Application of diphenyl heptane compound in tsaoko amomum flower in inhibition of alpha-glucosidase

Abstract

The invention discloses an application of a diphenyl heptane compound in tsaoko amomum flowers in inhibition of alpha-glucosidase. The natural component 3, 5-diacetoxy-1, 7-bis (4-hydroxyphenyl) heptane is separated and purified from tsaoko amomum flowers by adopting 95% ethanol extraction and petroleum ether and ethyl acetate extraction modes, and experiments find that the natural component 3, 5-diacetoxy-1, 7-bis (4-hydroxyphenyl) heptane can effectively inhibit the activity of alpha-glucosidase, so that the activity of the alpha-glucosidase is inhibited, and the activity of the alpha-glucosidase is inhibited. Therefore, the compound can be used as an alpha-glucosidase inhibitor for inhibiting alpha-glucosidase or reducing blood sugar.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biology, and particularly relates to the application of a diphenylheptane compound in Amomum tsaoko Crevost & Lem. flowers in inhibiting alpha-glucosidase. BACKGROUND

[0002] Alpha-glucosidase, as an important starch hydrolase, mainly functions to decompose starch and glycogen in food into glucose and maltose, and is widely used in the fields of food, chemistry and medicine. In medicine, alpha-glucosidase is used for the treatment of diabetes, by inhibiting the activity of alpha-glucosidase in the small intestine, delaying or inhibiting the absorption of glucose, thereby effectively reducing blood sugar. Common alpha-glucosidase inhibitors include acarbose and voglibose, but these drugs can cause adverse reactions such as abdominal distension, diarrhea, abdominal pain, etc. Therefore, the development of natural active ingredients has become a research hotspot, and these ingredients have mild drug efficacy and small side effects.

[0003] Amomum tsaoko Crevost & Lem. is a commonly used Chinese herbal medicine, and has various effects and functions in traditional Chinese medicine, such as invigorating the stomach and promoting digestion, dispelling cold and removing cold, promoting blood circulation and removing blood stasis, and anti-inflammatory and antibacterial effects. However, the research on the alpha-glucosidase activity inhibiting component in Amomum tsaoko Crevost & Lem. flowers is not clear. Therefore, the natural active ingredients in Amomum tsaoko Crevost & Lem. flowers still need to be further researched, so as to excavate new natural ingredients with the activity of inhibiting alpha-glucosidase. SUMMARY

[0004] The present application aims to overcome the shortcomings and deficiencies of the prior art, and provides the application of 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane in preparing a product with the activity of inhibiting alpha-glucosidase.

[0005] The object of the present application is achieved by the following technical solutions:

[0006] The application of 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane in preparing a product with the activity of inhibiting alpha-glucosidase.

[0007] The structural formula of 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane is shown as formula I:

[0008]

[0009] The product includes a drug or a food (functional food), such as an alpha-glucosidase inhibitor, etc.

[0010] The 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane is extracted and separated from Amomum tsaoko Crevost & Lem. flowers, and the specific steps are as follows:

[0011] (1) Ethanol extraction

[0012] The natural dried flower of Amomum tsao-ko is ground into powder, sieved, and then soaked in 95% (volume ratio) ethanol solution to extract, filtered, and concentrated to obtain an ethanol extract of the flower of Amomum tsao-ko;

[0013] (2) Petroleum ether and ethyl acetate extraction

[0014] The ethanol extract of the flower of Amomum tsao-ko obtained in step (1) is first extracted with petroleum ether to obtain a petroleum ether extract, and then extracted with ethyl acetate to obtain an ethyl acetate extract;

[0015] (3) Separation

[0016] The ethyl acetate extract obtained in step (2) is uniformly stirred with 100-mesh silica gel powder, and 300-400-mesh silica gel powder is uniformly mixed with dichloromethane and then wet-packed on a column. The column is continuously flushed with dichloromethane until the silica gel is fully packed. Gradient elution is then performed with dichloromethane:methanol = 100:0→0:100 (volume ratio), and 2 L of each gradient is taken as a fraction. All the fractions are then subjected to TLC spotting, and the same fractions are combined and concentrated under reduced pressure to obtain 21 fractions Fr.A1-Fr.A21.

[0017] The Fr.A8 fraction eluted with a concentration of dichloromethane:methanol = 100:5 is subjected to gel column elution with dichloromethane:methanol = 1:1 (volume ratio) as the eluent, and 200 mL of each fraction is taken as a fraction. All the fractions are then subjected to TLC spotting, and the same fractions are combined and concentrated under reduced pressure to obtain 19 fractions Fr.A8B1-Fr.A8B19.

