CN106565444A - Extraction method and application of phenanthrene compounds from overground part of Chinese yam - Google Patents

Abstract

The invention discloses an extraction method and application of phenanthrene compounds in the aerial part of yam and belongs to the technical field of separation and application of effective components in the aerial part of yam. The key points of the technical scheme of the present invention are: the coarse powder of the aboveground part of yam is extracted and concentrated by ethanol percolation, the extract is extracted with an organic solvent, and then separated by column chromatography such as macroporous adsorption resin, silica gel, dextran sugar gel, etc. to obtain phenanthrene class of compounds. The four phenanthrene compounds extracted from the aerial part of the yam of the present invention all have significant inhibitory activity on a-glucosidase, and are stronger than the activity of the hypoglycemic drug acarbose under the same conditions.

Description

Extraction method and application of phenanthrene compounds in aerial parts of yam

technical field

The invention belongs to the technical field of separation and application of effective components in aerial parts of yams, and in particular relates to an extraction method and application of phenanthrene compounds in aerial parts of yams.

Background technique

Due to many factors such as the improvement of people's living standards, changes in diet structure, and less active and more sedentary lifestyle, the incidence of diabetes worldwide has increased rapidly. Diabetes has become the third chronic disease that seriously threatens human health after tumors and cardiovascular diseases. . The first symptom in the pathogenesis of diabetes is often postprandial hyperglycemia, which is the early sign of diabetes, especially for patients with type Ⅱ diabetes. Postprandial hyperglycemia not only easily induces various complications, but also greatly increases the risk of diabetes. mortality rate. Therefore, reducing postprandial blood sugar is one of the important measures to prevent diabetes, reduce complications and reduce mortality. α-glucosidase is a key enzyme in the carbohydrate digestion process. It can hydrolyze polysaccharides or disaccharides into monosaccharides that can be absorbed by the human body. Therefore, inhibiting the activity of α-glucosidase can delay the digestion of carbohydrates in food. Decomposition, thereby reducing the intake of glucose, to achieve the purpose of inhibiting postprandial blood sugar rise. Currently clinical drugs of this type include acarbose, voglibose, and miglitol. There are few kinds of α-glucosidase inhibitors available and they are accompanied by intestinal side effects, so people are still working on the development of new α-glucosidase inhibitors.

Yam ( Disocorea opposita Thunb.) is the dry rhizome of Dioscorea yam, which has the effects of nourishing the spleen and lungs, strengthening the kidney and benefiting the essence. It is widely used clinically to treat symptoms such as diabetes, chronic diarrhea and consumptive cough. It is one of the traditional Chinese medicines with the same source of medicine and food. Since ancient times, Jiaozuo, Henan (ancient Huaiqing Prefecture) is the most authentic, so it is called "Huaiyam", and it is one of the famous "Four Huaiyao". In 2006, Wuzhi County, Henan Province was approved as a yam GAP planting base, which solved the shortage of wild yam resources and the sustainable utilization and development of resources. Chinese yam uses underground rhizomes as medicine, and the aboveground parts are often discarded as waste, resulting in a great waste of resources. At present, the research on yam mainly focuses on the underground rhizome (medicinal part), pharmacological studies have shown that it has hypoglycemic, hypolipidemic, antioxidative and antitumor activities. However, there are very few studies on the aerial part of yam, and there is no research report on its ability to lower blood sugar.

Contents of the invention

The technical problem to be solved by the present invention is to provide an extraction method and application of phenanthrene compounds in the aerial part of yam.

The present invention adopts following technical scheme for solving the above-mentioned technical problem, the extracting method of phenanthrene compound of yam aerial part, is characterized in that concrete steps are:

(1) After crushing the above-ground part of yam dried in the shade, extract by percolation with ethanol with a volume fraction of 90%-95%, combine the extracts, concentrate under reduced pressure to obtain the ethanol extract of the above-ground part of yam, and then suspend the ethanol extract in In distilled water, extract with petroleum ether and ethyl acetate successively, collect the ethyl acetate extract and concentrate under reduced pressure to obtain the ethyl acetate extract, the ethyl acetate extract is subjected to macroporous adsorption resin column chromatography, and the volume fraction is 60% ethanol, ethanol with a volume fraction of 80% and ethanol with a volume fraction of 95% were eluted, and the ethanol eluate with a volume fraction of 80% and the ethanol eluate with a volume fraction of 95% were concentrated under reduced pressure to obtain 80% ethanol elution product and 95% ethanol elution product;

