阅读说明：本技术 刺玫果中2个具有降血糖活性的新化合物及其制备方法 (New compound with hypoglycemic activity in 2 rosa davurica fruits and preparation method thereof ) 是由 钟方丽 张晓丽 王晓林 崔瑶 薛健飞 陈帅 于 2021-07-12 设计创作，主要内容包括：本发明公开了刺玫果中2个具有降血糖活性的新化合物及其制备方法,本发明提供的化合物I、化合物II分别命名为6-((庚-5-烯-3-基)氧基)-2-(甲氧基甲基)四氢吡喃-3,4,5-三醇、5-乙氧基-2-甲氧基甲基-6-(1-甲基-丁氧基)-四氢吡喃-3,4-二醇。本发明采用现代波谱技术～(1)HNMR和～(13)CNMR、化合物的理化性质对分离得到的单体化合物进行结构鉴定,推导出化合物I、化合物II的分子结构,为刺玫果的进一步质量控制和药效研究提供了物质基础。(The invention discloses 2 novel compounds with hypoglycemic activity in rose hip and a preparation method thereof, wherein the compound I and the compound II provided by the invention are respectively named as 6- ((hept-5-en-3-yl) oxy) -2- (methoxymethyl) tetrahydropyran-3, 4, 5-triol and 5-ethoxy-2-methoxymethyl-6- (1-methyl-butoxy) -tetrahydropyran-3, 4-diol. The invention adopts modern spectrum technology 1 HNMR and 13 CNMR, and the physicochemical property of the compound, the separated monomer compound is subjected to structural identification, the molecular structures of the compound I and the compound II are deduced, and a material basis is provided for further quality control and pharmacodynamic study of the rose hip.)

1. 2 new compounds with hypoglycemic activity in rose hip and preparation method thereof, characterized in that the chemical name of compound I is 6- ((hept-5-en-3-yl) oxy) -2- (methoxymethyl) tetrahydropyran-3, 4, 5-triol, and the chemical name of compound II is 5-ethoxy-2-methoxymethyl-6- (1-methyl-butoxy) -tetrahydropyran-3, 4-diol.

2. A process for the preparation of 6- ((hept-5-en-3-yl) oxy) -2- (methoxymethyl) tetrahydropyran-3, 4, 5-triol, 5-ethoxy-2-methoxymethyl-6- (1-methyl-butoxy) -tetrahydropyran-3, 4-diol as claimed in claim 1, characterized by carrying out the following steps:

(1) extraction and extraction: taking dried rose hip, crushing, repeatedly refluxing and extracting by 70% ethanol water solution, combining extracting solutions, concentrating under reduced pressure to obtain an extract, purifying by D101 macroporous adsorption resin to obtain a rose hip extract, sequentially extracting the rose hip extract by using petroleum ether, ethyl acetate and n-butyl alcohol to respectively obtain a petroleum ether extract, an ethyl acetate extract, an n-butyl alcohol extract and a water part, and selecting the n-butyl alcohol extract for separation;

(2) separation: taking a proper amount of the n-butanol extract of the rosa davurica pall, carrying out dry loading on the rosa davurica pall according to the mass ratio of 1:60 to silica gel for column chromatography, carrying out rough separation by normal phase silica gel column chromatography, carrying out detection by thin layer chromatography, combining elution fractions of methanol-ethyl acetate (1: 5, 1:3 and 1: 1) to obtain a component A, carrying out dry loading on the component A according to the mass ratio of 1:70 to silica gel for column chromatography, carrying out separation by normal phase silica gel column chromatography for the second time, carrying out detection by thin layer chromatography, combining the elution fractions of dichloromethane-ethyl acetate-methanol (4: 6:1, 2:6:1 and 1:6: 1) to obtain a component A1, carrying out dry loading on the component 1 according to the mass ratio of 1:100 to silica gel for column chromatography, carrying out separation by normal phase silica gel column chromatography for the third time, carrying out detection by thin layer chromatography, combining the elution fractions of dichloromethane-methanol (6: 1, 3:1 and 1: 1), obtaining a component A1-1, separating the component A1-1 by a semi-preparative liquid chromatograph, and carrying out methanol separation: eluting with water (1: 9) eluent, collecting the fractions with retention time of 9.2min and 11.5min, concentrating, crystallizing, filtering, drying to obtain compound I and compound II, and identifying compound I with chemical name of 6- ((hept-5-en-3-yl) oxy) -2- (methoxymethyl) tetrahydropyran-3, 4, 5-triol and compound II with chemical name of 5-ethoxy-2-methoxymethyl-6- (1-methyl-butoxy) -tetrahydropyran-3, 4-diol by NMR.