[0018] The 12th fraction Fr.A8B12 is subjected to preparative liquid chromatography with methanol-water = 98:2 (volume ratio) as the mobile phase, and the detection wavelengths are 280 nm and 254 nm. The flow rate is 4 mL / min, and 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane (with a retention time of 32.1 min on the preparative liquid chromatograph) is separated.

[0019] The sieving in step (1) is sieving through a 40-100-mesh sieve; preferably, sieving through a 60-mesh sieve.

[0020] In step (1), the ratio of the flower powder of Amomum tsao-ko to the ethanol solution is 1 g:5-10 mL; preferably, 1 g:10 mL.

[0021] The soaking time in step (1) is 2-4 days; preferably, 3 days, and the ethanol and the flower powder of Amomum tsao-ko are fully contacted by shaking every 8 h during the soaking.

[0022] The concentration in step (1) is performed by rotary evaporation.

[0023] The temperature of the rotary evaporation (concentration) is 40-45℃; preferably 40℃.

[0024] The number of times of petroleum ether extraction in step (2) is preferably 4 or more.

[0025] The number of times of ethyl acetate extraction in step (2) is preferably 3 or more.

[0026] The mass ratio of the ethyl acetate extract in step (3) to 100 mesh silica gel powder is 1:1-1.05; preferably 1:1.03.

[0027] The method for separating 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane from Amomum villosum in the application further comprises the step of further identifying by NMR (H-NMR, C-NMR) after step (3). 1 H-NMR, 13 C-NMR) after step (3).

[0028] 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane as an application of α-glucosidase in the preparation of a product for reducing blood sugar.

[0029] The blood sugar reduction includes reducing the blood sugar of a diabetic patient, and is used for treating diabetes (anti-diabetes).

[0030] The product includes a medicine or a functional food.

[0031] The present application has the following advantages and effects relative to the prior art:

[0032] 1. The present application uses 95% ethanol extraction, petroleum ether and ethyl acetate extraction to separate and purify a diphenylheptane compound from Amomum villosum flowers. Through experiments, it is found that the natural ingredient is 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane, which can effectively inhibit the activity of α-glucosidase, has good anti-α-glucosidase activity, and the IC50 of α-glucosidase is 2.824±0.109 μg / mL. Therefore, 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane can be used as a new natural α-glucosidase inhibitor for inhibiting α-glucosidase.

[0033] 2. The present application determines that 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane has good inhibitory activity on α-glucosidase through α-glucosidase activity test, and shows a dose-effect relationship. Therefore, it can be used in the preparation of a medicine or food for reducing blood sugar. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane 1 H-NMR chart.

[0035] Figure 2 is 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane 13 C-NMR chart.

[0036] Figure 3 is the total ion chromatogram of UPLC-QTOF-MS of Amomum tsao-ko flower.

[0037] Figure 4 is the high-resolution mass spectrum chart of 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane.

[0038] Figure 5 is the activity inhibition result chart of 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane on α-glucosidase. DETAILED DESCRIPTION

[0039] The application will be further described in conjunction with the examples below, but the embodiments of the application are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the application are the conventional reagents, methods and equipment in the technical field. The test methods in the following examples, unless otherwise specified, are usually carried out according to the conventional experimental conditions, or obtained according to the conventional culture and separation methods. Unless otherwise specified, the experimental materials used in the following examples are purchased from the conventional biochemical reagent manufacturers.

[0040] The α-glucosidase (CAS: 9001-42-7) and the substrate p-nitrophenyl-α-D-glucopyranoside (PNPG) (CAS: 3767-28-0) involved in the embodiments of the application are purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.

[0041] The Amomum tsao-ko flower involved in the embodiments of the application is collected from Sanhe Village, Luzhang Town, Lushui City, Nujiang Lisu Autonomous Prefecture, Yunnan Province.

[0042] The organic solvents involved in the embodiments of the application are specifically methanol, dichloromethane, petroleum ether, ethanol and ethyl acetate of analytical grade from Tianjin Damao Chemical Reagent Factory.

[0043] Example 1 Separation and extraction of the diphenylheptane compound of the application

[0044] (1) 95% (v / v) ethanol extraction

[0045] The collected Amomum tsao-ko flowers were dried naturally, ground into powder, and sieved through a 60-mesh sieve. Then, 95% (v / v) ethanol solution (1 g:10 ml) was added to soak the powder thoroughly (the soaking time was 3 days, and the ethanol solution was shaken every 8 hours to ensure full contact between the ethanol and the powder). After soaking, the solution was filtered and concentrated by rotary evaporation at 40°C to obtain an ethanol extract of Amomum tsao-ko flowers.

