Application and preparation method of rhizoma polygonati compound for inhibiting activity of alpha-glucosidase

Abstract

The invention discloses application and a preparation method of a polygonatum sibiricum compound for inhibiting alpha-glucosidase activity, and is characterized in that the preparation method comprises the following steps: S1, extraction: taking 21.97 kg of dried polygonatum sibiricum medicinal material, carrying out heating reflux extraction with 6, 5 and 5 times (M / M) of 95% ethanol for three times (3 hours each time), filtering with gauze, merging filtrates, and carrying out reduced pressure concentration at 60 DEG C to obtain 3.18 kg of extract; heating, refluxing and extracting decoction dregs twice by using 6-5 times (M / M) of 60% ethanol, 2 hours each time, filtering by using gauze, combining filtrates, and concentrating by using the same method to obtain 14.05 kg of extract; s2, extracting, namely dispersing the extract with water, sequentially extracting with petroleum ether, chloroform, ethyl acetate and n-butyl alcohol which are equal in volume, and extracting with 95% ethanol extract for three times. The invention relates to the technical field of GAA abnormal expression disease related drugs, the preparation method is simple and practical, good in reproducibility and high in extraction purity, and the natural alpha-glucosidase inhibitor is developed. The obtained compound has certain activity of inhibiting alpha-glucosidase.

Description

Technical Field

[0001] This invention relates to the field of drug technology for diseases related to abnormal GAA expression, specifically to the application of a Polygonatum compound that inhibits α-glucosidase activity and its preparation method. Background Technology

[0002] Currently, diabetes has become a common disease that seriously threatens human health. With the increasing number of patients, the types of drugs for treating diabetes are also constantly increasing. One type is alpha-glucosidase inhibitors, mainly used for patients who do not respond to other hypoglycemic drugs or those who require dietary control. However, long-term use can lead to increased bloating and, in severe cases, diarrhea. Therefore, developing natural alpha-glucosidase inhibitors is a necessary task with significant economic and social benefits.

[0003] Traditional Chinese medicine (TCM) has accumulated rich experience in treating diabetes. In TCM, diabetes falls under the category of "Xiao Ke Bing" (wasting and thirsting disease), and its occurrence is closely related to the spleen and kidneys. "The spleen is the foundation of acquired constitution and the source of qi and blood; the kidney is the foundation of innate constitution, storing primordial yin and yang." Treatment can be approached through three aspects: tonifying qi and strengthening the spleen to dispel dampness, nourishing the yin and yang of the kidneys, and treating both the spleen and kidneys simultaneously. Therefore, it is feasible to discover substances that regulate α-glucosidase activity from Chinese herbs with the effects of "strengthening the spleen and benefiting the kidneys" and "tonifying qi and nourishing yin." Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an application of a Polygonatum compound that inhibits α-glucosidase activity and its preparation method, which solves the problem that long-term use of existing α-glucosidase inhibitors can lead to increased gastrointestinal bloating and, in severe cases, diarrhea.

[0005] To achieve the above objectives, the present invention provides the following technical solution: an application of a Polygonatum compound that inhibits α-glucosidase activity and its preparation method, comprising the following preparation method:

[0006] S1: Extraction: Take 21.97 kg of dried Polygonatum sibiricum and extract it three times by heating and reflux with 6, 5, and 5 times the amount (M / M) of 95% ethanol, for 3 hours each time. Filter with gauze, combine the filtrates, and concentrate under reduced pressure at 60℃ to obtain 3.18 kg of extract. The residue is then extracted twice more by heating and reflux with 6 and 5 times the amount (M / M) of 60% ethanol, for 2 hours each time. Filter with gauze, combine the filtrates, and concentrate in the same way to obtain 14.05 kg of extract.

[0007] S2: Extraction: Take the aqueous dispersion of the extract and extract it sequentially with equal volumes of petroleum ether, chloroform, ethyl acetate, and n-butanol. Extract the 95% ethanol extract three times and the 60% ethanol extract twice. Combine the extracts and recover the solvent under reduced pressure at 60°C to obtain the extracted fractions.

