CN114213480A

CN114213480A - Novel glycoside compound, pharmaceutical composition, preparation method and application

Info

Publication number: CN114213480A
Application number: CN202111611492.4A
Authority: CN
Inventors: 张维库; 续洁琨; 赫军; 张佳; 李雅楠; 郭林波
Original assignee: China Japan Friendship Hospital
Current assignee: China Japan Friendship Hospital
Priority date: 2021-12-27
Filing date: 2021-12-27
Publication date: 2022-03-22
Anticipated expiration: 2041-12-27
Also published as: CN114213480B

Abstract

The invention provides a novel glycoside compound, a pharmaceutical composition, a preparation method and application, and relates to the technical field of medicines. The novel glycoside compound has a mother nucleus structure shown in formula I. The invention separates a glucoside compound with the mother nucleus structure from Euphorbia fischeriana (Euphorbia fischeriana stem), creatively discovers that the compound has obvious inhibition effect on alpha-glucosidase, can be used for preparing the medicine for treating diabetes related diseases,has wide application prospect.

Description

Novel glycoside compound, pharmaceutical composition, preparation method and application

Technical Field

The invention relates to the technical field of medicines, in particular to a novel glucoside compound, a pharmaceutical composition, a preparation method and application.

Background

With the increasing change of people's life and working style, the incidence rate of diabetes mellitus shows an increasing trend year by year. The continuing progression of the diabetic process may cause a series of complications, which in turn affect the quality of life of the patient and increase the economic burden on the patient and the country. Therefore, the development of drugs having the effect of preventing or treating diabetes is receiving increasing attention.

Euphorbia fischeriana (Euphorbia fischeriana Steud.) is a perennial herb of the genus Euphorbia of the family Euphorbiaceae, and research on this plant has focused on terpenoids and anti-tumor activity. For example, patent CN104622865A discloses the use of ingenus diterpenoids in the preparation of antitumor drugs, and patent CN103623033A discloses active substances of euphorbiaceae plants and their use for inhibiting cancer and/or tumor cell growth, all of which show the important role of active extract substances of euphorbiaceae plants in anticancer or antitumor (e.g. promoting tumor cell death).

There are currently little or no relevant studies and reports on the role of glycosides or euphorbiaceae plant extracts in diabetes.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a novel glucoside compound, a pharmaceutical composition, a preparation method and application, so as to enrich the types of diabetes-related medicaments in the prior art and provide a new treatment way for diabetes and related diseases thereof.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

in one embodiment, the present invention provides a novel class of glycosides compounds having a core structure of formula (I):

wherein R is₁And R₂Each independently selected from any one of hydrogen, ester group, methyl, ethyl, propyl, isopropyl, galloyl, amino, methylamino, dimethylamino, ethylamino, diethylamino, isopropylamino, diisopropylamino, saturated hydrocarbon group, unsaturated hydrocarbon group, substituted aromatic ring, unsubstituted aromatic ring, oligosaccharide, polysaccharide, metal ion, organic ester group, nitro group, or halogen.

The invention searches for high-efficiency low-toxicity bioactive components from euphorbia stellera and separates a glucoside compound from the plant, wherein the glucoside compound has obvious alpha-glucosidase inhibition activity.

In one embodiment, in the glycoside compound,

R₁is composed of

R₂Is composed of

This compound was named Euphorbiacetophenone E.

The molecular structural formula of the compound Euphorbiacetophenone E is as follows:

in one embodiment, in the glycoside compound,

R₁is composed of

R₂Is composed of

The compound is named as 1,2, 3-tri-O-galloyl-beta-D-glucopyranose.

The molecular structural formula of the compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose is as follows:

in another aspect, the present invention provides a pharmaceutical composition comprising the novel glycoside compound.

In one embodiment, the pharmaceutical composition comprises a combination drug; preferably, the combination comprises one or more of: insulin, tolbutamide, chlorpropamide, glyburide, glipizide, gliclazide, glimepiride, glibornuride, gliquidone, metformin hydrochloride, phenformin, pioglitazone hydrochloride, rosiglitazone maleate, troglitazone, ciglitazone, englitazone, alogliptin benzoate, saxagliptin, sitagliptin phosphate, vildagliptin, linagliptin, saccharide-100, acarbose, voglibose, miglitol, repaglinide, nateglinide, miglitide, exenatide, linagliptin, pramlintide acetate;

more preferably, the insulin comprises one or more of oligo-zinc insulin, globin zinc insulin or protamine zinc insulin.