[0046] (2) Extraction

[0047] The ethanol extract of Amomum tsao-ko flowers obtained in step (1) was sequentially extracted with water-insoluble organic reagents, petroleum ether and ethyl acetate. The petroleum ether extraction was performed four times to obtain a petroleum ether layer extract and a water layer extract. Then, the petroleum ether layer extract was extracted with ethyl acetate three times to obtain an ethyl acetate layer extract and a water layer extract of Amomum tsao-ko flowers.

[0048] (3) Separation

[0049] The ethyl acetate layer extract of Amomum tsao-ko flowers obtained in step (2) was uniformly mixed with 100-mesh silica gel powder (mass ratio 1:1.03). Meanwhile, 300-400-mesh silica gel powder was mixed with dichloromethane to form a wet column. The column was flushed with dichloromethane until the silica gel was fully saturated. Then, gradient elution was performed using dichloromethane / methanol (100:0→0:100, v / v), and 2 L was collected as a fraction. All the fractions were subjected to TLC spotting, and the same fractions were combined. After being concentrated under reduced pressure, 21 fractions Fr.A1-Fr.A21 were obtained.

[0050] The Fr.A8 fraction (dichloromethane / methanol elution concentration ratio 100:5) was subjected to gel column elution using dichloromethane:methanol (1:1, v / v), and 200 mL was collected as a fraction. All the fractions were subjected to TLC spotting, and the same fractions were combined. After being concentrated under reduced pressure, 19 fractions Fr.A8B1-Fr.A8B19 were obtained.

[0051] The Fr.A8B12 fraction (dichloromethane / methanol elution concentration ratio 1:1) was subjected to preparative liquid chromatography using 98%:2% methanol-water (v / v) as the mobile phase, and the detection wavelength was 280 nm and 254 nm. The flow rate was 4 mL / min. The retention time of the compound with biological activity of the present application on the preparative liquid chromatography was 32.1 min. The obtained solution was dried to obtain 384.3 mg of the compound of the present application.

[0052] Example 2 Structure confirmation of the compound of the present application

[0053] The compound obtained in the present application was a yellow oil. The compound was detected by UPLC-QTOF-MS ( Figure 3 ). Further, the HR-ESI-MS ( Figure 4) shows m / z: 399.1814 [M-H] - , molecular formula C 23 H 28 O6.

[0054] 1 H-NMR (600 MHz, CDC13): δ 6.97 (m, 4H), 6.72 (m, 4H), 4.94 (m, 1H), 4.90 (dd, J = 12.6, 6.7 Hz, 1H), 2.54 (m, 1H), 2.51 (m, 1H), 2.47 (m, 1H), 1.99 (d, J = 1.4 Hz, 6H), 1.93 (m, 1H), 1.86 (m, 1H), 1.80 (m, 4H), 1.74 (ddd, J = 14.3, 8.8, 3.8 Hz, 1H).

[0055] 13 C-NMR (151 MHz, CDC13): δ 154.17 (C-11, 11'), 133.26 (C-8, 8'), 129.60 (C-9, 9'), 115.49 (C-10, 10'), 70.68 (C-3, 5), 38.60 (C-2, 6), 36.31 (C-4), 30.51 (C-1, 7), 21.37 (C-15, 17).

[0056] The structure was confirmed as 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane (Formula I) by MS Figure 4 ), 1 H-NMR Figure 1 ) and 13 C-NMR data Figure 2 ).

[0057]

[0058] Example 3 Inhibition of α-glucosidase by the compound of the present application

[0059] (1) Solution preparation

[0060] ① 20 mg of p-nitrophenyl-α-D-glucopyranoside (PNPG) was dissolved in 4 mL of phosphate buffer (PBS) with pH = 7.0, concentration 0.2 M, to obtain a 5 mg / mL PNPG solution.

[0061] ② Dissolve 3 mg of 100 U α-glucosidase in 1 mL of PBS (pH = 7.0, 0.2 M) to obtain a 100 U / mL α-glucosidase stock solution; take 100 μL of the α-glucosidase stock solution and add it to 10 mL of PBS (pH = 7.0, 0.2 M) to dilute to 1 U / mL; then take 1 mL of the 1 U / mL enzyme solution and add it to 4 mL of PBS (pH = 7.0, 0.2 M) to dilute to 0.2 U / mL, finally obtaining an α-glucosidase solution with a concentration of 0.2 U / mL.

[0062] ③ Dissolve the test compound (3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane in Example 2) in methanol (MeOH) to prepare test compound solutions with concentration gradients.