[0008] S3: separation, combine the chloroform and ethyl acetate layer extraction parts, dissolve properly, mix sample; blank column chromatography silica gel 110 g, chloroform column, sample, chloroform-methanol system gradient elution (volume ratio 100:0.5 to 100:100), each flow 300 ml, each gradient at least 10 flow, a total of 236 flow;

[0009] S4: purification, using liquid chromatography and thin layer chromatography analysis, combine similar flow, a total of A ~ L group; C group (100:1.5, Fr. 35-57, 5.5 g) with ODS open column chromatography with methanol-water gradient elution (0%-100%), take the 2 / 3 flow of 40% methanol elution (P2-C-40-2, 0.17 g) with semi-preparative liquid chromatography purification, with methanol water (40%, tR=38.5 min) as the mobile phase to separate compound 1; take the 1st flow of 60% methanol elution (P2-C-60-1, 0.60 g) with semi-preparative liquid chromatography purification, with acetonitrile water (30.8%, tR=115.2 min) as the mobile phase to separate compound 2, further using reverse phase HPLC column methanol: acetonitrile 4:1-water (70%, tR=10.0, 10.7, 11.6 and 9.1 min) to obtain compounds 2a-2d.

[0010] Preferably: the compound 1 is a light yellow powder, easily soluble in methanol, high resolution mass spectrum gives quasi-molecular ion peak m / z 301.0723 [M-H]-, combined with 1 H-NMR spectrum and 13 C-NMR spectrum, determine the molecular formula of the compound as C 16 H 14 O6; the compound 2 is a light white powder, easily soluble in methanol, high resolution mass spectrum gives quasi-molecular ion peak m / z 354.1712 [M-H]-, combined with 1 H-NMR spectrum and 13 C-NMR spectrum, determine the molecular formula of the compound as C 21 H 25 NO4.

[0011] Huangji: Huangji is the dried rhizome of Polygonatum sibiricum, sweet, flat. Spleen, lung, kidney. Has the effect of spleen and kidney, tonic, nourishing yin, moistening the lungs. For internal heat and diabetes, spleen and stomach deficiency, body fatigue, stomach yin deficiency, dry mouth, lung deficiency, dry cough, coughing blood, lack of essence and blood, soreness of the knees and waist, premature graying. It is feasible to find α-glucosidase inhibitors from Huangji with the effect of spleen and kidney.

[0012] Beneficial effects

[0013] The present application provides a kind of inhibition of alpha-glucosidase activity of polygonatum compound application and its preparation method, with the following beneficial effects:

[0014] 1, the preparation method is simple and practical, and has good reproducibility, high extraction purity, and develops natural alpha-glucosidase inhibitor. The obtained compound has certain inhibition activity of alpha-glucosidase. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is the structure of compound 1 of the present application.

[0016] Figure 2 It is the structure of compound 2 of the present application.

[0017] Figure 3 It is the structure of compound 2a-2d of the present application.

[0018] Figure 4 It is the H-NMR (600MHz, CDOD3) spectrum of compound 1 of the present application. 1

[0019] Figure 5 It is the C-NMR (150MHz, CDOD3) spectrum of compound 1 of the present application. 13

[0020] Figure 6 It is the HMBC spectrum of compound 1 of the present application.

[0021] Figure 7 It is the CD spectrum of compound 1 of the present application.

[0022] Figure 8 It is the H-NMR (600MHz, CDOD3) spectrum of compound 2 of the present application. 1

[0023] Figure 9 It is the C-NMR (150MHz, CDOD3) spectrum of compound 2 of the present application. 13

[0024] Figure 10 It is the HMBC spectrum of compound 2 of the present application.

[0025] Figure 11 It is the HPLC spectrum of compound 2 of the present application.

[0026] Figure 12 It is the H-NMR (600MHz, CDOD3) spectrum of compound 2a of the present application. 1

[0027] Figure 13 It is the C-NMR (150MHz, CDOD3) spectrum of compound 2b of the present application. 1 ​​​​​H-NMR (600 MHz, CDOD3) spectrum.

[0028] Figure 14 Compound 2c of the present application is 1 H-NMR (600 MHz, CDOD3) spectrum.

[0029] Figure 15 Compound 2d of the present application is 1 H-NMR (600 MHz, CDOD3) spectrum. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0031] Please refer to Figures 1-15 The present application provides a technical solution: a polygonatum compound for inhibiting α-glucosidase activity and a preparation method thereof, which comprises the following preparation method.