(2) The product eluted with 80% ethanol is subjected to normal pressure silica gel column chromatography, and the petroleum ether-acetone solvent system is used in the volume ratio of 5:1-1:1 and the methylene chloride-methanol solvent system is used in the volume ratio of Gradient elution within the range of 2:1-1:1, identified by TLC and combined with the same components to obtain the initial target component, the initial target component was subjected to normal pressure silica gel column chromatography, and dichloromethane-acetone solvent system was used in volume ratio Gradient elution in the range of 50:1-10:1 respectively obtains compound 1, compound 4 and intermediate target components, and then recrystallizes the intermediate target components to obtain compound 2, wherein the structural formula of compound 1 is , the structural formula of compound 2 is , the structural formula of compound 4 is ;

(3) The product eluted with 95% ethanol is subjected to normal pressure silica gel column chromatography, and the petroleum ether-acetone solvent system is used in the volume ratio of 5:1-1:1 and the dichloromethane-methanol solvent system is in the volume ratio of Gradient elution within the range of 2:1-1:1, identified by TLC and combined with the same components to obtain the initial target component, the initial target component was subjected to normal pressure silica gel column chromatography, and dichloromethane-acetone solvent system was used in volume ratio Gradient elution in the range of 100:0-20:1, identified by TLC and combined with the same components to obtain the intermediate target component, the intermediate target component was purified by Sephadex column chromatography and semi-preparative HPLC to obtain compound 3, the The structural formula of compound 3 is , wherein the eluent of Sephadex column chromatography is methanol, and the mobile phase of semi-preparative HPLC is a methanol-water mixed solution with a volume ratio of 75:25.

Further preferably, the macroporous adsorption resin is Diaion HP-20 type macroporous adsorption resin, and the dextran gel is Sephadex LH-20 type dextran gel.

Application of the phenanthrene compounds from the aerial parts of yam extracted according to the above method in the preparation of hypoglycemic drugs or health care products.

Application of the aerial part phenanthrene compounds of Chinese yam extracted according to the above method in the preparation of α-glucosidase inhibitor drugs or health care products.

Application of compound 4 extracted according to the above method in the preparation of α-glucosidase inhibitor drugs or health care products.

In the present invention, through research on the inhibitory activity of α-glucosidase in vitro, it is found that the four phenanthrene compounds extracted from the aerial part of Chinese yam all have significant inhibitory effects on α-glucosidase, and their _IC50 are 3.04±0.25mM and 0.49±0.034mM respectively , 4.13±0.023mM and 0.07±0.0031mM, significantly stronger than the activity of the positive control drug acarbose (IC ₅₀ is 61.57±1.40mM), and the inhibition rate of α-glucosidase is dose-dependent.

Description of drawings

Fig. 1 is the variation curve of the compound 1 that embodiment 1 extracts to α-glucosidase inhibitory rate with drug concentration;

Fig. 2 is the compound 2 that embodiment 2 extracts to the change curve of α-glucosidase inhibitory rate with drug concentration;

Fig. 3 is the variation curve of the compound 3 that embodiment 3 extracts to α-glucosidase inhibitory rate with drug concentration;

Fig. 4 is the variation curve of compound 4 extracted in Example 1 on the inhibitory rate of α-glucosidase with drug concentration.

detailed description

The above-mentioned contents of the present invention are described in further detail below through the embodiments, but this should not be interpreted as the scope of the above-mentioned themes of the present invention being limited to the following embodiments, and all technologies realized based on the above-mentioned contents of the present invention all belong to the scope of the present invention.

