CN103012356B - Compound with alpha-glycosidase inhibitory activity, as well as preparation method and application of compound - Google Patents

Abstract

The invention discloses a kind of compound and its preparation method and application with alpha-glucoside inhibiting activity,The structure of the compound is shown in formula I,It is to extract 50~95% ethanol percolations of Swertia mussotii herb,Combined extract,Revolving removes ethyl alcohol,Medicinal extract is obtained after re-dry; Medicinal extract is soluble in water,It is extracted with dichloromethane; Methylene chloride is extracted product and is separated using silica gel column chromatography,With petroleum ether-ethyl acetate gradient elution,The compound polarity size shown according to thin-layer chromatography merges eluent according to the ascending sequence of polarity and obtains 8 thick fractions,It is respectively labeled as F1~F8; Compound ZYC-01 and ZYC-02 is prepared by the separation of silica gel column chromatography combination preparative high performance liquid chromatography in fraction F3; ZYC-04 is prepared by gel filtration chromatography in fraction F4; ZYC-03 and ZYC-05 is prepared by gel filtration chromatography in fraction F5. Traditionally Swertia mussotii is to research and develop Antihepatitis medicament as Main way. The compound of the present invention is to report for the first time to the inhibitory activity of alpha-glucosidase and the application in preparation treatment diabetes medicament.

Description

A compound with α-glucosidase inhibitory activity and its preparation method and application

technical field

The invention belongs to the field of medicaments, and in particular relates to a compound extracted from capillary chinensis and having the activity of inhibiting α-glucosidase enzymolysis, a preparation method thereof, and an application in preparation of medicines for treating diabetes.

Background technique

Tibetan Yinchen is a precious medicinal material in Tibetan medicine for the prevention and treatment of hepatitis, liver injury, liver fibrosis, fatty liver and other diseases. Chemical studies have shown that the main medicinal materials of Tibetan Capillary (Swertia in Western Sichuan, Indian Swertia, Swertia clam and Huamao) contain iridoid glycosides, flavonoids and their glycosides, xanone and its glycosides , triterpenoids and other components.

Chinese patent (Application No. 200710163097.8) discloses the extracts of Capum chinensis and its preparation methods, pharmaceutical compositions and uses. The extracts contain swertiamarin, gentiopicroside, swertiaroside, mangiferin, and isoorientin Five ingredients, mainly used to improve liver damage and promote the reversal of liver fibrosis process.

Chinese patent (Application No. 92108809.4) discloses an extraction process, equipment and production method of Tibetan capillary active ingredients.

Chinese patent CN101845037B discloses a method for isolating the ketone components of Capillary chinensis, which includes three process steps of extracting the aqueous solution of the extract of Caprena cypress, extracting the crude fraction according to the polarity gradient, and separating and purifying by column chromatography.

It has been reported in the literature that people in Sikkim, India and Darjeeling in the Himalayas traditionally used Swertia chirayita to treat diabetes (Chhetri, D.R., Parajuli, P., Subba, G.C. Antidiabetic plants used by Sikkim and Darjeeling Himalayan tribes, India. J. Ethnopharmacol., 2005, 99, 199-202). Studies have shown that the xanone compounds Swerchirin, methylswertianin and bellidifolin are its medicinal ingredients, which have a good hypoglycemic effect (Tian, L.-Y., Bai, X., Chen, X.-Y., et al. al. Anti-diabetic effect of methylswertianin and bellidifolin from Swertia punicea Hemsl. and its potential mechanism. Phytomedicine, 2010, 17, 533-539). On the other hand, mangiferin is a kind of shanone glycoside, which is also isolated from Capum chinensis. Miura et al. reported its hypoglycemic function (Miura, T., Ichiki, H., Hashimoto, I., et al. Antidiabetic activity of a xanthone compound, mangiferin. Phytomedicine, 2001, 8, 85-87).

Contents of the invention

The primary object of the present invention is to provide a compound having α-glucosidase inhibitory activity.

Another object of the present invention is to provide a method for preparing the above-mentioned compound having α-glucosidase inhibitory activity, and the above-mentioned compound is extracted from the whole herb of Capillary chinensis.

Another object of the present invention is to provide the application of the above-mentioned compound with α-glucosidase inhibitory activity in the preparation of drugs for treating disorders of glucose metabolism, especially the application in the preparation of drugs for treating type II diabetes.