[0032] S1: extraction, taking 21.97 kg of dried polygonatum medicinal materials, reflux extraction with 6, 5, 5 times (M / M) of 95% ethanol for three times, each time for 3 h, filtering with gauze, combining the filtrates, and concentrating at 60°C under reduced pressure to obtain 3.18 kg of extract; the residue is extracted with 6, 5 times (M / M) of 60% ethanol for two times, each time for 2 h, filtering with gauze, combining the filtrates, and concentrating in the same way to obtain 14.05 kg of extract;

[0033] S2: extraction, taking the extract and dispersing in water, extracting with equal volume of petroleum ether, chloroform, ethyl acetate, and n-butanol in turn, extracting the 95% ethanol extract for three times, and extracting the 60% ethanol extract for two times, respectively combining the extractives, and recovering the solvent at 60°C under reduced pressure to obtain each extract part;

[0034] S3: separation, combining the chloroform and ethyl acetate layer extract parts, appropriately dissolving, and stirring the sample; taking 110 g of silica gel for blank column chromatography, loading the column with chloroform, and loading the sample, gradient elution (volume ratio 100:0.5 to 100:100) with chloroform-methanol system, each flow 300 ml, at least 10 flow fractions for each gradient, and a total of 236 flow fractions;

[0035] S4: Purification, using liquid chromatography and thin layer chromatography analysis, combining similar fractions, total A~L groups; C group (100:1.5, Fr. 35-57, 5.5 g) was purified by ODS open column chromatography with methanol-water gradient elution (0%-100%), the 2 / 3 fraction eluted by 40% methanol (P2-C-40-2, 0.17 g) was purified by semi-preparative liquid chromatography, separated by methanol water (40%, tR=38.5 min) as mobile phase to obtain compound 1; the 1st fraction eluted by 60% methanol (P2-C-60-1, 0.60 g) was purified by semi-preparative liquid chromatography, separated by acetonitrile water (30.8%, tR=115.2 min) as mobile phase to obtain compound 2, further purified by reversed-phase HPLC column with methanol:acetonitrile 4:1-water (70%, tR=10.0, 10.7, 11.6 and 9.1 min) to obtain compounds 2a-2d.

[0036] Further: the compound 1 is light yellow powder, easily soluble in methanol, high resolution mass spectrum gives quasi-molecular ion peak m / z 301.0723 [M-H]-, combined with 1 H-NMR spectrum and 13 C-NMR spectrum, determine the molecular formula of the compound as C 16 H 14 O6; the compound 2 is light white powder, easily soluble in methanol, high resolution mass spectrum gives quasi-molecular ion peak m / z 354.1712 [M-H]-, combined with 1 H-NMR spectrum and 13 C-NMR spectrum, determine the molecular formula of the compound as C 21 H 25 NO4;

[0037] In the compound 1 1 H / 13 C-NMR spectrum (600 / 150MHz, CDOD3), the typical proton and carbon signals of dihydroflavonol 2 and 3 can be observed: (δH 4.53, 4.98, each d, J=11.4, 12.0 Hz, H-3, 2; δC 73.7, 84.7, C-3, 2); the aromatic proton signal δH 5.99, s, H-6 suggests that A ring has five substituents, the methyl carbon signal δ C 7.5 means direct connection with benzene ring. HMBC spectrum can see H-8-CH3(δ H 1.92) and C-8, 9, and 7 (δ C:104.8, 161.3, and 166.7) strongly correlated, suggesting the methyl group is located at the 8-position. In combination with other carbon hydrogen signals and CD curves, compound 1 is (2R, 3R)-5,7,4'-trihydroxy-8-methylflavanonol, named 8-methylaromadendrin;

[0038] Compound 2 of 1 H / 13 In the C-NMR (600 / 150 MHz, CDOD3) spectrum, the butyl protons and carbon signals can be seen in the high field region, δ H 3.26-3.36, 1.53, 1.35, and 0.87 (H-1"-4"), and δ C 69.5, 33.0, 20.4, and 14.2 (C-1"-4"). The p-coumaroyl fragment signals can be seen at δ H 7.44, 6.43, each d, each 15.8, H-7', 8'; δ H 7.40, 6.78, each d, each 8.6, H-2' / 6', 3' / 5'; and δ C 169.3, C-9'; δ C 141.9, 118.4, C-7', 8'; δ C 160.5, 130.6, 127.8, and 116.7, C-4', 2' / 6', 1', 3' / 5'. The tyramine derivative structure signals can be seen at δ H 3.39, 3.47, each dd, H a / b -8; δ H 4.34, dd, J = 8.3, 4.8 Hz, H-7; δ H 7.16, 6.77, each d, each 8.5, H-2 / 6, 3 / 5; and δ C 47.3, C-8; δ C 81.5, C-7; and δ C 158.4, 132.3, 129.1, 116.2, C-4, 1, 2 / 6, 3 / 5. The HMBC spectrum can see H-1" (δ H 3.26-3.36) and C-3", 2", 7 (δ C : 20.4, 33.0, 81.5) and H-7 (δ H 4.34) and C-8, 1", 2 / 6, 1 (δ C47.3, 69.5, 129.1, 132.3 long-range correlation, suggesting butyl group is in 7-position, and the trans structure of the compound is determined by other data.