Example 1

Shade-dried 8.5kg of aerial part of yam, crushed, extracted by percolation with ethanol with a volume fraction of 95%, combined the extracts, concentrated under reduced pressure to obtain 871.5g of total extract, suspended the extract in 2 times the amount of water, and washed with 5 Double the amount of petroleum ether and ethyl acetate for extraction, the ethyl acetate extract was collected and concentrated under reduced pressure to obtain 100.2 g of ethyl acetate extract. The ethyl acetate extract was subjected to Diaion HP-20 type macroporous adsorption resin column chromatography, eluted with 60% ethanol and 80% ethanol in sequence, and collected 80% ethanol for elution. liquid and concentrated under reduced pressure to obtain 45.7 g of 80% ethanol-eluted product. The product was eluted with 80% ethanol and subjected to normal pressure silica gel column chromatography, using petroleum ether-acetone (volume ratios of 5:1, 3:1, 2:1 and 1:1) and dichloromethane-methanol (volume ratios of 2:1 and 1:1) solvent system elution, identified by TLC and combined the same components to obtain 10 components (Fr.1-Fr.10). Fr.4 Normal pressure silica gel column chromatography, eluting with dichloromethane-acetone (50:1, 30:1, 20:1 and 10:1 by volume) solvent system, respectively, to obtain compound 1 (2.6mg) and compound 4 (2.1 mg).

Spectral data of compounds 1 and 4: 7-hydroxy-2,6-dimethoxy-1,4-phenanthrenedione (1): red amorphous powder, ESI-MS m/z 307 [M+Na] ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 10.35(1H, s, 7-OH), 9.01 (1H, s, H-5), 8.03 (1H, d, J = 8.5 Hz, H- 10), 7.87 (1H,d, J = 8.5 Hz, H-9), 7.26 (1H, s, H-8), 6.24 (1H, s, H-3), 3.96 (3H, s, 6-OCH ₃ ), 3.86 (3H, s, 2-OCH ₃ ); ¹³ C-NMR (400 MHz, DMSO- d ₆ ) δ: 188.6 (C-4), 180.5(C-1), 158.4 (C-2) , 152.5 (C-6), 149.6 (C-7), 134.4 (C-8a), 131.7 (C-9), 128.7 (C-10a), 124.9 (C-4b), 124.8 (C-4a), 120.0 (C-10), 111.3 (C-3), 110.3 (C-8), 106.1 (C-5), 56.4 (2-OCH ₃ ), 55.5 (6-OCH ₃ ).

Volucrin (4): Pale yellow amorphous powder, HRESI-MS m/z 561.1520 [M+Na] ⁺ (calcdfor C ₃₂ H ₂₆ O ₈ Na, 561.1525. ¹ H-NMR (400 MHz, acetone- d ₆ ) δ : 9.28 (2H, s, H-5, 5'),7.29 (2H, d, J = 9.1 Hz, H-10, 10'), 6.99 (2H, d, J = 9.1 Hz, H-9, 9 '), 6.92(2H, d, J = 2.5 Hz, H-1, 1'), 6.81 (2H, d, J = 2.5 Hz, H-3, 3'), 4.16 (6H, s,4,4 ¢-OCH ₃ ), 3.90 (6H, s, 2,2¢-OCH ₃ ).

Example 2

Shade-dried 8.5kg of aerial part of yam, crushed, extracted by percolation with ethanol with a volume fraction of 95%, combined the extracts, concentrated under reduced pressure to obtain 871.5g of total extract, suspended the extract in 2 times the amount of water, and washed with 5 Double the amount of petroleum ether and ethyl acetate for extraction, the ethyl acetate extract was collected and concentrated under reduced pressure to obtain 100.2 g of ethyl acetate extract. The ethyl acetate extract was subjected to Diaion HP-20 type macroporous adsorption resin column chromatography, eluted with 60% ethanol and 80% ethanol in sequence, and collected 80% ethanol for elution. liquid and concentrated under reduced pressure to obtain 45.7 g of 80% ethanol-eluted product. The product was eluted with 80% ethanol and subjected to normal pressure silica gel column chromatography, using petroleum ether-acetone (volume ratios of 5:1, 3:1, 2:1 and 1:1) and dichloromethane-methanol (volume ratios of 2:1 and 1:1) solvent system elution, identified by TLC and combined the same components to obtain 10 components (Fr.1-Fr.10). Fr.8 was recrystallized to give Compound 2 (7.0 mg).

Spectrum data of compound 2: 6,7-dihydroxy-2-methoxy-1,4-phenanthrenedione (2): red amorphous powder, ESI-MS m/z 293 [M+Na] ⁺ . ¹ H-NMR (400 MHz, DMSO- d ₆ ) δ: 8.93 (1H, s, H-5), 7.95 (1H, d, J = 8.4Hz, H-9), 7.78 (1H, d, J = 8.4Hz, H-10), 7.20(1H, s, H-8), 6.21 (1H, s, H-3), 3.84 (3H, s, 2-OCH ₃ ).

