Coffee atractylis glycoside with hypoglycemic activity and preparation method and application thereof

Abstract

The invention discloses caffeoside with hypoglycemic activity as well as a preparation method and application of the caffeoside. The preparation method of the caffeoside comprises the following steps: (1) taking roasted coffee beans, and extracting with hot water to obtain a hot water extract; (2) suspending the hot water extract in water, and extracting with ethyl acetate to respectively obtain an ethyl acetate extract and a water layer; (3) extracting the water layer with n-butyl alcohol to obtain an n-butyl alcohol extract; (4) carrying out silica gel column chromatography on the n-butyl alcohol extract, carrying out gradient separation and elution by using a mobile phase CH2Cl2 / MeOH with the volume ratio of (30: 1)-(1: 1), respectively collecting separated fractions with the volume ratios of CH2Cl2 / MeOH of (30: 1), (20: 1), (10: 1), (5: 1) and (1: 1), concentrating to obtain extractum of five fractions, and respectively marking the extractum as Fr.A, Fr.B, Fr.C, Fr.D and Fr.E; and (5) the Fr.D is separated and purified again through reverse silica gel PR-C18 column chromatography, and the caffein is obtained. The method has the advantages that the caffeoside extracted from the hot water of the coffee has high hypoglycemic activity, and the preparation method is simple and feasible.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to a coffee atractylodesin with hypoglycemic activity and a preparation method and application thereof. Background Art

[0002] Coffee ranks first among the world's three famous beverage plants. Coffee affects the life of human society and has become an indispensable necessity in life.

[0003] The endosperm oil extract of coffee beans was found to contain about 19% of coffee diterpenes. In the 1930s, scientists identified the structures of famous coffee diterpenes such as cafestol and kahweol. The inventor has been deeply exploring the new structure and activity of coffee diterpenes, and has combined the published coffee diterpenes and similar compounds. 13 C NMR, build a coffee diterpene database; conduct 13 C NMR was compared with the database to quickly analyze the main diterpenoid compounds in the crude extract, selectively purify the crude extract, and quickly discover and separate new active diterpenoid compounds. Using this technology, not only the known coffee diterpenoid compounds discovered by scientists were determined, but also more than 150 new coffee diterpenoid structures were discovered from Yunnan coffee, including oxidized diterpenoids, rearranged diterpenoids, furan diterpenoids, lactam diterpenoids, lactone diterpenoids, Δ4,18 diterpenoids, degraded diterpenoids, Villanova diterpenoids and atissenane diterpenoids and their glycoside compounds. This is one of the basic studies to interpret the rich content of coffee, but because most coffee diterpenoids are fat-soluble compounds, there is no relevant literature report on the extraction of coffee diterpenoids in roasted coffee with hot water, so it is very necessary to study the preparation technology of water extraction methods in roasted coffee beans. Summary of the invention

[0004] In view of the above problems, the purpose of the present invention is to provide a kind of coffee atractylodes glycosides with hypoglycemic activity and its preparation method and application.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] A use of atractylodesin in the preparation of a hypoglycemic drug and / or a health food, wherein the chemical structural formula of the atractylodesin is as follows:

[0007]

[0008] Preferably, the medicine contains a pharmaceutically acceptable carrier and / or excipient.

[0009] Preferably, the dosage form of the drug is one of injection, tablet, pill, capsule, solution, suspension or emulsion.

[0010] The present invention also provides a method for preparing the coffee atractylodesin, comprising the following steps:

[0011] (1) taking roasted coffee beans, extracting with hot water to obtain a hot water extract;

[0012] (2) suspending the hot water extract in water, and extracting with ethyl acetate to obtain an ethyl acetate extract and an aqueous layer;

[0013] (3) extracting the aqueous layer with n-butanol to obtain an n-butanol extract;

[0014] (4) The n-butanol extract was subjected to silica gel column chromatography, and the mobile phase was separated and eluted with a gradient of CH2Cl2 / MeOH in a volume ratio from 30:1 to 1:1, and the separated fractions with a CH2Cl2 / MeOH volume ratio of 30:1, 20:1, 10:1, 5:1 and 1:1 were collected respectively, and after concentration, five extracts were obtained, which were marked as Fr.A, Fr.B, Fr.C, Fr.D and Fr.E respectively;

[0015] (5) The Fr.D is separated and purified again by reverse silica gel PR-C18 column chromatography to obtain atractylodes lancea glycoside.

[0016] Preferably, the temperature of the hot water in step (1) is 60° C. to 90° C.; and the number of hot water extractions is 1 to 3 times.

[0017] Preferably, the number of times of ethyl acetate extraction in step (2) is 3 times.

