Hexahydropyrroloindole alkaloid A extracted from wolfberry fruit, and preparation method and application thereof

Abstract

A novel hexahydropyrroloindole alkaloid extracted from wolfberry fruit can effectively solve the problems of extracting and preparing novel hexahydropyrroloindole alkaloid A from wolfberry fruit and realizing its application in preparing drugs for preventing and treating type 2 diabetes. The dried wolfberry fruit is heated under reflux for extraction, concentrated, and applied to a macroporous adsorption resin column. The product is eluted with water, ethanol, and acetone in sequence. The ethanol fraction is concentrated under reduced pressure, extracted, mixed with silica gel, gradient eluted, the same fractions are combined, the target component is eluted, mixed with silica gel, gradient eluted, the fractions within the retention time are collected, concentrated, and dried. The application of the prepared novel hexahydropyrroloindole alkaloid A in preparing drugs for preventing and treating type 2 diabetes can significantly promote glucose consumption in insulin-resistant HepG2 cells, improve glucose uptake capacity, promote glycogen synthesis, and enhance the activities of key glycolytic enzymes hexokinase and pyruvate kinase. The development and application prospects are broad and the economic and social benefits are significant.

Description

Technical Field

[0001] The present invention relates to the field of medicine, in particular to a hexahydropyrroloindole alkaloid A extracted from wolfberry fruit, and a preparation method and application thereof. Background Art

[0002] The incidence of type 2 diabetes mellitus (T2DM) has increased dramatically worldwide due to multiple factors, including changes in diet and lifestyle. It has become a chronic disease that poses a serious threat to human health, following cancer and cardiovascular and cerebrovascular diseases. Although a variety of drugs targeting different targets are currently available, they all carry side effects such as weight gain, hypoglycemia, and gastrointestinal disturbances. Therefore, the search and discovery of novel, safe, and highly effective lead compounds for glucose-lowering is urgent.

[0003] Lycium barbarum (Lycium barbarum L.), the dried mature fruit of the Solanaceae family Lycium genus, is sweet and neutral in nature. It enters the liver and kidney meridians, nourishing the liver and kidneys, improving vital energy and vision, and possesses significant medicinal value in the treatment of diabetes. However, current reports on its hypoglycemic active ingredients have primarily focused on Lycium barbarum polysaccharides and total flavonoids. Hexahydropyrroloindole alkaloids have garnered significant attention from phytochemists and medicinal chemists due to their unique tricyclic fused ring system and significant biological activity. Approximately 400 hexahydropyrroloindole alkaloids have been discovered in nature, and reports on their biological activities primarily focus on antiviral, antitumor, anti-plasmodial, antibacterial, and acetylcholinesterase inhibition, while research on their hypoglycemic activity has been limited. Lyciumine A is a novel hexahydropyrroloindole alkaloid first discovered from wolfberry fruit. It has a complex 6 / 5 / 5 / 6 tetracyclic fused ring system connected to a pyrrole ring. Currently, there are no public reports on the use of lyciumine A in the prevention and treatment of type 2 diabetes mellitus (T2DM). Summary of the Invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present invention is to provide a hexahydropyrroloindole alkaloid extracted from wolfberry fruit and its preparation method and application, which can effectively solve the problem of extracting and preparing hexahydropyrroloindole alkaloid A from wolfberry fruit and realizing its application in the preparation of drugs for the prevention and treatment of type 2 diabetes mellitus (T2DM).

[0005] The technical solution provided by the present invention is a hexahydropyrroloindole alkaloid extracted from wolfberry fruit, the chemical molecular structure of which is:

[0006]

[0007] The preparation method is:

[0008] The dried wolfberry fruit is extracted with ethanol under heating and refluxing to obtain a concentrated solution; the dried wolfberry fruit is subjected to a macroporous adsorption resin column and eluted with water, ethanol, and acetone in sequence to obtain the corresponding fraction; the ethanol fraction is concentrated under reduced pressure to obtain a first extract; the first extract is extracted with ethyl acetate and concentrated to obtain a second extract;

[0009] The second extract was mixed with an equal amount of silica gel, and eluted with a gradient ratio of petroleum ether-ethyl acetate and dichloromethane-methanol. Each fraction was concentrated under reduced pressure, and the fractions were analyzed by thin layer spectrophotometry. The same fractions were combined to obtain multiple first group fractions.