Example 3

Shade-dried 8.5kg of aerial part of yam, crushed, extracted by percolation with ethanol with a volume fraction of 95%, combined the extracts, concentrated under reduced pressure to obtain 871.5g of total extract, suspended the extract in 2 times the amount of water, and washed with 5 Double the amount of petroleum ether and ethyl acetate for extraction, the ethyl acetate extract was collected and concentrated under reduced pressure to obtain 100.2 g of ethyl acetate extract. The ethyl acetate extract was subjected to Diaion HP-20 type macroporous adsorption resin column chromatography, eluted with 80% ethanol and 95% ethanol in sequence, and collected 95% ethanol for elution. liquid and concentrated under reduced pressure to obtain 25.0 g of 95% ethanol-eluted product. The product was eluted with 95% ethanol and subjected to normal pressure silica gel column chromatography, using petroleum ether-acetone (volume ratios of 5:1, 3:1, 2:1 and 1:1) and dichloromethane-methanol (volume ratios of 2:1 and 1:1) solvent system elution, identified by TLC and combining the same components to obtain 7 components (Fr.1-Fr.7). Fr.2 Normal pressure silica gel column chromatography, eluted with dichloromethane-acetone (100:0, 50:1 and 20:1 by volume ratio) solvent system, identified by TLC and combined the same components to obtain 6 components (Fr.2.1-Fr.2.6), Fr.2.6 was purified by Sephadex LH-20 Sephadex column chromatography (elution with methanol) and semi-preparative HPLC (mobile phase: methanol-water, volume ratio 75:25) Compound 3 (2.4 mg).

Spectrum data of compound 3: 7-hydroxy-2,4,6-trimethoxyphenanthrene (3): white powder, ESI-MS m/z 307 [M+Na] ⁺ . ¹ H-NMR (400 MHz, acetone- d ₆ ) δ: 9.04 (1H, s, H-5), 7.66 (1H, d, J = 8.8 Hz, H-9), 7.52 (1H, d, J = 8.8 Hz, H-10), 7.37 (1H, s, H-8), 6.98 (1H, d, J = 2.5 Hz, H-1), 6.78 (1H, d, J = 2.5 Hz, H-3) , 4.11 (3H,s, 2-OCH ₃ ), 3.99 (3H, s, 6-OCH ₃ ), 3.92 (3H, s, 4-OCH ₃ ); ¹³ C-NMR (100 MHz,acetone- d ₆ ) δ: 160.4 (C-2), 158.7 (C-4), 147.6 (C-6), 147.3 (C-7), 135.9 (C-10a), 128.6 (C-9), 127.7 (C-4b) , 126.5 (C-8a), 125.2 (C-10), 116.2 (C-4a), 113.5 (C-8), 109.2 (C-5), 102.2 (C-1), 99.7 (C-3), 56.1 (6, 4-OCH ₃ ), 55.7 (2-OCH ₃ ).

Example 4

This example is a test of the inhibitory activity of compounds 1-4 extracted from the above examples on α-glucosidase.

Method: microplate method.

Instruments: microplate reader, constant temperature incubator, analytical balance, various types of pipette guns.

Reagents: α-glucosidase, 4-nitrophenyl-α-D-glucopyranoside, acarbose, phosphate buffer, and dimethylsulfoxide.

Test method: Take a 96-well plate, add 80 μL of 2.0 mmol/L PNPG solution (dissolved in PBS) to each well, and then add 10 μL of sample solutions of different concentrations (dissolved in DMSO), use DMSO as the blank control group, and set 3 parallels in each group Wells, the absorbance (A value) of the background group was measured at 400 nm in a microplate reader. Then add 10 μL 1.62U/mL α-glucosidase solution (dissolved in PBS), place in a constant temperature incubator, and incubate at 37°C for 30 minutes, and measure the absorbance (A value) of the test group. Inhibition rate (%) was calculated with the following formula:

Inhibition rate (%) = (ΔA _blank - ΔA _sample ) / ΔA _blank × 100%

ΔA _blank is the A value after the blank group enzyme reacts with the substrate - the background A value;

ΔA _sample is the A value after the reaction of the sample group enzyme with the substrate - the background A value.