[0018] Preferably, the number of extractions with n-butanol in step (3) is 3 times.

[0019] The present invention has the following beneficial effects:

[0020] (1) Atractylodes macrocephala glycosides with good hypoglycemic activity were extracted and isolated from roasted coffee beans. Its hypoglycemic effect in mice is comparable to that of the positive first-line clinical drug metformin.

[0021] (2) The method for preparing coffee atractylodesin is simple and easy, and can be applied to large-scale industrial production processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the chemical structural formula of atractylodesin;

[0023] Figure 2 The process flow chart for preparing coffee atractylodesin;

[0024] Figure 3 Effects of GPAD on food intake, water consumption, body weight and lean body mass of mice. (A) Food intake, (B) Water consumption, (C) Body weight, (D) Lean body mass;

[0025] Figure 4 Effects of GPAD on fasting blood glucose and glucose tolerance. (A) Fasting blood glucose during treatment, (B) Fasting blood glucose at week 0 and week 7, (C) OGTT test, (D) Area under the curve (AUC) of OGTT test;

[0026] Figure 5 Effects of GPAD on serum insulin and insulin resistance. (A) Serum insulin, (B) insulin tolerance test (ITT), (C) area under the curve (AUC) of ITT test;

[0027] Figure 6 Effects of GPAD on lipid parameters: (A) triglyceride (TG), (B) total cholesterol (TC), (C) low-density lipoprotein (LDL), and (D) high-density lipoprotein (HDL). DETAILED DESCRIPTION

[0028] The following is further described in conjunction with the specific embodiments of the present invention. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0029] Example 1

[0030] The preparation method of coffee atractylodesin (GPAD) comprises the following steps:

[0031] Take 20 kg of roasted coffee beans, grind them, extract them twice with hot water at 80°C, filter the extract, and concentrate it to obtain 3.6 kg of hot water extract Aq-EC. The concentrated extract is resuspended in distilled water, extracted three times with ethyl acetate, and concentrated to obtain 350 g of ethyl acetate extract (EtOAc-EC). The remaining aqueous layer is extracted three times with n-butanol, and concentrated to obtain 500 g of n-butanol extract (BuOH-EC). The n-butanol extract (BuOH-EC) is chromatographed on a silica gel column, and the mobile phase CH2Cl2 / MeOH is used for gradient separation and elution from 30:1 to 1:1, and five fractions (Fr.A-Fr.E) are obtained after concentration. The fourth fraction Fr.D is about 65 g, which is separated and purified again by reverse silica gel PR-C18 column chromatography to obtain 25.2 g of coffee atractylodes glycosides (GPAD). See Figure 1-Figure 2 .

[0032] The chemical structure of coffee atractylodesin (GPAD) is as follows:

[0033]

[0034] It is a coffee diterpene compound: 2-glucosyl atractyligenin (2-O-[β-D-glucopyranosyl]atractyligenin, GPAD), also known as coffee atractyligenin.

[0035] Chemical structure characterization of GPAD

[0036] GPAD is a white powdery solid with a molecular formula of C 25 H 38 O9; molecular weight: 482.564.

[0037] 1 H NMR (CD3OD, 600MHz)δ H :2.17(1H,dd,J=11.8,3.1Hz,H-1a),0.69(1H,t,J=11.8Hz,H-1b),4.16(1H,m,H-2),2.34-2.4(1H,m,H-3a),1.23-1.27(1H,m,H-3b),2.65( 1H,brs,H-4),1.45-1.54(1H,m,H-5),1.61-1.71(1H,m,H-6a),1.88-1 .98(1H,m,H-6b),1.45-1.55(1H,m,H-7a),1.60-1.70(1H,m,H-7a),1. 05(1H,d,J=6.9Hz,H-9),1.61-1.71(2H,m,H-11),1.61-1.71(2H,m,H- 12),2.70(1H,brs,H-13),1.86-1.91(1H,m,H-14a),1.38-1.42(1H,m, H-14b),3.76(1H,s,H-15),5.07(1H,brs,H-17a),5.18(1H,brs,H-17b),1.00(3H,s,H-20),4.43(d,J=11.8Hz,H-1'),3.3-4.3(H-2'-H-6'); 13 CNMR (CD3OD, 150MHz)δ C:50.4(C-1),75.1(C-2),38.3(C-3),44.9(C-4),26.6(C-6),36.2(C-7),48.9 (C-8),54.5(C-9),41.8(C-10),19.2(C-11),33.6(C-12),43.7(C-13),37.2( C-14),83.5(C-15),160.3(C-16),109.1(C-17),178.9(C-19),17.3(C-20),1 02.8(C-1'),77.3(C-2'),73.5(C-3'),71.6(C-4'),78.1(C-5'),62.7(C-6')

[0038] Purity of GPAD: analyzed by liquid chromatography: chromatographic column ZORBAX SB-C18 (250×9.4 mm, 5.0 μm), mobile phase CH3CN / H2O (10:90), purity was 96% (RT=5.6 min).