In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient; "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. Generally, a carrier is not an essential active ingredient and is not unduly toxic after administration. Pharmaceutically acceptable carriers in the compositions may include liquids such as water, saline, glycerol and ethanol. In some cases, the excipients include types of fillers, binders, lubricants, disintegrants, solubilizers, surfactants, and the like. Suitable carriers and/or excipients are well known to those of ordinary skill in the art and may be selected depending on the circumstances. The pharmaceutical active ingredients of the present invention may be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants and formulated into a suitable administration form or dosage form for use as a human or veterinary medicament.

In the present invention, the dosage form of the pharmaceutical composition includes any one of tablets, capsules, granules, oral liquids, syrups, pastes, granules, dripping pills or pellets. For example, tablets include, but are not limited to, plain tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets; capsules include, but are not limited to, hard capsules, soft capsules, enteric capsules; the injection includes but is not limited to water injection, powder injection and infusion solution. The medicaments of the invention can also be prepared into other dosage forms which are common in the field, such as solutions, including but not limited to true solutions and colloidal solutions; emulsions include, but are not limited to, o/w, w/o and multiple emulsions; semisolid dosage forms include, but are not limited to, ointments, gels, pastes, and the like; suspension, powder, suppository, pellicle, patch, aerosol, spray, etc.

In one embodiment, the pharmaceutical composition comprises an effective amount of the glycoside compound; for example, in the pharmaceutical composition, the glycoside compound is present in an amount of 0.01 to 95% by weight, including but not limited to 0.01, 0.1, 0.2, 0.5, 1, 1.5, 2,3, 4, 5, 6, 7, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 and 95%.

In a preferred embodiment, the weight ratio of the glycosides compounds in the unit dosage form of the pharmaceutical composition is 0.01 to 50mg, including but not limited to 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mg; more preferably, the weight ratio is 0.01 to 10 mg.

In another aspect, the present invention provides a method for preparing the glycoside compound, which is isolated from Euphorbia fischeriana (Euphorbia fischeriana stem.);

further, drying and crushing roots of euphorbia stellera chamaejasme L, extracting with a solvent, collecting an extracting solution, and concentrating to obtain a concentrated solution; extracting the concentrated solution with an organic solvent to obtain an extract; performing silica gel column chromatography on the extract, and performing reversed phase silica gel column chromatography on the first target fraction obtained after elution; subjecting the second target fraction to Sephadex column chromatography, and purifying by reverse phase or normal phase preparative liquid chromatography to obtain the final product.

In one embodiment, the preparation method specifically comprises:

(a) taking roots of euphorbia stellera, drying and crushing, extracting by using an extraction solvent, combining extracting solutions, filtering, and concentrating under reduced pressure to obtain a concentrated solution;

(b) extracting the concentrated solution by using an organic solvent, and combining organic phases to obtain an extract;

(c) performing silica gel column chromatography on the extract, performing gradient elution by using an eluent, and performing reversed phase silica gel column chromatography elution on the obtained first target fraction to obtain a second target fraction; subjecting the second target fraction to sephadex column chromatography and elution;

(d) purifying by reverse phase or normal phase preparative liquid chromatography to obtain the compound.

In one embodiment, in the step (a), the mass of the extraction solvent is 8 to 15 times, preferably 10 times that of the euphorbia stellera root raw material; preferably, the extraction is reflux extraction;

further, the reflux extraction time is 1-3 hours, the temperature is 65-85 ℃, and the reflux extraction frequency is 2-4 times; more preferably, the extraction solvent is selected from one or more of water, methanol or ethanol; most preferably, the extraction solvent is 60% to 95% ethanol;

in the step (b), after dispersing with water, extracting for 2-5 times by using an organic solvent; preferably, the organic solvent comprises one or more of dichloromethane, trichloromethane, cyclohexane, ethyl acetate, propyl acetate and petroleum ether, preferably ethyl acetate, propyl acetate; preferably, the dosage of the organic solvent is 1.5-2 times of the volume of the concentrated solution;

in the step (c), the eluent for gradient elution comprises any one of chloroform-methanol, petroleum ether-acetone, petroleum ether-ethyl acetate, cyclohexane-acetone or cyclohexane-ethyl acetate; preferably petroleum ether-ethyl acetate, cyclohexane-ethyl acetate; the eluent for the reverse phase silica gel column chromatography is selected from any one of ethanol-water or methanol-water; the eluent for the sephadex column chromatography is selected from any one of methanol, chloroform-methanol, dichloromethane-methanol and petroleum ether-chloroform-methanol, preferably methanol and dichloromethane-methanol; preferably, the Sephadex is selected from Sephadex LH-20, Sephadex G-10, G-15, G-25, G-50, G-75, G-100, G-150 and G-200, preferably Sephadex LH-20;

in step (d), the chromatographic column in the chromatographic purification treatment is selected from any one of C4, C8, C6 or C18; among them, C18 is preferable; the mobile phase is selected from any one of methanol-water or acetonitrile-water.