The results of the inhibitory activity of compounds 1-4 on α-glucosidase are as follows (Table 1):

Table 1 Inhibitory activity of compounds 1-4 on α-glucosidase

compound _IC50 (mM) 1 3.04±0.25 2 0.49±0.034 3 4.13±0.023 4 0.07±0.0031 Acarbose 61.57±1.40

As can be seen from Table 1, the inhibitory activity of compounds 1-4 extracted in Examples 1-3 to α-glucosidase is stronger than that of the positive control drug acarbose, and the activity of compound 4 is the strongest, far superior to The activity of the positive control drug acarbose.

The above embodiments have described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What are described in the above embodiments and description are only to illustrate the principles of the present invention. Without departing from the scope of the principle of the present invention, there will be various changes and improvements in the present invention, and these changes and improvements all fall within the protection scope of the present invention.

Claims (5)

1. the extracting method of Rhizoma Dioscoreae aerial partss phenanthrene class compound, it is characterised in that concretely comprise the following steps：

（1）After the Rhizoma Dioscoreae aerial partss for drying in the shade are crushed, with the ethanol percolate extraction that volume fraction is 90%-95%, united extraction Liquid, is concentrated under reduced pressure to give Rhizoma Dioscoreae aerial partss ethanol extraction, then the ethanol extraction is suspended in distilled water, use stone successively Oily ether and ethyl acetate extraction, collect acetic acid ethyl acetate extract and are concentrated under reduced pressure to give acetic acid ethyl ester extract, by ethyl acetate Extract Jing macroporous adsorbent resin column chromatographies, successively with the ethanol that volume fraction is 60%, the ethanol that volume fraction is 80% and body Fraction is 95% ethanol elution, the ethanol elution that collected volume fraction is 80% ethanol elution and volume fraction is 95% Liquid is concentrated under reduced pressure to give 80% ethanol elution product and 95% ethanol elution product respectively；

（2）By 80% ethanol elution product Jing normal pressure silica gel columns chromatograph, with petroleum ether-acetone solvent system volume ratio be 5:1- 1:In the range of 1 and methylene chloride-methanol solvent system volume ratio be 2:1-1:Gradient elution in the range of 1, Jing TLC identifications Merge same composition and obtain initial target component, initial target component Jing normal pressure silica gel column is chromatographed, it is molten with dichloromethane-acetone Agent system is 50 in volume ratio:1-10:In the range of 1, gradient elution respectively obtains compound 1, compound 4 and intermediate objective group Point, then intermediate objective component is recrystallized to give into compound 2, the structural formula of wherein compound 1 is, The structural formula of compound 2 is, the structural formula of compound 4 is；

（3）By 95% ethanol elution product Jing normal pressure silica gel columns chromatograph, with petroleum ether-acetone solvent system volume ratio be 5:1- 1:In the range of 1 and methylene chloride-methanol solvent system volume ratio be 2:1-1:Gradient elution in the range of 1, Jing TLC identifications Merge same composition and obtain initial target component, initial target component Jing normal pressure silica gel column is chromatographed, it is molten with dichloromethane-acetone Agent system is 100 in volume ratio:0-20:Gradient elution in the range of 1, Jing TLC identifications merge same composition and obtain intermediate objective Component, intermediate objective component Jing dextran gel column chromatography and half preparation HPLC purification obtain compound 3, the knot of the compound 3 Structure formula is, the wherein eluent of dextran gel column chromatography is methanol, and half prepares HPLC Mobile phase be volume ratio 75:25 methanol-water mixed solution.

2. the extracting method of Rhizoma Dioscoreae aerial partss according to claim 1 phenanthrene class compound, it is characterised in that：Described is big Macroporous adsorbent resin be Diaion HP-20 type macroporous adsorbent resins, polydextran gel be Sephadex LH-20 types glucosan coagulate Glue.

3. the Rhizoma Dioscoreae aerial partss phenanthrene class compound that the method described in claim 1 or 2 is extracted is preparing hypoglycemic drug or guarantor Application in strong product.

4. the Rhizoma Dioscoreae aerial partss phenanthrene class compound that the method described in claim 1 or 2 is extracted is preparing alpha-glucosidase suppression Application in preparation class medicine or health product.

5. the compound 4 that the method described in claim 1 or 2 is extracted is preparing alpha-glucosidase inhibitor medicine or health care Application in product.