[0010] The target first group fraction was loaded onto an ODS column and eluted with a gradient ratio of methanol:water to obtain multiple second group fractions;

[0011] The target second group of components was mixed with silica gel and eluted with a gradient ratio of dichloromethane:methanol to obtain multiple third group components;

[0012] The target third group of components was eluted with acetonitrile at a flow rate of 3 mL / min and a volume of 1 L. The retention time t R The fraction at 12.8 min was concentrated and dried to obtain 14.3 mg of hexahydropyrroloindole alkaloid A (lycium barbarum alkaloid A, the same below).

[0013] The invention relates to an application of the hexahydropyrroloindole alkaloid extracted from wolfberry fruit prepared by the above method in the preparation of drugs for preventing and treating type 2 diabetes mellitus (T2DM).

[0014] The present invention discloses a hexahydropyrroloindole alkaloid extracted from wolfberry fruit, a preparation method thereof, and an application thereof. Experimental studies have shown for the first time that wolfberry alkaloid A, which has a completely new skeleton type, can significantly promote glucose consumption in IR-HepG2 cells, improve glucose uptake capacity, promote glycogen synthesis, and enhance the activities of key glycolysis enzymes hexokinase (HK) and pyruvate kinase (PK). This provides a completely new chemical structure type for the development of new diabetes prevention and treatment drugs, has broad development and application prospects, can be conveniently prepared into a variety of pharmaceutical dosage forms, is convenient for clinical use, and has significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure is a chemical molecular formula structure diagram of the hexahydropyrroloindole alkaloid A (lycium barbarum alkaloid A) of the present invention;

[0016] Figure 2 This is a high-resolution mass spectrum of the hexahydropyrroloindole alkaloid A of the present invention;

[0017] Figure 3 is the infrared spectrum of the hexahydropyrroloindole alkaloid A of the present invention;

[0018] Figure 4 is the H NMR spectrum of the hexahydropyrroloindole alkaloid A of the present invention;

[0019] Figure 5 is the carbon nuclear magnetic resonance spectrum of the hexahydropyrroloindole alkaloid A of the present invention;

[0020] Figure 6 This is the test result of glucose consumption of IR-HepG2 cells by the hexahydropyrroloindole alkaloid A of the present invention;

[0021] Figure 7 This is the test result of the hexahydropyrroloindole alkaloid A of the present invention on glucose uptake in IR-HepG2 cells;

[0022] Figure 8 The results are the test results of the hexahydropyrroloindole alkaloid A of the present invention on the glycogen content, PK and HK activities of IR-HepG2 cells. DETAILED DESCRIPTION

[0023] The specific implementation methods of the present invention are described in detail below with reference to examples and specific situations.

[0024] Example 1:

[0025] A hexahydropyrroloindole alkaloid extracted from wolfberry fruit, having the chemical molecular structure formula:

[0026]

[0027] The preparation method comprises the following steps:

[0028] (1) 50 kg of dried wolfberry was extracted three times with 95% ethanol by volume, with the amount of ethanol used each time being 3-5 times the weight volume of the wolfberry, for 2 hours each time. The weight volume refers to the solid in kg and the liquid in L. The three extracts were filtered and combined; the extracts were concentrated under reduced pressure to a concentrate equivalent to 0.5 g crude drug / ml;

[0029] (2) adding the concentrate to a 2-fold volume of a macroporous adsorption resin column and adsorbing the concentrate at a flow rate of 4 BV / h. Elution was first performed with 8-fold volume of distilled water, then with 4 BV of 95% ethanol, and finally with 3 BV of acetone. The 95% ethanol eluate was collected and concentrated under reduced pressure to a first extract (i.e., a 95% ethanol eluate concentrate) having a volume of 1 / 10-12 of the 95% ethanol eluate.