The object of the present invention is achieved through the following technical solutions:

A compound with α-glucosidase inhibitory activity, its structure is shown in formula I:

(Formula I)

In formula I, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ are H, OH or OCH ₃ ;

Preferably, the above-mentioned compounds with α-glucosidase inhibitory activity are 1,7,8-trihydroxy-3,4-dimethoxyxanone (code ZYC-01), 1,3,5,8-tetra Hydroxyxanone (ZYC-02), 1,3,7,8-tetrahydroxyxanone (ZYC-03), 1,7-dihydroxy-3-methoxyxanone (ZYC-04) and 1,3- Dihydroxy-7,8-dimethoxyxanone (ZYC-05);

The preparation method of the above-mentioned compound with α-glucosidase inhibitory activity comprises the following steps:

Grind the whole herb of Capillary chinensis into powder, extract it several times by percolating with 50-95% ethanol, combine the extracts, filter and remove the residue, remove the ethanol by rotary evaporation, and then dry to obtain the extract; dissolve the extract in water, wash with dichloro Methane extraction; dichloromethane extraction products were separated by silica gel column chromatography, eluted with petroleum ether-ethyl acetate gradient, the initial volume ratio of petroleum ether to ethyl acetate was 30:1, and finally transitioned to 0:1; according to TLC According to the polarity of the compounds shown by chromatography (TLC), the eluents were combined in order of polarity from small to large to obtain 8 crude fractions, which were marked as F1-F8; Compounds ZYC-01 and ZYC-02 were prepared by chromatographic separation; fraction F4 was prepared by gel column chromatography to obtain ZYC-04; fraction F5 was prepared by gel column chromatography to obtain ZYC-03 and ZYC-05;

The said Tibetan yinchen refers to Swertia mussotii Franch in western Sichuan, Swertia mussotii Franch in western Sichuan, Swertia mussotii Franch in India, Swertia mussotii Franch.

The present invention uses the following experiments to prove that the above-mentioned compounds have excellent inhibitory activity on α-glucosidase: 4-nitrophenol-α-D-glucopyranoside (PNPG) is used as a substrate to determine xanone compounds (that is, the present invention The inhibitory rate of the compound) on the enzymatic hydrolysis activity of α-glucosidase, and the clinical drug acarbose (Acarbose) was used as a positive control. The results showed that 1,7,8-trihydroxy-3,4-dimethoxyxanone (ZYC-01) and 1,3,5,8-tetrahydroxyxanone (ZYC-02) had no effect on human α-glucoside The enzyme inhibitory activity was the best, with IC ₅₀ values of 1.43mmol/L and 1.64mmol/L; close to the level of acarbose (IC ₅₀ =0.99mmol/L).

In vivo experiments in experimental diabetic NOD mice showed that 1,7,8-trihydroxy-3,4-dimethoxyxanone (ZYC-01) and 1,3,5,8-tetrahydroxyxanone (ZYC- 02) It can significantly reduce the concentration of blood sugar, TC and TG, and increase the level of serum insulin.

Therefore, the above-mentioned compounds with α-glucosidase inhibitory activity can be used for the preparation of drugs for treating disorders of glucose metabolism, especially for the preparation of drugs for treating type II diabetes.

Compared with the prior art, the present invention has the following advantages and effects:

Traditionally, the main direction of Tibetan Yinchen is to develop anti-hepatitis drugs. The inhibitory activity of the compound of the present invention on alpha-glucosidase and its application in the preparation of medicines for treating diabetes are reported for the first time.

Description of drawings

Figure 1 is the mass spectrum of 3,4-dimethoxy-1,7,8-trihydroxyxanone (ZYC-01).

Figure 2 is the mass spectrum of 1,3,5,8-tetrahydroxyxanone (ZYC-02).

Figure 3 is the mass spectrum of 1,3,7,8-tetrahydroxyxanone (ZYC-03).

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1

The extraction, separation and purification of compounds with alpha-glucosidase inhibitory activity comprises the following steps:

2.5kg of the whole herb of Swertia chinensis in western Sichuan was ground into powder, extracted by percolation with 75% ethanol three times, each time for 2h, 2h and 1h, filtered, the extracts were combined, removed by rotary evaporation, and then placed in a vacuum box at 60°C After drying, 509.7g of dry extract was obtained. Grind the extract, dissolve in water, and extract with equal volumes of dichloromethane several times to obtain 92 g of dichloromethane extract. The dichloromethane extract was separated by silica gel column chromatography, eluted with petroleum ether-ethyl acetate gradient, the initial volume ratio of petroleum ether and ethyl acetate was 30:1, and finally transitioned to 0:1; according to TLC According to the polarity of the compounds shown by the analysis (TLC), the eluents were combined in order of polarity from small to large to obtain 8 crude fractions, which were marked as F1-F8. Fraction F3 was separated by silica gel column chromatography combined with preparative high performance liquid chromatography to obtain compounds ZYC-01 and ZYC-02. Fraction F4 was prepared by gel column chromatography to obtain ZYC-04. Fraction F5 was prepared by gel column chromatography to obtain ZYC-03 and ZYC-05.