In a particular embodiment, the process for the preparation of the compounds Euphorbiacetophenone E and 1,2,3-tri-O-galloyl- β -D-glucopyranose comprises the following steps:

step 1: taking roots of euphorbia stellera, drying and crushing, performing reflux extraction for a plurality of times by using an extraction solvent, combining extracting solutions, filtering, and concentrating under reduced pressure to obtain a concentrated solution;

step 2: extracting the concentrated solution obtained in the step S10 for several times by using an organic solvent, combining organic phases, and recovering the solvent to obtain an extract III;

and step 3: performing silica gel column chromatography on the extract III, performing gradient elution by using an eluent, and merging to obtain a fraction A, a fraction B, a fraction C, a fraction D and a fraction E in sequence after the silica gel thin-layer plate is used for identification;

subjecting fraction D to reverse phase silica gel column chromatography, and detecting with silica gel thin layer plate to obtain (6 fractions D1-D6) fraction D1, fraction D2, fraction D3, fraction D4, fraction D5 and fraction D6; subjecting fraction D3 to Sephadex column chromatography;

and 4, step 4: purifying by reverse phase or normal phase preparative liquid chromatography respectively to obtain compound Eucalyptus topophenone E and 1,2, 3-tri-O-galloyl-beta-D-glucopyranose.

In another aspect, the invention provides an application of the glucoside compound or the pharmaceutical composition containing the glucoside compound in preparing a medicament for preventing or treating diabetes and complications thereof.

The invention provides an application of the glucoside compound in preparing an alpha-glucosidase inhibitor hypoglycemic medicinal preparation (such as an alpha-glucosidase inhibitor).

According to the invention, the glucoside compounds are used as raw materials, and the results of alpha-glucosidase inhibition activity screening tests show that the activity of European iectophenone E and 1,2, 3-tri-O-galloyl-beta-D-glucopyranose for inhibiting alpha-glucosidase is better than that of positive drug acarbose (IC)₅₀286.23 ± 0.86 μ M). Among them, the compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose has the most significant inhibitory activity, IC₅₀The value was 15.48. + -. 0.19. mu.M. The compound is an alpha-glucosidase mixed inhibitor by enzyme inhibition kinetics test of 1,2, 3-tri-O-galloyl-beta-D-glucopyranose.

Has the advantages that: the invention provides a glucoside compound, and alpha-glucosidase inhibition activity screening test and inhibition kinetics test show that the compound has obvious inhibition effect on alpha-glucosidase and can be applied as a medicament for treating diabetes. Can also be used for preparing health food or functional food for lowering blood sugar.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the inhibitory effect of 1,2,3-tri-O-galloyl- β -D-glucopyranose of the present invention on α -glucosidase activity;

FIG. 2 is a graph showing the inhibition kinetics of 1,2,3-tri-O-galloyl- β -D-glucopyranose activity against α -glucosidase activity according to the present invention;

FIG. 3 is a Michaelis-Menten graph (Michelia-Menten graph, also known as Michelia-Menten curve) showing the activity of 1,2,3-tri-O-galloyl- β -D-glucopyranose according to the present invention on α -glucosidase;

FIG. 4 is a Lineweaver-Burk plot (i.e., double reciprocal plot) of 1,2,3-tri-O-galloyl- β -D-glucopyranose activity of the present invention versus α -glucosidase activity.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 preparation of glycoside Compounds, European beta-D-glucopyranosone E and 1,2, 3-tri-O-galloyl-beta-D-glucopyranose

Step S1: taking roots of euphorbia stellera, drying and crushing, adding 8-15 times of solvent for reflux extraction for 1-3 hours, repeating for 2-4 times, combining extracting solutions, filtering, and concentrating under reduced pressure to obtain a concentrated solution; the extraction temperature is 65-85 ℃;

the extraction solvent can be selected from water, methanol, ethanol or their mixture, preferably 60-95% ethanol; (in this example, 10 times the amount of 80% ethanol solvent was added, and the extraction was repeated 3 times for 2 hours under reflux).