3. The method for preparing a novel compound having hypoglycemic activity in rose hip according to claim 2, wherein: when the silica gel column chromatography separation is carried out in the separation step, 200-mesh 300-mesh silica gel is selected and subjected to three times of silica gel column chromatography separation, and an eluent in the first separation is a methanol-ethyl acetate system with the ratio of 1:5, 1:3 and 1: 1; the eluent in the second separation is a dichloromethane-ethyl acetate-methanol system, and the mixture ratio of the dichloromethane to the ethyl acetate to the methanol is 4:6:1, 2:6:1 and 1:6: 1; the eluent used in the third separation is a dichloromethane-methanol system, and the mixture ratio of the dichloromethane to the methanol is 6:1, 3:1 and 1: 1; the mobile phase of the semi-preparative liquid chromatograph is a methanol-water system, the ratio of the two is 1:9, and the retention time of the compound I and the compound II is respectively 9.2min and 11.5 min.

Technical Field

The invention relates to the field of extraction and separation of traditional Chinese medicines, in particular to 2 new compounds with hypoglycemic activity in rosa davurica pall and a preparation method thereof.

Background

The rose hip is dry mature fruit of Rosa davurica pall of Rosa davurica of rosaceae, is harvested when the fruit is mature in autumn, is mainly produced in northeast, northChina, inner Mongolia and other places, contains chemical components such as general flavone, vitamin, pectin, sugar, saponin and the like, is commonly used as a natural additive of human anti-aging products, and can obviously improve the anti-aging and anti-oxidation capability of human bodies. Pharmacological experiments show that the aqueous extract and the alcohol extract of the rose hip can reduce the blood pressure and the cerebrovascular resistance of anesthetized rats, slow the heart rate of anesthetized cats, increase the coronary flow and reduce the coronary resistance; can also reduce oxygen consumption of mice, prolong the survival time of anoxic mice, and resist the effect of isoproterenol on increasing myocardial oxygen consumption; the fructus Rosae Davuricae water extract can be injected intravenously for inhibiting thrombosis in rat. A great deal of domestic research in recent years shows that the health care tea has the effects of resisting aging, enhancing immunity, resisting fatigue, protecting liver and the like.

Disclosure of Invention

The invention aims at 2 novel compounds with hypoglycemic activity in rose hip and a preparation method thereof.

The preparation method is characterized in that 2 new compounds with hypoglycemic activity in the rose hip are prepared by taking the rose hip as a raw material through the steps of extraction, extraction and separation, the 2 new compounds have the hypoglycemic activity, the chemical name of the compound I is 6- ((hept-5-en-3-yl) oxy) -2- (methoxymethyl) tetrahydropyran-3, 4, 5-triol, and the structural formula is as follows:compound II, having the chemical name 5-ethoxy-2-methoxymethyl-6- (1-methyl-butoxy) -tetrahydropyran-3, 4-diol, has the formula:。

2 new compounds with hypoglycemic activity in rose hip and preparation method thereof, comprising the following steps:

(1) extraction and extraction: pulverizing dried fructus Rosae Davuricae, reflux-extracting with 70% ethanol water solution repeatedly, mixing extractive solutions, concentrating under reduced pressure to obtain extract, and purifying with D101 macroporous adsorbent resin to obtain fructus Rosae Davuricae extract. Sequentially extracting the rose hip extract with petroleum ether, ethyl acetate and n-butanol to respectively obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water part, and selecting n-butanol extract for separation;