[0063] (2) Acarbose (Bayer) dissolved in PBS (pH = 7.0, 0.2M) was used as a positive control. 20 μL of 0.2 u / mL α-glucosidase and 30 μL of solutions containing the test compounds at concentrations of 50, 25, 12.5, 6.25, and 3.175 μg / mL were sequentially added to 96-well plates and mixed, with three replicates for each sample. The plates were incubated at 37°C for 5 min, then 20 μL of PNPG solution was added to initiate the reaction, followed by incubation at 37°C for 15 min. Finally, 40 μL of 0.1M NaOH solution was added to stop the reaction.

[0064] (3) Record the absorbance (OD value) at 405 nm using a multi-functional microplate reader (Epoch, BioTek, USA). The negative control was prepared using the same method as the experiment, but with a mixture of MeOH and PBS (50:50, v / v) instead of the sample. A blank was prepared using PBS phosphate buffer instead of α-glucosidase, following the same method. Inhibition rate (%) = (OD value) 空白 -OD 样品 ) / OD 空白 ×100%. All data were analyzed using SPSS 27.0.1, and graphs were plotted using Graphpad software. The results are shown in Table 1 and 2000. Figure 5 As can be seen, 3,5-diacetoxy-1,7-di(4-hydroxyphenyl)heptane has a good inhibitory activity against α-glucosidase.

[0065] Table 1. Half-inhibitory concentration of 3,5-diacetoxy-1,7-di(4-hydroxyphenyl)heptane on α-glucosidase

[0066] Drug IC50(μg / mL) 3,5-diacetoxy-1,7-di(4-hydroxyphenyl)heptane 2.824±0.109 Acarbose 131.475±29.189

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane in the preparation of a product for inhibiting the activity of α-glucosidase.

2. Use according to claim 2, characterized in that: The product is an α-glucosidase inhibitor.

3. Use according to claim 1 or 2, characterized in that: The 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane is obtained by extraction and separation from Amomum tsao-ko flowers, and the specific steps are as follows: (1) Ethanol extraction The Amomum tsao-ko flowers are naturally dried, ground into powder, sieved, and then soaked in ethanol solution with a concentration of 95% by volume ratio for extraction, filtered, and concentrated to obtain Amomum tsao-ko flower ethanol extract; (2) Petroleum ether and ethyl acetate extraction The Amomum tsao-ko flower ethanol extract obtained in step (1) is first extracted with petroleum ether, and then extracted with ethyl acetate; (3) Separation The ethyl acetate extract obtained in step (2) is stirred with 100-mesh silica gel powder, and 300-400-mesh silica gel powder is stirred with dichloromethane to form a wet column, which is flushed with dichloromethane until the silica gel is fully flushed; then gradient elution is performed with dichloromethane:methanol = 100:0→0:100, 2 L for each gradient as a fraction, and all the fractions are subjected to TLC spotting, and the same fractions are combined and concentrated under reduced pressure to obtain 21 fractions Fr.A1-Fr.A21; The Fr.A8 fraction with a concentration of dichloromethane:methanol = 100:5 is subjected to gel column elution with dichloromethane:methanol = 1:1, 200 mL for each fraction, and all the fractions are subjected to TLC spotting, and the same fractions are combined and concentrated under reduced pressure to obtain 19 fractions Fr.A8B1-Fr.A8B19; The 12th fraction Fr.A8B12 is subjected to preparative liquid chromatography with methanol-water = 98:2 by volume ratio as the mobile phase, and the detection wavelength is 280 nm and 254 nm, and the flow rate is 4 mL / min, to separate 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane.

4. The use according to claim 3, characterized in that: In step (1), the ratio of Amomum tsao-ko flower powder to ethanol solution is 1 g:5-10 mL; In step (3), the mass ratio of ethyl acetate extract to 100-mesh silica gel powder is 1:1-1.

05.

5. The use according to claim 3, characterized in that: In step (1), the ratio of Amomum tsao-ko flower powder to ethanol solution is 1 g:10 mL; In step (3), the mass ratio of ethyl acetate extract to 100-mesh silica gel powder is 1:1.

03.

6. The use according to claim 3, characterized in that: In step (1), the sieving is through a 40-100-mesh sieve; In step (1), the soaking time is 2-4 days.

7. The use according to claim 3, characterized in that: In step (1), the concentration is performed by rotary evaporation at a temperature of 40-45°C; In step (2), the petroleum ether extraction is performed more than 4 times; In step (2), the ethyl acetate extraction is performed more than 3 times.

8. Use of 3,5-diacetoxy-1,7-bis(4-hydroxyphenyl)heptane as an alpha-glucosidase in the manufacture of a product for lowering blood sugar.

9. Use according to claim 8, characterized in that: The lowering of blood sugar includes lowering blood sugar in diabetic patients for the treatment of diabetes.

10. Use according to claim 8, characterized in that: The product includes a pharmaceutical or a functional food.

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