Step S2: extracting the concentrated solution with organic solvent for 2-5 times, mixing organic phases, and recovering organic solvent to obtain extract III; the extraction temperature is from 65 ℃ to the boiling temperature of the extraction solvent;

the organic extraction solvent can be dichloromethane, chloroform, cyclohexane, ethyl acetate, propyl acetate and petroleum ether, preferably ethyl acetate, propyl acetate; (in this example, the concentrated solution was extracted 4 times with an organic solvent, ethyl acetate).

Step S3: subjecting the extract III to silica gel column chromatography, performing gradient elution with organic reagent, detecting with silica gel thin layer plate, and mixing to obtain 5 fractions (A-E); then selecting D for reverse phase silica gel column chromatography, and identifying with silica gel thin layer plate to obtain 6 fractions (D1-D6); performing sephadex column chromatography on the D3;

the silica gel column chromatography eluent is petroleum ether-ethyl acetate, petroleum ether-acetone, cyclohexane-ethyl acetate, cyclohexane-acetone, chloroform-methanol, preferably petroleum ether-ethyl acetate, cyclohexane-ethyl acetate; the eluent for the reverse phase silica gel column chromatography can be selected from ethanol-water or methanol-water; the Sephadex LH-20, Sephadex G-10, G-15, G-25, G-50, G-75, G-100, G-150 and G-200 can be selected as the material for the Sephadex column chromatography; the eluent for Sephadex column chromatography can be selected from methanol, chloroform-methanol, dichloromethane-methanol, and petroleum ether-chloroform-methanol, preferably methanol, and dichloromethane-methanol (in this example, the eluent for Sephadex LH-20 is petroleum ether-ethyl acetate, the eluent for reverse phase silica gel column chromatography is ethanol-water, and the eluent is methanol).

Step S4: purifying by reversed phase preparative liquid chromatography to obtain compounds of Euphorbiacetophenone E and 1,2, 3-tri-O-galloyl-beta-D-glucopyranose;

the reversed phase preparative liquid chromatography column may be selected from C4, C6, C8 or C18, preferably C18; the mobile phase can be methanol-water or acetonitrile-water (in this example, C18 liquid chromatography column is selected, and methanol-water is selected as the mobile phase).

Example 2

The rest is the same as example 1 except that:

step S1: in this example, an 8-fold amount of 50% ethanol solvent was added, and the extraction was repeated 2 times for 1 hour under reflux.

Step S2: in this example, the concentrate was extracted 2 times with an organic solvent, propyl acetate.

Step S3: in this embodiment, the eluent for silica gel column chromatography is petroleum ether-acetone, the eluent for reverse phase silica gel column chromatography is methanol-water, the material for Sephadex G-10 and the eluent is dichloromethane-methanol.

Step S4: in this example, a C4 liquid chromatography column was selected, and acetonitrile-water was selected as the mobile phase.

Example 3

The rest is the same as example 1 except that:

step S1: in this example, a 15-fold amount of 95% ethanol solvent was added, and the extraction was repeated 4 times with reflux for 3 hours.

Step S2: in this example, the concentrate was extracted 5 times with an organic solvent, propyl acetate.

Step S3: in this example, the eluent for silica gel column chromatography was cyclohexane-ethyl acetate, the eluent for reverse phase silica gel column chromatography was methanol-water, the material for Sephadex G-10 column chromatography was Sephadex G-10, and the eluent was petroleum ether-chloroform-methanol.

Step S4: in this example, a C6 liquid chromatography column was selected, and acetonitrile-water was selected as the mobile phase.

It should be noted that examples 2 to 3 of the present invention and other examples of the production method not shown in the present invention were similar to the production result of example 1 of the present invention; the glycoside compounds of Euphorbiacetophenone E and 1,2, 3-tri-O-galloyl-beta-D-glucopyranose of the present invention can be prepared as long as they are within the parameters defined in the preparation process of the present invention.

Example 4

physical properties of euphone E are white amorphous powder; the nuclear magnetic data are as follows:

UV(MeOH)λ_max 215,280nm；IR(KBr)ν_max 3396,1715,1619cm^–1；HR-ESI-MS:m/z 647.1254[M-H]^-(cal.647.1254)；¹H NMR(500MHz,DMSO-d₆):δ_H 5.72(1H,d,J＝8.0Hz,H-1),5.18(1H,dd,J＝9.8,8.0Hz,H-2),5.42(1H,t,J＝9.5Hz,H-3),3.65(1H,t,J＝9.5Hz,H-4),3.81(1H,overlap,H-5),3.42(1H,dd,J＝12.0,3.0Hz,H-6α),3.33(1H,dd,J＝12.0,6.5Hz,H-6β),6.89(1H,s,H-2’),6.89(1H,s,H-6’),6.83(1H,s,H-2”),6.83(1H,s,H-6”),6.17(1H,d,J＝2.3Hz,H-3”’),6.10(1H,d,J＝2.3Hz,H-5”’),2.53(3H,s,H-8”’),3.80(3H,s,OCH₃)；¹³C NMR(125MHz,DMSO-d₆):δ_c 96.7(C-1),73.3(C-2),74.9(C-3),67.7(C-4),77.1(C-5),60.4(C-6),119.1(C-1’),108.8(C-2’),145.5(C-3’),138.6(C-4’),145.5(C-5’),108.8(C-6’),165.1(C-7’),118.5(C-1”),108.8(C-2”),145.5(C-3”),139.0(C-4”),145.5(C-5”),108.8(C-6”),164.7(C-7”),106.9(C-1”’),162.7(C-2”’),92.0(C-3”),162.7(C-4”’),96.2(C-5”’),165.2(C-6”’),203.2(C-7”’),32.9(C-8”’),56.2(OCH₃)。

example 5

the physical properties of 1,2,3-tri-O-galloyl- β -D-glucopyranose are pale white amorphous powder; the nuclear magnetic data are as follows:

UV(MeOH)λ_max 215,280nm；HR-ESI-MS:m/z 635.0889[M-H]^-(cal.635.0890)；¹H NMR(500MHz,DMSO-d₆):δ_H 6.05(1H,d,J＝8.3Hz,H-1),5.41(1H,dd,J＝9.9,8.2Hz,H-2),5.55(1H,t,J＝9.5Hz,H-3),3.73-3.97(4H,m,H-4,H-5,H-6),7.03(1H,s,H-2’),7.02(1H,s,H-6’),7.03(1H,s,H-2”),7.02(1H,s,H-6”)；¹³C NMR(125MHz,DMSO-d₆):δ_c 93.9(C-1),72.4(C-2),76.8(C-3),69.3(C-4),79.1(C-5),61.9(C-6),119.9(C-1’),110.0(C-2’),146.4(C-3’),146.6(C-4’),146.4(C-5’),110.4(C-6’),166.4(C-7’),120.5(C-1”),110.0(C-2”),146.4(C-3”),146.4(C-4”),146.4(C-5”),110.4(C-6”),167.2(C-7”),121.1(C-1”’),110.4(C-2”’),146.6(C-3”),146.6(C-4”’),146.6(C-5”’),110.6(C-6”’),167.8(C-7”’)。

example 6

The embodiment provides a tablet taking Euphorbiacetophenone E as a raw material medicine, which comprises the following components:

100 pieces were prepared.

Mixing Euphorbiacetophenone E with hydroxypropyl methylcellulose, pulvis Talci, lactose, and magnesium stearate, adding anhydrous ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and tabletting.

Example 7

The embodiment provides a tablet taking 1,2, 3-tri-O-galloyl-beta-D-glucopyranose as a raw material medicine, which comprises the following components:

1,2,3-tri-O-galloyl-β-D-glucopyranose	24.0mg
		hydroxypropyl methylcellulose	16g
Talcum powder	0.3g
		Lactose	0.2g
Magnesium stearate	0.2g
		Anhydrous ethanol	0.1mL

100 pieces were prepared.

Mixing 1,2, 3-tri-O-galloyl-beta-D-glucopyranose with hydroxypropyl methylcellulose, pulvis Talci, lactose and magnesium stearate, adding anhydrous ethanol to obtain soft mass, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and tabletting.

Example 8

The embodiment provides a capsule taking a compound Euphorbiacetophenone E as a raw material medicine, which comprises the following components:

making into 100 granules.

Mixing Euphorbiacetophenone E with starch, microcrystalline cellulose and sodium metabisulfite, adding anhydrous ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and making into capsule.

Example 9

The embodiment provides a capsule taking a compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose as a raw material medicine, which comprises the following components:

1,2,3-tri-O-galloyl-β-D-glucopyranose	19.0mg
		microcrystalline cellulose	0.2g
Starch	5.0g
		Sodium metabisulfite	0.2g
Magnesium stearate	0.2g
		Anhydrous ethanol	0.1mL

Making into 100 granules.

Mixing 1,2, 3-tri-O-galloyl-beta-D-glucopyranose with starch, microcrystalline cellulose and sodium pyrosulfite, adding anhydrous ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and making into capsule.