(2) separation: taking a proper amount of the n-butanol extract of the rosa davurica pall, carrying out dry loading on the rosa davurica pall according to the mass ratio of 1:60 to silica gel for column chromatography, carrying out rough separation by normal phase silica gel column chromatography, carrying out detection by thin layer chromatography, combining elution fractions of methanol-ethyl acetate (1: 5, 1:3 and 1: 1) to obtain a component A, carrying out dry loading on the component A according to the mass ratio of 1:70 to silica gel for column chromatography, carrying out separation by normal phase silica gel column chromatography for the second time, carrying out detection by thin layer chromatography, combining the elution fractions of dichloromethane-ethyl acetate-methanol (4: 6:1, 2:6:1 and 1:6: 1) to obtain a component A1, carrying out dry loading on the component 1 according to the mass ratio of 1:100 to silica gel for column chromatography, carrying out separation by normal phase silica gel column chromatography for the third time, carrying out detection by thin layer chromatography, combining the elution fractions of dichloromethane-methanol (6: 1, 3:1 and 1: 1), obtaining a component A1-1, separating the component A1-1 by a semi-preparative liquid chromatograph, and carrying out methanol separation: eluting with water (1: 9) eluent, collecting the fractions with retention time of 9.2min and 11.5min, concentrating, crystallizing, filtering, drying to obtain compound I and compound II, and identifying compound I with chemical name of 6- ((hept-5-en-3-yl) oxy) -2- (methoxymethyl) tetrahydropyran-3, 4, 5-triol and compound II with chemical name of 5-ethoxy-2-methoxymethyl-6- (1-methyl-butoxy) -tetrahydropyran-3, 4-diol by NMR.

In a preferred embodiment of the present invention, when performing silica gel column chromatography separation in the separation step, 200-300 mesh silica gel is selected, and subjected to silica gel column chromatography separation for three times, wherein an eluent in the first separation is a methanol-ethyl acetate system, and the ratio of the two is 1:5, 1:3, and 1: 1; the eluent in the second separation is a dichloromethane-ethyl acetate-methanol system, and the mixture ratio of the dichloromethane to the ethyl acetate to the methanol is 4:6:1, 2:6:1 and 1:6: 1; the eluent used in the third separation is a dichloromethane-methanol system, and the mixture ratio of the dichloromethane to the methanol is 6:1, 3:1 and 1: 1; the mobile phase of the semi-preparative liquid chromatograph is a methanol-water system, the ratio of the two is 1:9, and the retention time of the compound I and the compound II is respectively 9.2min and 11.5 min.

The invention has the following technical effects: the invention adopts modern spectrum technology¹HNMR and¹³CNMR, the physicochemical properties of the compound carry out structural identification on the separated monomer compound, deduces the molecular structure of the compound, and provides a material basis for further quality control and pharmacodynamic study of the rosa davurica pall.

The present invention is further illustrated by the following examples, but the scope of the invention as claimed is not limited to the following embodiments.

Example 1: 2 new compounds with hypoglycemic activity in rose hip and preparation method thereof, comprising the following steps:

(1) extraction and extraction: taking 15.0kg of dried rose hip, crushing, sieving with a 20-mesh sieve, extracting with 6 times of 75% ethanol aqueous solution in a multifunctional dynamic extraction and concentration machine set for 3 times, controlling the temperature at 85 ℃ and 5 hours each time, filtering the extracting solution, merging, concentrating under reduced pressure to obtain a thick extract, adding 10 times of distilled water for dilution, and preparing the rose hip column-loading solution. Adding dilute hydrochloric acid into the rose hip column-loading liquid to adjust the pH value to 2-4, diluting with distilled water until the concentration of crude drugs is 0.025 g/mL, adsorbing by a D-101 macroporous adsorption resin chromatographic column, eluting with 6 times of column volume of distilled water, eluting with 3 times of column volume of 70% ethanol water solution, collecting the eluent, concentrating, and drying to obtain the rose hip extract. Taking a proper amount of rose hip extract, adding 2 times of distilled water, dispersing uniformly, extracting for 3 times by using petroleum ether (boiling range is 60-90 ℃), ethyl acetate and water saturated n-butyl alcohol which are equal in volume respectively, combining extraction liquids, concentrating the extraction liquids under reduced pressure respectively until no alcohol smell exists, and performing vacuum spray drying to obtain petroleum ether extract, ethyl acetate extract, n-butyl alcohol extract and water parts;