[0030] (3) 1.2 kg of the first extract was suspended in 2.5 L of distilled water and extracted with ethyl acetate, 2.5 L each time for 1 h. The extraction was repeated 8 times. The extracts were combined and concentrated under reduced pressure to a volume of 1 / 9-12 of the volume of the second extract;

[0031] (4) 200 g of the second extract was mixed with an equal amount of 100-200 mesh silica gel and gradient eluted with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 100:1, 70:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1 and a dichloromethane-methanol mixed solvent with a volume ratio of 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1, with each gradient using 5-10 L. The flow rate was 10-15 ml / min, and each 500 ml was a fraction. Each fraction was concentrated under reduced pressure and analyzed by silica gel thin layer chromatography using GF254 thin layer plates with petroleum ether-ethyl acetate in a volume ratio of 2:1 and dichloromethane-methanol in a volume ratio of 10:1 as developing solvents, respectively. The analysis was detected at 254 nm under an ultraviolet lamp. Identical fractions were combined based on thin layer spots to obtain 11 first group components Fr. A, B, C, D-K;

[0032] (5) 20.6 g of the first group fraction Fr.D was passed through an ODS column and eluted with a methanol:water gradient of 10:90, 30:70, 50:50, 70:30, and 90:10 by volume, with each gradient volume of 5 L and a flow rate of 10-15 ml / min to obtain five second group fractions Fr.D1, Fr.D2, Fr.D3, Fr.D4, and Fr.D5;

[0033] (6) 608 mg of the second subgroup, Fr.D3, was mixed with an equal amount of 100-200 mesh silica gel and eluted with a dichloromethane:methanol mixed solvent with a volume ratio of 100:0, 70:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1, in sequence. Each gradient volume was 100-200 mL, the flow rate was 10-15 mL / min, and each 50 mL was a fraction. Each fraction was concentrated under reduced pressure and analyzed by silica gel thin layer chromatography using GF254 thin layer plates with dichloromethane:methanol in a volume ratio of 10:1 as the developing solvent. The analysis was detected at 254 nm by ultraviolet light. The same fractions were combined according to the thin layer spots to obtain four third subgroups, Fr.D3.1-D3.4);

[0034] (7) The third group of components Fr.D3.1 125 mg was eluted with 30% acetonitrile at a flow rate of 3 mL / min and a volume of 1 L. The elution time t R The fraction at 12.8 min was concentrated and dried to obtain 14.3 mg of hexahydropyrroloindole alkaloid A (lycium barbarum alkaloid A).

[0035] The invention relates to an application of the hexahydropyrroloindole alkaloid extracted from wolfberry fruit prepared by the above method in the preparation of drugs for preventing and treating type 2 diabetes mellitus (T2DM).

[0036] The medicine is a medicine containing hexahydropyrroloindole alkaloid A (lycium barbarum alkaloid A), or a pharmaceutically acceptable salt or halide for preventing and treating type 2 diabetes (T2DM).

[0037] The drug is a tablet, capsule, injection, powder injection, granule, powder, pill, fat emulsion, microcapsule, dripping pill, ointment, sustained-release agent or controlled-release drug prepared from hexahydropyrroloindole alkaloid A (lycium barbarum alkaloid A) and a pharmaceutically acceptable carrier.

[0038] The compounds given in the above examples can be prepared in any amount as needed. The examples given are only used to illustrate the specific implementation methods of the present invention, rather than to limit the scope of protection of the present invention. The technical core protected by the present invention is the compound.