Example 10

The embodiment provides a granule taking compound Euphorbiacetophenone E as a raw material medicine, which comprises the following components:

100 bags are made.

Mixing Euphorbiacetophenone E with starch and sodium bisulfite, adding anhydrous ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and packaging.

Example 11

The embodiment discloses a granule taking a compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose as a raw material medicine, which comprises the following components:

1,2,3-tri-O-galloyl-β-D-glucopyranose	34.0mg
		starch	5g
Sodium metabisulfite	0.2g
		Magnesium stearate	0.2g
Anhydrous ethanol	0.1mL

100 bags are made.

Mixing 1,2, 3-tri-O-galloyl-beta-D-glucopyranose with starch and sodium bisulfite, adding anhydrous ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and packaging.

Example 12

The embodiment discloses an oral liquid taking compound Euphorbiacetophenone E as a raw material medicine, which comprises the following components:

100 pieces are made. Mixing the above materials, and packaging by conventional method.

Example 13

The embodiment provides an oral liquid taking a compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose as a raw material medicine, which comprises the following components:

1,2,3-tri-O-galloyl-β-D-glucopyranose	24.0mg
		sucrose	2.0g
Sodium bisulfite	0.2g
		P-hydroxybenzoic acid methyl ester	0.2g
Sodium bicarbonate	0.1mL
		Water for injection	1000mL

Example 14

The embodiment provides an injection taking compound Euphorbiacetophenone E as a raw material medicine, which comprises the following components:

Euphorbiacetophenone E	45.0mg
		vitamin C	0.2g
Sodium chloride	6.0g
		Sodium bicarbonate	0.1mL
Water for injection	1000mL

100 pieces are made. Mixing the above materials, and making into 100 pills by conventional injection preparation method.

Example 15

The embodiment discloses an injection taking a compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose as a raw material medicine, which comprises the following components:

1,2,3-tri-O-galloyl-β-D-glucopyranose	44.0mg
		vitamin C	0.2g
Sodium chloride	5.0g
		Sodium bicarbonate	0.1mL
Water for injection	1000mL

Example 16

The embodiment provides a tablet taking compounds of euphone E and acarbose as raw material medicines, and the tablet comprises the following components:

Euphorbiacetophenone E	20.0mg
		acarbose	5g
Hydroxypropyl methylcellulose	18g
		Talcum powder	0.4g
Lactose	0.2g
		Magnesium stearate	0.2g
Anhydrous ethanol	0.1mL

100 pieces were prepared. Mixing Euphorbiacetophenone E, acarbose, hydroxypropyl methylcellulose, pulvis Talci, lactose, and magnesium stearate, adding anhydrous alcohol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and tabletting.

Example 17

This example discloses a tablet using 1,2,3-tri-O-galloyl- β -D-glucopyranose compound and acarbose as raw material, which comprises the following components:

100 pieces were prepared.

Mixing 1,2, 3-tri-O-galloyl-beta-D-glucopyranose, acarbose, hydroxypropyl methylcellulose, talcum powder, lactose and magnesium stearate, adding anhydrous ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and tabletting.

Example 18

The embodiment provides a capsule taking compound Euphorbiacetophenone E and voglibose as raw material medicines, which comprises the following components:

Euphorbiacetophenone E	18.0mg
		voglibose	2.0g
Microcrystalline cellulose	0.2g
		Starch	6.0g
Sodium metabisulfite	0.2g
		Magnesium stearate	0.2g
Anhydrous ethanol	0.1mL

Making into 100 granules.

Mixing Euphorbiacetophenone E, voglibose and starch, microcrystalline cellulose, and sodium pyrosulfite, adding anhydrous ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and making into capsule.

Example 19

The embodiment provides a capsule taking a compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose and voglibose as raw material medicaments, which comprises the following components:

1,2,3-tri-O-galloyl-β-D-glucopyranose	17.0mg
		voglibose	2.0g
Microcrystalline cellulose	0.2g
		Starch	5.0g
Sodium metabisulfite	0.2g
		Magnesium stearate	0.2g
Anhydrous ethanol	0.1mL

Making into 100 granules.

Mixing 1,2, 3-tri-O-galloyl-beta-D-glucopyranose, voglibose, starch, microcrystalline cellulose and sodium pyrosulfite, adding anhydrous ethanol to obtain soft material, sieving with 24 mesh sieve, granulating, drying, adding magnesium stearate, mixing, and making into capsule.

