food consumption; the fasting blood glucose measurement results of the mice are shown in fig. 4.

The experimental result shows that the

compounds

1 and 2, like the positive control drug metformin, can obviously reduce the fasting blood glucose content of db/db mice after administration, act faster than metformin, and improve padding obviously;

3) effect of

Compounds

1 and 2 of the invention on oral glucose tolerance (OGTT) in db/db diabetic mice:

after fasting for 12h to detect blood sugar of each group of mice, glucose is given at 0.25g/kg by gastric lavage, blood sugar of each group is measured at five time points of 15min,30min,60min,90min and 120min respectively by tail vein, an OGTT curve is drawn, and Area under the curve (AUC) is calculated. The oral glucose tolerance curves and area under the curves statistics for the mice are shown in figure 5.

4) Effect of

Compounds

1 and 2 of the invention on insulin tolerance (ITT) in db/db diabetic mice:

after fasting for 6h to detect blood sugar of each group of mice, 0.8IU/kg of insulin is injected into the abdominal cavity, and the blood sugar of each group is measured by tail vein at four time points of 30min,60min,90min and 120 min. The ITT curve and area under the curve statistics for each group of mice are shown in figure 6.

The experimental results show that compounds 1 and 2 significantly improved insulin tolerance (. about.. about.p <0.01vs Control,. about.p <0.001vs Control) in db/db mice, increasing insulin sensitivity.

5) The influence of the

compounds

1 and 2 on various indexes of the serum of db/db diabetic mice is as follows:

on day 34 of administration, serum of each group of mice was subjected to correlation measurement of triglyceride level (TG), Glucose content (Glucose), glutamic-oxalacetic transaminase Activity (AST), and glutamic-pyruvic transaminase Activity (ALT). The measurement results of the serum-related indices of the mice in each group are shown in FIG. 7.

The experimental results showed that mice in

compound

1 and 2 administration group had a significant decrease in serum glucose levels (× p <0.001) and decreased serum triglyceride levels (× p <0.001) compared to the control group; has certain increasing trend on the activity of glutamic-oxaloacetic transaminase and the activity of glutamic-pyruvic transaminase, but has no obvious statistical difference with a control group, which indicates that the liver toxicity is not generated.

Example 3

Preparation of tablets:

the

compounds

1 and 2 are prepared according to the method of example 1, and salts prepared by organic acids (tartaric acid, citraconic acid, formic acid, oxalic acid, etc.) or inorganic acids (hydrochloric acid, sulfuric acid, phosphoric acid, etc.) are added with excipients according to the weight ratio of the salts to the excipients of 1:5-1:10, and then the mixture is granulated and tabletted.

Example 4

Preparation of oral liquid preparation:

the

compounds

1 and 2 were prepared according to the method of example 1, and salts prepared with organic acids (tartaric acid, citraconic acid, formic acid, oxalic acid, etc.) or inorganic acids (hydrochloric acid, sulfuric acid, phosphoric acid, etc.) were used to prepare oral liquids according to conventional oral liquid preparation methods.

Example 5

Preparation of capsules, granules or medicinal granules:

the

compounds

1 and 2 are prepared according to the method of example 1, and salts prepared by organic acids (tartaric acid, citraconic acid, formic acid, oxalic acid, etc.) or inorganic acids (hydrochloric acid, sulfuric acid, phosphoric acid, etc.) are added with excipients according to the weight ratio of 5:1 to the excipients to prepare capsules, granules or granules.

Claims

1. A clerodane-type diterpene compound 1 represented by the following structural formula (1),

2. the application of the clerodane diterpenoid compound 1 or 2 shown in the following structural formula in the preparation of the drugs for treating or preventing the II type diabetes and the hyperlipidaemia,

3. use of the clerodane-type diterpene compound 1 or 2 represented by the structural formula in claim 2 for the preparation of health foods for treating or preventing type II diabetes and hyperlipidemia.

4. A process for producing a clerodane-type diterpene compound 1 or 2 represented by the structural formula in claim 2, characterized by comprising the steps of: drying and crushing the overground part of the chia, extracting with acetone for three times, combining the three extracting solutions, concentrating under reduced pressure to obtain a total extract, carrying out silica gel column chromatography on the extract, carrying out TLC (thin layer chromatography) identification on the extract with petroleum ether/acetone ratios of 9:1, 8:2, 7:3 and 6:4, combining the extracts according to main spots to obtain 5 components, separating the 5 th component by using an inverse medium-pressure liquid chromatography (MPLC MCI), eluting with ethanol water system gradients of 70:30, 75:25, 80:20, 85:15, 90:10 and 95:5, and combining the components into 5 subcomponents Fr.5.1-F.5.5 through detection; a large amount of crystals are separated out from the component Fr.5.1 by standing to obtain a compound 1, and the component 4 is subjected to silica gel column chromatography, and the ratio of petroleum ether to chloroform to ethyl acetate is 4:4:1 to obtain a compound 2.

5. A pharmaceutical composition comprising the clerodane-type diterpene compound 1 represented by the structural formula in claim 1 and a pharmaceutically acceptable carrier.