[0039] The wolfberry alkaloid A of the present invention is prepared from wolfberry fruit and can significantly promote glucose consumption in IR-HepG2 cells, improve glucose uptake capacity, promote glycogen synthesis, enhance the activities of key glycolysis enzymes PK and HK, lower blood sugar and regulate sugar metabolism. Experiments have achieved very good beneficial technical effects. The relevant information is as follows:

[0040] 1. Instruments and reagents

[0041] Bruker AVANCE III 500 nuclear magnetic resonance spectrometer (TMS internal standard) (Bruker), infrared spectrometer using Nicolet is10 Microscope Spectrometer (Thermo Scientific, USA), high-resolution mass spectrometer using Bruker maxis HDmass spectrometer, ultraviolet spectrometer using Shimadzu UV-2401PC apparatus, Waters Alliance series 2695 high-performance liquid chromatography system equipped with 2998 diode array detector, Empower3 chromatography data workstation, LC50 high-pressure preparative liquid chromatograph, UV200 ultraviolet detector (Sepurus (Beijing) Technology Co., Ltd.), YMC-Pack ODS-A chromatographic column (250×10mm.DS-5mm,12mm) (YMC Co., Ltd.), and the rest include N-1100 rotary evaporator (Shanghai Ailang Instrument Co., Ltd.), A-1000S water flow aspirator (Shanghai Ailang Instrument Co., Ltd.), N-1111 chilled water circulation device (Shanghai Ailang Instrument Co., Ltd.), FDU-2110 freeze dryer (Shanghai Ailang Instrument Co., Ltd.), DFZ-60508 vacuum drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.), and AB204-N 1 / 10,000 precision analytical balance (METTLER TOLEDO).

[0042] Column chromatography packing materials included Diaion HP-20, MCI Gel CHP-20 (Mitsubishi Chemical Corporation, Japan), Toyopearl HW-40 (TOSOH Corporation, Japan), and Sephadex LH-20 (Parmacia Biotech). Silica gel H (100-200 mesh, 200-300 mesh) and thin-layer chromatography silica gel (GF254) were produced by Qingdao Ocean Chemical Plant. Chromatographic reagents were produced by Tianjin Siyou Fine Chemicals Co., Ltd., and analytical reagents were produced by Beijing Chemical Plant and Tianjin Third Chemical Reagent Plant. Wolfberry fruit was collected in Ningxia and identified as the dried mature fruit of Lycium barbarum L.

[0043] 2. Structural Identification

[0044] The wolfberry alkaloid A of the present invention is a white or light yellow powder, and the spectrum is shown in FIG. Figure 2-5 , the specific data are as follows:

[0045] HR-ESI-MS spectrum gave the quasi-molecular ion peak m / z 394.138 1[M+H] + (calculated value is 394.139 0), the molecular formula of compound 1 is determined to be C 21 H19 N3O5, the calculated unsaturation degree is 14. The infrared spectrum shows that the compound has free hydroxyl groups (3358cm -1 ) and associated carbonyl groups (1702 and 1662 cm -1 ). Analyze its 1 H-NMR (600MHz, CD3CN) spectrum shows that the compound contains one amino hydrogen proton [δ H 9.84 (1H, s, H-1′)]; 1 aldehyde hydrogen proton [δ H 9.31 (1H, s, 7′-CHO)]; 4 aromatic hydrogen protons [δ H 7.33 (1H, d, J = 7.6, H-5), 7.16 (1H, td, J = 7.6, 1.0, H-6), 7.28 (1H, td, J = 7.8, 1.0, H-7), 7.70 (1H, d, J = 7.8, H-8)]. Based on its chemical shift and coupling constant characteristics, it is inferred that it is an ortho-disubstituted benzene ring unit; [δ H 6.77 (1H, dd, J = 3.6, 2.6, H-3′), 5.77 (1H, dd, J = 3.6, 2.6, H-4′)] are characteristic hydrogen proton signals on the pyrrole ring; in addition, there are two methine [δ H 5.87 (1H, s, H-2), 4.68 (1H, dd, J=9.0, 3.6, H-11)]; 3 methylene groups [δ H 2.99 (1H, d, J = 16.4, H-15a, 3.87 (1H, d, J = 16.4, H-15b), 3.38 (1H, d, J = 15.2, H-6′a), 3.16 (1H, d, J = 15.2, H-6′b), 2.63 (1H, dd, J = 13.8, 3.6, H-10a), 2.59 (1H, dd, J = 13.8, 9.0, H-10b)]; and 1 methoxy group [δ H 3.75 (3H, s, 17-OCH3)]. 13 C-NMR (150 MHz, CD3CN) and HSQC spectra showed a total of 21 carbon signals, including three carbonyl carbon signals [δ C 172.8 (C-12), 166.2 (C-14), 165.7 (C-16)], and one saturated quaternary carbon signal [δ C57.4 (C-3)]. In order to confirm the structure of the compound, its 2D-NMR spectrum was further analyzed, and the HMBC correlations of H-2 with C-3, C-4, C-9 and C-11, H-5 with C-3, H-8 with C-9, H-11 with C-3, H2-10 with C-2 and C-3, and the HMBC correlations of H-5 / H-6 / H-7 / H-8 and H2-10 / H-11 were obtained. 1 H- 1 HCOSY correlation indicated the presence of a hexahydropyrrolo[2,3-b]indole fragment. In addition, the residues from H-2 to N-1 and N-13, H-8 to N-1, and H2-10 to N-13 were 1 H- 15 The presence of this fragment was further confirmed by HMBC correlation of N. Further HMBC correlations of H-3′ with C-2′ and C-5′, H-4′ with C-2′ and C-5′, H2-6′ with C-3, C-4, C-10, C-5′ and N-1′, and H-7′ with C-2′, combined with the H-3′ / H-4′ 1 H- 1 H COSY correlation confirmed the presence of a 5-methyl-2-formylpyrrole fragment, linked to the hexahydropyrrolo[2,3-b]indole fragment via C-3. HMBC correlations of H-10 and H-11 with C-12 indicate that the ester carbonyl (C-12) is linked to ring C via C-11. The remaining structure is a pyrimidine-4,6-dione fragment composed of atoms from rings B and C. HMBC correlations from H2-15 to C-14, C-16, N-1, and N-13 confirm the presence of this fragment. The planar structure of the compound was determined. The relative configuration of the compound was determined by NOESY spectroscopy and a series of NOE experiments. NOE correlations of H-2 / H-6′b, H-2 / H-1′, and H-2 / H-10a indicate that H-2 and H-6′b lie on the same face of the cis-fused rings B and C, consistent with a rigid fusion of the two five-membered rings. Furthermore, the absence of a significant NOE effect for H-2 / H-11 suggests that they are located on opposite sides. Furthermore, the relative configuration of the compound was further confirmed by NMR and DP4+ probability analysis. Finally, the absolute configurations of the compound's 2R, 3R, and 11S were determined by ECD calculations. The structure of the compound was ultimately determined and named Lycium barbarum alkaloid A. Its molecular formula is:

[0046]

[0047] Table 1. 1 H, 13 C,and 15 N NMR spectroscopic data of compounds 1

[0048]

[0049] a Recorded in CD3CN,600MHz for 1 H,150MHz for 13 C, and 60MHz for 15 N.

[0050] 3. Activity Experiment

[0051] 1. Effect of Lycium barbarum alkaloid A on glucose consumption in IR-HepG2 cells

[0052] Experimental methods:

[0053] HepG2 cells in good growth condition were cultured at a rate of 2×10 5 HepG2 cells were seeded at a density of 100 μL / mL in a 96-well plate, with 100 μL of cell suspension per well. The cells were incubated at 37°C with 5% CO₂. After cell attachment, the culture medium was discarded and the cells were washed twice with PBS. Serum-free medium was added for starvation for 24 hours. The culture medium was then discarded and 100 μL of serum-free medium containing 1 μmol / L insulin was added to induce the HepG2 cell insulin resistance model. After successful model establishment, a blank control group, a model control group, a positive drug metformin group (1 mmol / L), and groups with different concentrations of each compound were set up. A blank control group containing only culture medium without cells was also set up. 36 hours after drug intervention, the supernatant from each well was aspirated for glucose content measurement. Glucose content was measured using a glucose assay kit, and cell number was determined using a CCK-8 assay. Relative glucose consumption was calculated. The experiment was repeated three times with six replicate wells per group.