Example 20

The embodiment discloses an injection taking compound Euphorbiacetophenone E and insulin as raw material medicines, which comprises the following components:

Euphorbiacetophenone E	40.0mg
		insulin	2.0g
Vitamin C	0.2g
		Sodium chloride	6.0g
Sodium bicarbonate	0.1mL
		Water for injection	1000mL

Example 21

The embodiment discloses an injection taking a compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose and insulin as raw material medicines, which comprises the following components:

1,2,3-tri-O-galloyl-β-D-glucopyranose	39.0mg
		insulin	2.0g
Vitamin C	0.2g
		Sodium chloride	5.0g
Sodium bicarbonate	0.1mL
		Water for injection	1000mL

The pharmaceutical preparations prepared in examples 6 to 21 all have an obvious inhibitory effect on α -glucosidase, and can be applied as a medicament for treating diabetes.

In order to better understand the essence of the present invention, the following will be described the new application of the glycoside compounds in the pharmaceutical field in combination with the pharmacological test and the results.

Test example 1

This test example provides the α -glucosidase inhibitory activity test of the compounds Euphorbiacetophenone E, 1,2,3-tri-O-galloyl- β -D-glucopyranose and the known compounds 3,5-dihydroxy-2,4-dimethyl-1-O- (6' -O-galloyl-1- β -D-glucopyranosol) -benzophenone, salaicoside A, 3,4,6-tri-O-galloyl- β -D-glucopyranose and 1-O-gallicacy-6-O- (3-methoxy) caffeicyl- β -D-glucopyranose.

(1) Experimental materials and instruments

Alpha-glucosidase (from yeast), 4-nitrophenyl-alpha-D-glucopyranoside (p-NPG), acarbose were purchased fromFrom Shanghai-derived leaf Biotech, Inc.; PBS (beijing solibao technologies ltd); anhydrous Na reagent₂CO₃For analytical purification.

Carbon dioxide cell culture chamber (Thermo Fisher Scientific, usa); a full-wavelength microplate reader (Thermo Fisher Scientific, USA); pipettor (Eppendorf, germany).

(2) Experimental methods

The experiment was divided into a blank group, a blank control group, a sample blank group and a sample group. The experiment is carried out in a 96-well plate, 20 mu L of samples with different concentrations or the positive drug acarbose is added into each well in a sample group and a sample blank group, 3 parallel multiple wells are arranged in each group, 100 mu L of PBS buffer solution is respectively added into each well, 120 mu L of PBS buffer solution is added into a blank control group and a sample blank group, and 140 mu L of PBS buffer solution is added into a blank group; adding 20 μ L of alpha-glucosidase phosphate buffer solution (0.25U/mL) into the sample group and blank control group, incubating at 37 deg.C for 15min, taking out, adding 20 μ L of 2.5mmol/Lp-NPG solution, mixing, incubating at 37 deg.C for 15min, adding 40 μ L of 0.2mol/L Na₂CO₃The reaction was stopped with the solution. The absorbance was measured at 405nm according to the following formula:

inhibition ratio (%) [ (OD control-OD control blank) - (OD sample-OD sample blank) ]/(OD control-OD control blank) × 100%

And calculating the inhibition rate of each sample on the alpha-glucosidase. Using acarbose as positive control to determine corresponding IC₅₀The value is obtained.

(3) As a result, the experimental results are shown in Table 1:

TABLE 1 screening of compounds such as European tocopheryl E, 1,2, 3-tri-O-galloyl-beta-D-glucopyranose for alpha-glucosidase inhibitory activity

The results in Table 1 show that glycoside compounds Euphorbiacetophenone E and 1,2, 3-tri-O-galloyl-beta-D-glucopyranose have good inhibition effect on alpha-glucosidase, the inhibition activity is better than that of positive drug Acarbose and other known compounds, the inhibition effect is from large to small, the arrangement is that 1,2, 3-tri-O-galloyl-beta-D-glucopyranose is larger than that of Euphoraceae tocopherol E larger than 3,4, 6-tri-O-galloyl-beta-D-glucopyranose larger than 3,5-dihydroxy-2,4-dimethyl-1-O- (6' -O-galloyl-1-beta-D-glucopyranose) -benzophenone is larger than that of Acrylase larger than 1-O-gallocy-6-O- (3-galloyl-beta-D-glucopyranose), the glucoside compound is a molecule with stronger alpha-glucosidase inhibitory activity in a natural product, so that the glucoside compound can be applied to medicines for treating diabetes.

Test example 2

The test example discloses a kinetic test of the compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose on the inhibition of alpha-glucosidase.