[0039] Example 2

[0040] Study on the hypoglycemic activity of coffee atractylodesin (GPAD)

[0041] Blood sugar lowering test method

[0042] 1) Animal experiment design

[0043] A total of fifty specific pathogen-free (SPF) male mice, including forty db / db diabetic mice and eight C57BL / 6J mice, were purchased from Changzhou Kevins Experimental Animal Co., Ltd. (Jiangsu, China), all of which were eight weeks old. After a one-week adaptation period, C57BL / 6J mice were used as normal control group (NC, n = 8), while db / db mice were randomly divided into five groups (n = 8 in each group): diabetic model group (DM): given distilled water; diabetic positive control group (PC): given metformin (200 mg / kg); diabetic high-dose group (HD): given high-dose 2-glucopyranoside (GPAD, 60 mg / kg); diabetic medium-dose group (MD): given medium-dose GPAD (40 mg / kg); diabetic low-dose group (LD): given low-dose GPAD (20 mg / kg). All treatments were given by oral gavage once a day for 8 weeks. Mice were housed under standard conditions, maintained with a 12-h light-dark cycle and a temperature of 25 ± 2 °C, with free access to water and mouse chow. Animal experiments were performed in accordance with the National Guide for the Care and Use of Animals and approved by the Animal Care and Use Committee of the Kunming Institute of Botany, Chinese Academy of Sciences (SYXK K2024-0003).

[0044] 2) Food intake, water consumption, and body weight measurement

[0045] The body weight of mice was monitored weekly during the experiment. Water consumption and food intake were measured on consecutive days each week.

[0046] 3) Fasting blood sugar measurement

[0047] Fasting blood glucose was measured weekly using a glucometer, provided that the mice had fasted for 12 h the previous night.

[0048] 4) Lean body mass (LBM) measurement

[0049] After seven weeks of treatment, lean body mass (LBM) was measured using magnetic resonance imaging (MRI, EchoMRI).

[0050] 5) Oral glucose tolerance test (OGTT)

[0051] OGTT was performed by fasting the mice for 12 hours the previous night to ensure baseline blood glucose levels. Then, 1 g / kg of glucose solution was orally administered. Afterwards, blood glucose levels were measured regularly, usually 15, 30, 60, 90, and 120 minutes after administration. The area under the curve (AUC) of the glucose tolerance test (GTT) was calculated according to the standard trapezoidal rule. 6) Insulin tolerance test (ITT)

[0052] ITT is performed by fasting mice for 6 hours to ensure baseline blood glucose levels. Then, 1U / kg of insulin is injected intraperitoneally. Blood glucose levels are measured regularly (usually 15, 30, 60, 90 and 120 minutes) after administration. The area under the curve (AUC) of the insulin tolerance test (ITT) is calculated according to the standard trapezoidal rule.

[0053] 7) Blood and organ collection

[0054] Under anesthesia, blood was collected from mice from the retro-orbital plexus into centrifuge tubes. Blood samples were stored at room temperature for 2 hours and then centrifuged at 5000 rpm for 10 minutes at 4°C. Serum was drawn and stored at -80°C for subsequent analysis. Mice were euthanized by cervical dislocation and their organs (liver, kidney, pancreas, spleen, white adipose tissue and muscle) were collected. The liver index was calculated according to the following formula: Liver index (LI) = (liver weight / body weight) × 100. A small part of the liver was fixed in formalin for 48 hours and embedded in paraffin for histopathological analysis. The rest was stored at -80°C for further analysis.

[0055] 8) Serum biochemical analysis

[0056] Insulin in serum was measured using a mouse insulin (INS) ELISA kit (Jiangsu Meike Biotechnology Co., Ltd., China). Total cholesterol (T.CHO), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) in serum were determined using different kits from Nanjing Jiancheng Bioengineering Institute.

[0057] Results of hypoglycemic experiment

[0058] 1) Effects of GPAD on food intake, water consumption, body weight and lean body mass in mice

[0059] like Figure 3 As shown in Figure 2, water consumption, food intake, and body weight were recorded weekly during the experimental period. The mean water consumption and food intake in the diabetes model group were significantly higher than those in the NC group during all seven weeks (p<0.001). The food intake in the PC and HD groups was significantly higher than that in the DC model group at weeks 6 and 7 ( Figure 3 A). From week 3 to week 7, water consumption in the PC and HD groups was lower than that in the DC group ( Figure 3 B). The body weight of the diabetes group was significantly higher than that of the NC group (p<0.001) ( Figure 3 C). Although the body weight differences among the groups were not significant, the results of lean body mass (LBM) showed that the LBM values ​​of the PC, HD, and MD groups were significantly higher than those of the DC group (p<0.05) ( Figure 3 D).