[0054] The experimental results are as follows Figure 6 As shown, compared with the model group, the positive drug metformin group and the wolfberry alkaloid A group can significantly increase the glucose consumption of IR-HepG2 cells (P<0.001), and the effect of wolfberry alkaloid A at a concentration of 1-10 μmol / L is better than that of the positive drug metformin (1 mmol / L). As the concentration of wolfberry alkaloid A gradually increases, the glucose consumption of wolfberry alkaloid A gradually increases, and the effect is best at a concentration of 10 μmol / L. The difference is statistically significant.

[0055] 2. Effects of Lycium barbarum alkaloid A phenolic amides on glucose uptake in IR-HepG2 cells

[0056] Experimental methods:

[0057] HepG2 cells in good growth condition were cultured at a rate of 1×10 5Cells were seeded at a density of 100 μL / mL in a black transparent-bottom 96-well plate with 100 μL of cell suspension per well. After cell attachment, an insulin resistance model was established and drug treatment was performed for 36 h. 2 μL of 5 mmol / L 2-NBDG solution was directly added to the original culture medium and incubated in a 37°C, 5% CO2 incubator for 60 min. The 2-NBDG solution was discarded and the plate was washed twice with pre-cooled PBS. 100 μL of PBS was added and fluorescence images were obtained using a fluorescence microscope. The fluorescence intensity of the images was analyzed using Image J software.

[0058] The experimental results are as follows Figure 7 As shown, Lycium barbarum alkaloid A at 10 μmol / L can significantly increase the ability to absorb glucose (P<0.05).

[0059] 3. Effects of Lycium barbarum alkaloid A on glycogen content, PK and HK activities in IR-HepG2 cells

[0060] Experimental methods:

[0061] The cells were plated at 5×10 5 Cells were seeded at a density of 100 μL / mL in a 12-well plate, with 1 mL of cell suspension per well. After cell attachment, an insulin resistance model was established and drug treatment was performed for 24 hours. Cells were washed twice with PBS and centrifuged at 1000 rpm / min for 5 minutes. The cell pellet was collected in a 1.5 mL centrifuge tube. 500 μL of pre-chilled PBS was added to each EP tube, and cells were disrupted by ultrasound. Protein concentration was measured with a BCA protein concentration assay kit, and glycogen content, PK, and HK activity were measured using glycogen content, PK, and HK activity assay kits, respectively.

[0062] The experimental results are as follows Figure 8 As shown in the results, Lycium barbarum alkaloid A at 10 μmol / L can significantly promote glycogen synthesis and increase the activities of HK and PH (P<0.001).

[0063] As can be seen from the above, the beneficial effects of the present invention are:

[0064] 1. The present invention has discovered new medical uses for Lycium barbarum alkaloid A and opened up a new application field for Lycium barbarum alkaloid A. The invention provides the use of Lycium barbarum alkaloid A in the preparation of a drug for preventing and / or treating diabetes.

[0065] 2. The wolfberry alkaloid A of the present invention has abundant sources, simple preparation process, and can be made into tablets, capsules, powders, granules, injections, etc., which are easy to use.

[0066] 3. Through activity experiments, it was found that the product discovered, Lycium barbarum alkaloid A, has a new skeleton type. It can significantly promote glucose consumption in IR-HepG2 cells at a concentration of 1 μmol / L, and its effect is better than that of the positive drug metformin, with the best effect at 10 μmol / L. It can significantly improve glucose uptake capacity, promote glycogen synthesis, and enhance the activity of key enzymes PK and HK in the glycolysis process. This product lowers blood sugar and regulates sugar metabolism by promoting the absorption and utilization of glucose.