[0039] In addition, its cis structure can be seen in its hydrogen spectrum and carbon spectrum. The optical rotation test result is zero, suggesting a racemic mixture; further separation by chiral chromatographic column into compounds 2a-2d, the absolute configuration is determined by analyzing the characteristics of hydrogen spectrum and circular dichroism spectrum, respectively identified as (7R)-N-trans-p-coumaroyl-7-butoxytyramine, (7S)-N-trans-p-coumaroyl-7-butoxytyramine, (7R)-N-cis-p-coumaroyl-7-butoxytyramine, and (7S)-N-cis-p-coumaroyl-7-butoxytyramine.

[0040] Activity test

[0041] (1) Test materials: purified water, α-glucosidase (10 U / mg), PNPG (substrate), anhydrous sodium carbonate, DMSO, PBS (pH 6.8, 2.5 mM).

[0042] (2) Preparation of test solution

[0043] Positive control acarbose: weigh 8.09 mg, dissolve in DMSO, then dilute to different concentrations in a step-by-step manner with a 2-fold decrease.

[0044] Preparation of test compound solution: weigh 0.32 mg and 44 mg of compounds 1 and 2 respectively, dissolve in DMSO, then dilute to different concentrations in a step-by-step manner with a 2-fold decrease.

[0045] Preparation of enzyme solution: weigh 0.50 mg of enzyme, dissolve in 1 ml of PBS to make a stock solution, mix well, aliquot, and store at -20°C. Before use, dilute with PBS to 0.3 U / ml of working solution.

[0046] Preparation of substrate solution: weigh 11.30 mg of substrate, dissolve in PBS to prepare a 2.5 mM solution, mix well, and store in the refrigerator.

[0047] Preparation of termination solution: weigh 852.00 mg of anhydrous sodium carbonate, dissolve in water to prepare a 200 mM solution, mix well, and obtain.

[0048] Activity test method

[0049] Add each test solution in the order shown in the table in a 96-well plate. After the reaction is complete, measure the absorbance at 405 nm on the enzyme marker. The blank group should be prepared in the same way.

[0050]

[0051]

[0052] Inhibition rate (%) = (ΔADMSO- ΔA sample) / ΔADMSO x 100%

[0053] Test results:

[0054] IC of acarbose 50 is 472.2 μM, IC of compound 1 50 is 396.2 μM, IC of compound 2 50 is 611.2 μM.

[0055] The above results show that compounds 1-2 have certain inhibitory effect on α-glucosidase and can be used for related diseases of type II diabetes.

[0056] The materials in the case are processed by those skilled in the art, and the specific processing process and operation sequence should be referred to the following working principles, and the detailed connection means are the commonly known technologies in the art, and the following mainly introduces the working principles and processes.

[0057] Example: in the preparation process:

[0058] Firstly, 21.97 kg of dried Huangjing medicinal material is refluxed with 6, 5, 5 times (M / M) of 95% ethanol for three times, 3 h each time, filtered with gauze, combined the filtrate, concentrated at 60°C under reduced pressure to obtain 3.18 kg of extract; the residue is refluxed with 6, 5 times (M / M) of 60% ethanol for two times, 2 h each time, filtered with gauze, combined the filtrate, concentrated in the same way to obtain 14.05 kg of extract;

[0059] Then, the extract is dispersed with water, extracted with equal volume of petroleum ether, chloroform, ethyl acetate and n-butanol in turn, the 95% ethanol extract is extracted three times, and the 60% ethanol extract is extracted two times, and the extracted liquids are combined respectively, and the solvent is recovered at 60°C under reduced pressure to obtain each extract part;

[0060] Then, the chloroform and ethyl acetate layer extract parts are combined, appropriately dissolved and mixed; 110 g of silica gel is used for blank column chromatography, 110 g of silica gel is used for blank column chromatography, chloroform is loaded into the column, the sample is loaded, chloroform-methanol system gradient elution (volume ratio 100:0.5 to 100:100), 300 ml each flow, at least 10 flow fractions for each gradient, a total of 236 flow fractions;