1,7,8-Trihydroxy-3,4-dimethoxyxanone (ZYC-01): Pale yellow solid powder. _{1H NMR} ^{_ _ _} (300MHz, DMSO-d ₆ )δ: 7.27(1H, d, J=9Hz, H-6), 6.90(1H, d, J=9Hz, H-5), 6.55(1H, s, H-2) , 3.93 (3H, s, -OCH ₃ ), 3.77 (3H, s, -OCH ₃ ); ¹³ C NMR (300MHz, DMSO-d ₆ ) δ: 157.67 (C-1), 95.03 (C-2), 160.25(C-3), 128.24(C-4), 148.97(C-4a), 148.01(C-4b), 106.04(C-5), 124.02(C-6), 140.65(C-7), 147.34 (C-8), 101.21 (C-8a), 107.16 (C-8b), 184.29 (C-9), 60.91, 56.62 (-OCH ₃ ); see Figure 1 for its mass spectrum. Controlled references (Silveira, ER, , MJC, Menezes, et al.Pentaoxygenated xanthones from BredemeyeraFloribunda.Phytochemistry, 1995, 39(6), 1433-1436), proved that the product was 1,7,8-trihydroxy-3,4-dimethoxyxanthone (ZYC -01).

1,3,5,8-Tetrahydroxyxanone (ZYC-02): Pale yellow solid powder. _{1H NMR} ^{_ _ _} (300MHz, CD ₃ OD) δ: 7.15 (1H, d, J=9Hz, H-6), 6.56 (1H, d, J=9Hz, H-7), 6.16 (1H, d, J=3Hz, H -2), 6.30 (1H, d, J=3Hz, H-4); ¹³ CNMR (300MHz, CD ₃ OD) δ: 164.26(C-1), 99.45(C-2), 167.93(C-3) , 95.41(C-4), 159.26(C-4a), 145.08(C-4b), 138.25(C-5), 124.63(C-6), 110.41(C-7), 154.28(C-8), 108.63(C-8a), 102.73(C-8b), 185.71(C-9); their mass spectra are shown in Figure 2. Compared with references (Qing, Z. Liu, Y.-W., 2003. Studies on the constituents of Swertia kauitchensis Franch. Hubei College of Traditional Chinese Medicine, Hubei), it was proved that the product was 1,3,5,8-tetrahydroxyl Ketones (ZYC-02).

1,3,7,8-Tetrahydroxyxanone (ZYC-03): Pale yellow solid powder. UV(MeOH)λ _max :238,265,331,386nm; IR(KBr)υ _max :3355,1661,1612,1518,1475cm ^-1 ; ESI-MS (m/z): 259.2[MH] ^- ; ¹ H NMR(300MHz, DMSO-d ₆ ) δ: 11.87 (1H, s, -OH), 11.70 (1H, s, -OH), 11.16 (1H, s, -OH), 9.36 (1H, s, -OH), 7.26 (1H ,d, J=6Hz, H-6), 6.87 (1H,d, J=6Hz, H-5), 6.20 (1H,d, J=3Hz, H-2), 6.34 (1H,d, J= 3Hz, H-4); ¹³ C NMR (300MHz, DMSO-d ₆ ) δ: 162.15(C-1), 98.15(C-2), 166.37(C-3), 94.01(C-4), 157.77( C-4a), 147.86(C-4b), 105.95(C-5), 123.92(C-6), 140.37(C-7), 147.01(C-8), 107.19(C-8a), 100.80(C -8b), 183.89 (C-9); its mass spectrum is shown in Figure 3. Controlled references (Li, S., Shi, Y.Shang, X.–Y., et al. Triterpenoids from the roots of Pterospermum heterophyllum Hance.J Asian Nat.Prod.Res., 2009, 11(7), 652- 657), proving that the product is 1,3,7,8-tetrahydroxyxanone (ZYC-03).