(2) separation: taking a proper amount of the n-butanol extract of the rosa davurica pall, carrying out dry sampling according to the mass ratio of the n-butanol extract to silica gel for column chromatography of 1:60, and carrying out methanol: eluting with ethyl acetate (1: 8-1: 64) in gradient, and detecting by thin layer chromatography, wherein the ratio of methanol: carrying out dry loading on the component A and silica gel for column chromatography in a mass ratio of 1:70, carrying out normal phase silica gel column chromatography for the second time, carrying out thin-layer chromatography detection, combining elution components of dichloromethane-ethyl acetate-methanol (4: 6:1, 2:6:1 and 1:6: 1) to obtain a component A1, carrying out dry loading on the component 1 and silica gel for column chromatography in a mass ratio of 1:100, carrying out normal phase silica gel column chromatography for the third time, carrying out thin-layer chromatography detection, combining elution components of dichloromethane-methanol (6: 1, 3:1 and 1: 1) to obtain a component A1-1, separating the component A1-1 by using a semi-preparative liquid chromatograph, carrying out methanol: eluting with water (1: 9) eluent, collecting fractions with retention time of 9.2min and 11.5min respectively, concentrating, crystallizing, filtering, and drying to obtain compound I and compound II.

Conditions for TLC detection according to the present invention: and (3) color developing agent a: observing fluorescence under an ultraviolet lamp (354nm, 360 nm); and (3) color developing agent b: iodine color development; and (3) a color developing agent c: 10% sulfuric acid ethanol.

And (3) structural identification: using modern wave spectrum techniques¹HNMR and¹³CNMR, physical and chemical Properties of the Compounds the isolated monomeric compounds were structurally characterized.

Compound I is a white solid, readily soluble in water, insoluble in methanol, chloroform, at m.p.142.2 ℃, and determined by spectroscopic techniques to be 6- ((hept-5-en-3-yl) oxy) -2- (methoxymethyl) tetrahydropyran-3, 4, 5-triol; the compound II is white solid, is easy to dissolve in water, is insoluble in solvents such as petroleum ether, methanol and the like, and is identified as 5-ethoxy-2-methoxymethyl-6- (1-methyl-butoxy) -tetrahydropyran-3, 4-diol by nuclear magnetism.

Nuclear magnetic data and structural identification: nuclear magnetic data for 6- ((hept-5-en-3-yl) oxy) -2- (methoxymethyl) tetrahydropyran-3, 4, 5-triol are as follows:

¹H NMR(D₂o) shows two sets of methyl hydrogen signals, respectively:δ0.952（t，-CH₃），1.245（d，-CH₃) (ii) a One set of methoxy hydrogen signals:δ2.743（s，-OCH₃) (ii) a Two sets of methylene hydrogen signals:δ1.788（m，-CH₂），1.743（m，-CH₂) (ii) a Two olefin hydrogen signals:δ5.108（m，=CH-），5.049（m，=CH-）；δ3.0-4.0 (m) is the proton signal on carbon attached to oxygen.¹³C NMR(D₂O) gives a total of 14 carbon signals,δ55.597（-OCH₃) A carbon signal that is methoxy;δ76.747（-CH₂OCH₃) Is the methylene carbon signal in methoxymethyl;δ14.012（-CH₃) Andδ17.132（-CH₃) Is two methyl carbon signals in hept-5-en-3-yloxy;δ28.066（-CH₂-) andδ41.827（-CH₂-) are the two methylene carbon signals in hept-5-en-3-yloxy;δ63.570 (-CH) is the methine carbon signal in hept-5-en-3-yloxy;δ132.780 (= CH-) andδ131.586 (= CH-) is two alkene carbon signals in hept-5-ene-3-yloxy;δ106.519 (C-6), 79.365 (C-5), 78.740 (C-3), 78.523 (C-4), 72.418 (C-2) are tetrahydropyran carbon signals;