[0060] 2) Effects of GPAD on fasting blood glucose (FBG) and glucose tolerance

[0061] like Figure 4 As shown, fasting blood glucose was measured once a week. At the beginning of the experiment, the blood glucose levels of all diabetic model groups exceeded 15mmol / L, which is the diagnostic standard for diabetes and meets the characteristics of the diabetic model. Figure 4 The results showed that the FBG of the PC, MD and HD groups at week 7 was significantly higher than the initial FBG (p<0.05). In addition, the FBG of the PC, MD and HD groups at week 7 was significantly lower than that of the DC group (p<0.05 or p<0.01). Figure 4 B.

[0062] To determine the effect of GPAD on glucose homeostasis and glucose tolerance, OGTT test was performed in the last week of treatment. Compared with the NC group, the blood glucose level in the diabetes model group was significantly higher at all time points (p<0.01). The results showed that the glucose tolerance of the PC group and the HD group was significantly improved compared with that of the DC model group (p<0.05). Figure 4 C and 4D.

[0063] 3) Effects of GPAD on serum insulin and insulin sensitivity

[0064] like Figure 5 As shown in Figure 2, compared with DC mice, serum insulin levels in PC, HD, and MD groups were significantly decreased (p<0.01 or p<0.05). Figure 5 A. An insulin tolerance test (ITT) was performed to detect improvements in insulin sensitivity. Figure 5 As shown in B and 5C, GPAD significantly improved insulin resistance in MD and HD mice compared with DC mice (p<0.05).

[0065] 4) Effects of GPAD on lipid parameters

[0066] like Figure 6 As shown in the figure, in the diabetic model group, the levels of triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL) were generally significantly higher than those in the NC group (p<0.001). These are common features of type 2 diabetes associated with insulin resistance and lipid metabolism impairment. Figure 6 A shows that the TG levels of PC, MD, and HD mice were significantly decreased compared with those of DC mice (p<0.01 or p<0.05). The results showed that GPAD had a dose-dependent effect on TG levels in diabetic mice. As the dose of GPAD increased, the TG levels decreased accordingly, indicating improved lipid metabolism. In addition, compared with the DC group, the TC and LDL levels in the HD and PC groups decreased significantly (p<0.01 or p<0.05), while the high-density lipoprotein (HDL) level increased significantly (p<0.05). In the HD group, the efficacy of GPAD in reducing TG, TC, and LDL levels was comparable to that of metformin, indicating its potential as an effective treatment for diabetes-related lipid metabolic abnormalities.

[0067] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the scope of protection of the present invention.

Claims

1. An application of atractylodesin in the preparation of hypoglycemic drugs and / or health foods, wherein the chemical structural formula of atractylodesin is as follows:

2. The use according to claim 1, characterized in that: The medicine contains a pharmaceutically acceptable carrier and / or excipient.

3. The use according to claim 1, characterized in that: The dosage form of the drug is one of injection, tablet, pill, capsule, solution, suspension or emulsion.

4. The method for preparing atractylodesin according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) taking roasted coffee beans, extracting with hot water to obtain a hot water extract; (2) suspending the hot water extract in water, and extracting with ethyl acetate to obtain an ethyl acetate extract and an aqueous layer; (3) extracting the aqueous layer with n-butanol to obtain an n-butanol extract; (4) The n-butanol extract was subjected to silica gel column chromatography, and the mobile phase was separated and eluted with a gradient of CH2Cl2 / MeOH in a volume ratio from 30:1 to 1:1, and the separated fractions with a CH2Cl2 / MeOH volume ratio of 30:1, 20:1, 10:1, 5:1 and 1:1 were collected respectively, and after concentration, five extracts were obtained, which were marked as Fr.A, Fr.B, Fr.C, Fr.D and Fr.E respectively; (5) The Fr.D is separated and purified again by reverse silica gel PR-C18 column chromatography to obtain atractylodes lancea glycoside.

5. The preparation method according to claim 4, characterized in that: The temperature of the hot water in step (1) is 60° C. to 90° C.; the number of hot water extractions is 1 to 3 times.

6. The preparation method according to claim 4, characterized in that: The number of extractions with ethyl acetate in the step (2) is 3 times.

7. The preparation method according to claim 4, characterized in that: The number of times of n-butanol extraction in the step (3) is 3 times.

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