1,7-Dihydroxy-3-methoxyxanone (ZYC-04): Pale yellow solid powder. UV(MeOH)λ _max :239,262,325,382nm; IR(KBr)υ _max :3382,1657,1606,1478cm ^-1 ; ESI-MS(m/z):259.2[M+H] ⁺ ; ¹ H NMR(300MHz, DMSO-d ₆ )δ: 12.86 (1H, s, -OH), 7.42 (1H, d, J=3Hz, H-8), 7.30 (1H, d, J=9Hz, H-6), 7.49 (1H , d, J=9Hz, H-5), 6.58 (1H, d, J=3Hz, H-4), 6.36 (1H, d, J=3Hz, H-2) δ3.87 (3H, s, - OCH ₃ ); ¹³ C NMR (300MHz, DMSO-d ₆ ) δ: 162.47(C-1), 96.84(C-2), 166.30(C-3), 92.46(C-4), 149.00(C-4a ), 157.38(C-4b), 119.02(C-5), 124.77(C-6), 154.07(C-7), 107.95(C-8), 120.39(C-8a), 102.79(C-8b) , 180.06 (C-9). Controlled references (Hidehiro, A., Yasuaki, H., Mikio, F., et al. The chemical constituents of fresh Gentian root. J. Nat. Med., 2007, 61 (3), 269-279), proved The product is 1,7-dihydroxy-3-methoxyxanone (ZYC-04).

1,3-dihydroxy-7,8-dimethoxyxanone (ZYC-05): pale yellow solid powder. UV(MeOH)λ _max :237,264,328,385nm; IR(KBr)υ _max :3390,1645,1608,1479cm ^-1 ; ESI-MS(m/z):289[M+H] ⁺ ; ¹ H NMR (300MHz , DMSO-d ₆ ) δ: 13.26 (1H, s, -OH), 7.5 (1H, d, J=9Hz, H-6), 7.28 (1H, d, J=9Hz, H-5), 6.07 ( 1H, s, H-2), 6.22 (1H, s, H-4), 3.85 (3H, s, -OCH ₃ , 3.79 (3H, s, -OCH ₃ ); ¹³ C NMR (300MHz, DMSO-d ₆ ), δ: 163.13(C-1), 97.89(C-2), 165.57(C-3), 93.24(C-4), 156.78(C-4a), 149.90(C-4b), 112.76(C -5), 120.60(C-6), 147.53(C-7), 149.01(C-8), 114.81(C-8a), 102.29(C-8b), 180.17(C-9), 60.99(-OCH ₃ ), 56.56 (-OCH ₃ ); Control reference (Cortez, DAG, Young, MCM, Marston, A., et al.Xanthones, triterpenes and a biphenylfrom Kielmeyera coriacea.Phytochemistry, 1998,47 (7), 1367- 1374), proving that the product was 1,3-dihydroxy-7,8-dimethoxyxanone (ZYC-05).

Example 2

Inhibitory activity of the compound extracted and separated in embodiment 1 to α-glucosidase

In this example, human α-glucosidase was purchased from Sigma; 4-nitrophenol-α-D-glucopyranoside (PNPG) was purchased from Merk.

First, add 5 μl of human α-glucosidase ( Concentration: 20mg/mL), keep warm at 37°C for 10min, add 0.116mol/L PNPG50μl, react at 37°C for 10min, add Na ₂ CO ₃ to terminate the reaction, measure the absorbance of nitrophenol released under the action of the enzyme at 400nm, and then Take the compound extracted and separated in Example 1 and add it to the enzyme activity assay system. First, keep the enzyme at 37°C for 5 minutes, then add PNPG and react at 37°C for 10 minutes, add Na ₂ CO ₃ to terminate the reaction, and measure the enzyme released under the action of the enzyme at 400nm. Absorbance of nitrophenols. Finally, the percent inhibition was obtained by comparing the two absorbance values.

The experimental results are shown in Table 1.

Table 1 Inhibitory activity of medicinal ingredients of Zang Yin Chen on human α-glucosidase

It can be seen from Table 1 that the compounds extracted and separated in Example 1 have good inhibitory activity on human α-glucosidase, among which 1,7,8-trihydroxy-3,4-dimethoxyxanone ( ZYC-01) and 1,3,5,8-tetrahydroxyxanone (ZYC-02) had the best inhibitory activity, which was close to the positive control drug acarbose.