(1) Experimental materials and instruments

Alpha-glucosidase (from yeast), 4-nitrobenzene-alpha-D-glucopyranoside (p-NPG), acarbose were purchased from Shanghai-derived leaf Biotech Ltd; PBS (beijing solibao technologies ltd); the reagent anhydrous Na2CO3 was analytical pure.

(2) Determination of the binding Pattern of alpha-glucosidase

The initial reaction rates at different concentrations of 1,2,3-tri-O-galloyl- β -D-glucopyranose (0,12.5,25,50 μmol/L) and α -glucosidase of 0.125, 0.25, 0.5, 1.0U/mL, the final concentration of p-NPG as a substrate was 2.5mmol/L, three parallel wells were prepared for each concentration, the enzyme concentration E (U/mL) was plotted on the abscissa and the initial reaction rate v (. DELTA.OD/min) was plotted on the ordinate, and the enzyme binding pattern was deduced from the characteristics of the graph, according to the "test 1".

(3) Determination of the type of alpha-glucosidase inhibition

The initial rates of the reactions were measured according to "test 1" at different concentrations of 1,2,3-tri-O-galloyl- β -D-glucopyranose (0,12.5,25, 50. mu. mol/L) and at different concentrations of substrate p-NPG of 0.625, 1.25, 2.5, 5mmol/L, respectively, and a concentration of α -glucosidase of 0.25U/mL was added in three parallel wells per concentration, and the inhibition kinetics curves were plotted statistically according to Michaelis-Menten and Lineweaver-Burk equations to determine the type of inhibition.

(4) Statistical analysis

Data were processed using GraphPad Prism 6.02 software and all experimental results were measured 3 times and expressed as mean ± standard deviation (x ± SD).

(5) Results

As shown in the figure 1 and the figure 2, the compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose is an alpha-glucosidase inhibitor with good inhibitory activity, and is a reversible inhibitor which loses or reduces the activity of the alpha-glucosidase inhibitor by non-covalent bond with enzyme

As can be seen from FIGS. 3 and 4, the inhibition types of the compound 1,2, 3-tri-O-galloyl-beta-D-glucopyranose and alpha-glucosidase are mixed type inhibition, and V is increased with the increase of the substrate concentration_maxDecrease of K_mAn increase, i.e.the compound not only competes with the substrate for binding to the active center of the enzyme, but also binds to free enzyme or enzyme-substrate complex, thereby reducing or eliminating the activity of the enzyme.

In conclusion, the glycoside compound has the inhibiting activity superior to acarbose which is a first-line hypoglycemic medicament, and can be used as a medicament for treating diabetes.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Novel glycoside compounds, characterized by having a parent nucleus structure of formula (I):

2. The glycoside compound according to claim 1, wherein,

R₁is composed of

R₂Is composed of

3. The glycoside compound according to claim 1, wherein,

R₁is composed of

R₂Is composed of

4. A pharmaceutical composition comprising the glycoside compound according to any one of claims 1 to 3.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition comprises a combination drug; preferably, the combination comprises one or more of: insulin, tolbutamide, chlorpropamide, glyburide, glipizide, gliclazide, glimepiride, glibornuride, gliquidone, metformin hydrochloride, phenformin, pioglitazone hydrochloride, rosiglitazone maleate, troglitazone, ciglitazone, englitazone, alogliptin benzoate, saxagliptin, sitagliptin phosphate, vildagliptin, linagliptin, saccharide-100, acarbose, voglibose, miglitol, repaglinide, nateglinide, miglitide, exenatide, linagliptin, pramlintide acetate;

6. The pharmaceutical composition of claim 4 or 5, further comprising a pharmaceutically acceptable carrier or excipient; further, the dosage form of the pharmaceutical composition comprises any one of tablets, capsules, granules, oral liquid, syrup, paste, granules, dripping pills or pellets.

7. The process for preparing the glycosides of claim 2 or 3 wherein the glycosides are isolated from Euphorbia fischeriana (Euphorbia fischeriana Steud);

8. The preparation method according to claim 7, wherein the preparation method specifically comprises:

9. The preparation method according to claim 8, wherein in the step (a), the mass of the extraction solvent is 8-15 times, preferably 10 times that of the euphorbia stellera root raw material; preferably, the extraction is reflux extraction;

10. Use of a glycoside compound according to any one of claims 1 to 3 or a pharmaceutical composition according to any one of claims 4 to 6 for the preparation of a medicament for the prevention or treatment of diabetes and its complications; preferably the application in preparing alpha-glucosidase inhibitor hypoglycemic drugs.