nuclear magnetic data for 5-ethoxy-2-methoxymethyl-6- (1-methyl-butoxy) -tetrahydropyran-3, 4-diol are as follows:

¹H NMR(D₂o) shows three groups of methyl hydrogen signals, respectivelyδ0.914（t，-CH₃），1.242（t，-CH₃），1.324（d, -CH₃) (ii) a One set of methoxy hydrogen signals:δ3.147（s，-OCH₃) (ii) a A group of-CH₂-CH₂-hydrogen signal:δ0.938（m，-CH₂），0.925（m，-CH₂）；δ3.2-4.0 (m) is the proton signal on carbon attached to oxygen.¹³C NMR(D₂O) gives a total of 14 carbon signals,δ58.316（-OCH₃) Is the carbon signal of the methoxy group(s),δ78.552（-CH₂OCH₃) Is a methylene carbon signal attached to an oxygen,δ13.859（-CH₃) Andδ27.931（-CH₃) Two methyl carbon signals in 1-methyl-butoxy,δ22.928（-CH₂-) andδ44.493（-CH₂-) are the two methylene carbon signals in 1-methyl-butoxy,δ66.993 (-CH) is the oxygen-bonded methine carbon signal in 1-methyl-butoxy,δ17.011（-CH₃) Is the signal of the methyl carbon in the ethoxy group,δ63.453（-OCH₂CH₃) Is the signal of the methylene carbon in the ethoxy group,δ106.292（C-6），δ87.804（C-5），δ76.089（C-3），δ72.382（C-4），δ68.624（C-2）。

test for alpha-glucosidase inhibition

(1) Preparing a test solution: accurately weighing appropriate amounts of the compound I and the compound II, respectively placing in 10mL volumetric flasks, adding distilled water for dissolving, and fixing the volume to the scale to obtain the standard solution of the test sample. Sucking standard solutions of the test articles with different volumes, respectively placing the standard solutions in different volumetric flasks, adding distilled water to dilute the standard solutions to a scale, and obtaining test article solutions with different concentration gradients;

(2) the experimental method comprises the following steps: adding 80 μ L phosphate buffer solution (pH 6.8) with concentration of 0.1mmol/L and 20 μ L4-nitrobenzene- α -D galactopyranoside solution with concentration of 2.5mmol/L in sequence into a 96-well enzyme-labeled plate, adding 20 μ L sample solution with different concentration gradients, heating in 37 deg.C constant temperature water bath for 10min, adding 20 μ L α -glucosidase solution, heating in 37 deg.C constant temperature water bath for 1h, adding 100 μ L Na with concentration of 0.2mol/L₂CO₃Stopping the reaction of the solution, standing at room temperature for 15min, measuring the absorbance at 405nm by using an enzyme-labeling instrument, and respectively calculating the inhibition rate and IC (integrated Circuit) of the test solution with different concentrations on the alpha-glucosidase₅₀；

(3) As a result: experimental results show that the IC of the compound I and the compound II provided by the invention on the alpha-glucosidase inhibition rate₅₀0.255mg/mL and 0.561mg/mL, respectively, and IC of the inhibition rate of the control drug acarbose on alpha-glucosidase₅₀The content of the alpha-glucosidase in the compound I and the compound II is 10.000mg/mL, which shows that the in vitro inhibition capacity of the compound I and the compound II provided by the invention on the alpha-glucosidase is obviously stronger than that of acarbose.