Example 3

The hypoglycemic effect of the compound extracted and separated in embodiment 1

Experimental materials: NOD mice were purchased from Guangdong Experimental Animal Center.

experimental method:

After the mice were purchased, they were fed with high-fat food, including 40% carbohydrates, 15% protein, 40% fat, and 5% other. After continuous feeding for 3 months, streptozotocin (STZ, 60mg/kg) was injected intraperitoneally once. After 72 hours, blood was taken from the tail vein, and blood glucose and insulin were measured respectively. Those with blood sugar concentration > 13mmol/L, positive urine sugar, polydipsia, polyphagia and polyuria are diabetic models.

Diabetic mice were randomly divided into 8 groups, 10 in each group, and diabetic model group, positive control group (acarbose), ZYC-01 (1,7,8-trihydroxy-3,4-dimethyl Oxyxanone) treatment group, ZYC-02 (1,3,5,8-tetrahydroxyxanone) treatment group. The model group was fed with common feed, free to drink water, and gavage with distilled water every day; each mouse in the positive control group was given 50 mg/kg by gavage; each mouse in the drug treatment group was given 20, 40, and 60 mg/kg by gavage, respectively . The mice in each group were fasted for venous blood on 1d, 10d, and 20d respectively, and blood glucose and insulin concentrations were measured.

Diabetic mice were fasted for 8 hours after the last administration, and the eyeballs were picked to take blood. After centrifugation, the plasma was taken to measure blood sugar by GOD-PAP method, and serum insulin TC and TG were measured by radioimmunoassay. The determination was performed according to the instructions on the kit.

All data are used Said, data statistics were processed by SPSS10.0 software, and t test was used for comparison between groups.

Experimental results: see Table 2. It can be seen from the analysis in Table 2 that the compounds extracted and isolated in Example 1 (ZYC-01 and ZYC-02) can significantly reduce the blood glucose, TC and TG concentrations of experimental diabetic mice, and significantly increase the plasma insulin level. This indicates that the compounds of the present invention have the effects of regulating glucose metabolism and treating experimental diabetes.

Table 2 Each group of biochemical indicators after the experiment ( mmol/L; n=10)

Example 4

The acute toxicity test of the compound that embodiment 1 extracts separation

Oral administration

Healthy male mice, weighing 18-23g, were given 1.0g/kg of ZYC-01 and ZYC-02 in aqueous solution orally for 1 week, and no mice died.

injection

Healthy male mice, weighing 18-23g, were intraperitoneally injected with ZYC-01 and ZYC-02 in aqueous solution, respectively, for 1 week, and observed the death of the mice.

Experimental results: LD50 of ZYC-01 is 5.32±0.26g/kg; LD50 of ZYC-02 is 4.82±0.74g/kg.

The experimental results reveal that ZYC-01 and ZYC-02 have less toxicity, stronger pharmacological effects, and have better medicinal prospects.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (1)

1. A method for preparing a compound with alpha-glucosidase inhibitory activity, characterized in that it may further comprise the steps: Grind the whole herb of Capillary chinensis into powder, extract it several times by percolation with 50~95% ethanol, combine the extracts, filter and remove the residue, remove the ethanol by rotary evaporation, and then dry to obtain the extract; dissolve the extract in water, wash with dichloro Methane extraction; dichloromethane extraction products were separated by silica gel column chromatography, eluted with petroleum ether-ethyl acetate gradient, the initial volume ratio of petroleum ether to ethyl acetate was 30:1, and finally transitioned to 0:1; according to TLC The polarity of the compounds shown by chromatography was combined in order of polarity from small to large to obtain 8 crude fractions, which were marked as F1~F8; fraction F3 was separated and prepared by silica gel column chromatography combined with preparative high performance liquid chromatography Compounds ZYC-01 and ZYC-02 were obtained; fraction F4 was prepared by gel column chromatography to obtain ZYC-04; fraction F5 was prepared by gel column chromatography to obtain ZYC-03 and ZYC-05; The said Tibetan Yinchen is Swertia chinensis in Western Sichuan; The ZYC-01 is 1,7,8-trihydroxy-3,4-dimethoxy Ketone, ZYC-02 is 1,3,5,8-tetrahydroxy Ketone, ZYC-03 is 1,3,7,8-tetrahydroxy Ketone, ZYC-04 is 1,7-dihydroxy-3-methoxy Ketone, ZYC-05 is 1,3-dihydroxy-7,8-dimethoxy ketone.