[0061] Finally, the fractions were analyzed by HPLC and TLC and similar fractions were combined into groups A to L; group C (100:1.5, Fr. 35-57, 5.5 g) was purified by ODS open column chromatography eluting with a gradient of methanol-water (0% to 100%) and the second third fraction eluting with 40% methanol (P2-C-40-2, 0.17 g) was purified by semi-preparative HPLC using methanol-water (40%, tR= 38.5 min) as mobile phase to give compound 1; the first fraction eluting with 60% methanol (P2-C-60-1, 0.60 g) was purified by semi-preparative HPLC using acetonitrile-water (30.8%, tR= 115.2 min) as mobile phase to give compound 2, which was further purified by reverse phase chiral column using methanol:acetonitrile 4:1-water (70%, tR= 10.0, 10.7, 11.6 and 9.1 min) to give compounds 2a-2d.

[0062] It has to be noted that, as used herein, the terms "first", "second", etc. do not imply any physical or logical relationship between the elements denoted by these terms. Furthermore, the terms "comprise", "comprising", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus. In other words, the terms "comprise", "comprising", or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0063] While the embodiments of the application have been shown and described herein, it is understood that various modifications, substitutions, combinations, and alterations can be made to the embodiments without departing from the spirit and scope of the application, which is defined by the following claims and their equivalents.

Claims

1. An application of a Polygonatum compound that inhibits α-glucosidase activity and its preparation method, characterized in that, The preparation methods include the following: S1: Extraction: Take 21.97 kg of dried Polygonatum sibiricum and extract it three times by heating and reflux with 6, 5, and 5 times the amount (M / M) of 95% ethanol, for 3 hours each time. Filter with gauze, combine the filtrates, and concentrate under reduced pressure at 60℃ to obtain 3.18 kg of extract. The residue is then extracted twice more by heating and reflux with 6 and 5 times the amount (M / M) of 60% ethanol, for 2 hours each time. Filter with gauze, combine the filtrates, and concentrate in the same way to obtain 14.05 kg of extract. S2: Extraction: Take the aqueous dispersion of the extract and extract it sequentially with equal volumes of petroleum ether, chloroform, ethyl acetate, and n-butanol. Extract the 95% ethanol extract three times and the 60% ethanol extract twice. Combine the extracts and recover the solvent under reduced pressure at 60°C to obtain the extracted fractions. S3: Separate, combine the extracts from the chloroform and ethyl acetate layers, dissolve appropriately, and mix; for blank column chromatography, use 110g of silica gel, pack the column with chloroform, load the sample, and perform gradient elution with a chloroform-methanol system (volume ratio 100:0.5 to 100:100), 300ml per fraction, at least 10 fractions per gradient, for a total of 236 fractions. S4: Purification was performed using liquid chromatography and thin-layer chromatography. Similar fractions were combined and divided into groups A to L. Group C (100:1.5, Fr. 35-57, 5.5g) was eluted with a methanol-water gradient (0%-100%) using ODS open column chromatography. The second / third fraction eluted with 40% methanol (P2-C-40-2, 0.17g) was purified by semi-preparative liquid chromatography with methanol-water (40%, tR = 38.5 min). Compound 1 was obtained by separation using methanol as the mobile phase; the first fraction (P2-C-60-1, 0.60 g) eluted with 60% methanol was purified by semi-preparative liquid chromatography and separated using acetonitrile-water (30.8%, tR = 115.2 min) as the mobile phase to obtain compound 2. Compound 2 was further obtained by separation using a reversed-phase chiral column with methanol:acetonitrile 4:1-water (70%, tR = 10.0, 10.7, 11.6 and 9.1 min) to obtain compounds 2a-2d.

2. The method for preparing a Polygonatum compound that protects α-glucosidase activity according to claim 1, characterized in that: Compound 1 is a pale yellow powder, readily soluble in methanol. High-resolution mass spectrometry yielded a quasi-molecular ion peak at m / z 301.0723 [MH]-. 1 H-NMR spectrum and 13 C10-NMR spectroscopy determined the molecular formula of the compound to be C10-C20. 16 H 14 O6; Compound 2 is a pale white powder, readily soluble in methanol, and high-resolution mass spectrometry yields a quasi-molecular ion peak at m / z 354.1712 [MH]-. 1 H-NMR spectrum and 13 C10-NMR spectroscopy determined the molecular formula of the compound to be C10-C20. 21 H 25 NO4.

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