[0067] Experiments have fully demonstrated that Lycium barbarum alkaloid A has potential hypoglycemic activity and can regulate glucose metabolism, thus being used to prevent and treat diabetes. Therefore, the present invention provides a new chemical structure type for the development of new antidiabetic drugs, expands the medicinal value of Lycium barbarum, and opens up a new path for the preparation of drugs for the treatment and prevention of type 2 diabetes mellitus (T2DM). It has excellent development and application prospects and significant economic and social benefits.

Claims

1. A hexahydropyrroloindole alkaloid extracted from wolfberry fruit, having the chemical molecular formula: 。 2. The method for preparing a hexahydropyrroloindole alkaloid extracted from wolfberry fruit according to claim 1, characterized in that: The following steps are involved: (1) 50 kg of dried wolfberry fruit was extracted three times with 95% ethanol by volume, with the amount of ethanol used each time being 3-5 times the weight volume of the wolfberry fruit. Each extraction was performed for 2 hours. The weight volume refers to the solid in kg and the liquid in L. The three extracts were filtered and combined. The extracts were concentrated under reduced pressure to a concentrate containing 0.5 g crude drug / ml. (2) Add the concentrated solution to a macroporous adsorption resin column with a volume of 2 times, adsorb at a flow rate of 4BV / h, first elute with 8 times the volume of distilled water, then with 4BV of 95% ethanol, and finally with 3BV of acetone, collect the 95% ethanol eluate, and concentrate under reduced pressure to a first extract of 1 / 10-12 of the volume of the 95% ethanol eluate, i.e., the 95% ethanol eluate concentrate; (3) The first extract was suspended in 2.5 L of distilled water and extracted with ethyl acetate, 2.5 L each time for 1 h. The extraction was repeated 8 times. The extracts were combined and concentrated under reduced pressure to a second extract with a volume of 1 / 9-12 of the extract volume. (4) The second extract was mixed with an equal amount of 100-200 mesh silica gel, and gradient eluted with a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 100:1, 70:1, 50:1, 30:1, 20:1, 10:1, 5:1, and 1:1 and a dichloromethane-methanol mixed solvent with a volume ratio of 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1, with each gradient dosage of 5-10 L and a flow rate of 10-15 ml / min. Each 500 mL was a fraction, and each fraction was concentrated under reduced pressure. The fractions were analyzed by silica gel thin layer chromatography using GF254 thin layer plates with petroleum ether-ethyl acetate with a volume ratio of 2:1 and dichloromethane-methanol with a volume ratio of 10:1 as the developing solvent, respectively. The analysis was detected at 254 nm by ultraviolet light, and the same fractions were merged according to the thin layer spots to obtain 11 first group components Fr.A, B, C, D-K; (5) The first group fraction Fr.D was passed through an ODS column and eluted with a methanol:water gradient of 10:90, 30:70, 50:50, 70:30, and 90:10 by volume, with each gradient volume of 5 L and a flow rate of 10-15 ml / min, to obtain five second group fractions Fr.D1, Fr.D2, Fr.D3, Fr.D4, and Fr.D5; (6) The second group fraction Fr.D3 was mixed with an equal amount of 100-200 mesh silica gel and eluted with a dichloromethane:methanol mixed solvent with a volume ratio of 100:0, 70:1, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, and 0:1 in sequence by gradient elution. The amount of each gradient was 100-200 mL, the flow rate was 10-15 ml / min, and each 50 mL was a fraction. Each fraction was concentrated under reduced pressure and analyzed by silica gel thin layer chromatography using GF254 thin layer plates with dichloromethane:methanol with a volume ratio of 10:1 as the developing solvent. The analysis was detected at 254 nm by ultraviolet light. The same fractions were merged according to the thin layer spots to obtain four third group fractions Fr.D3.1-D3.

4. (7) The third component Fr.D3.1 was eluted with 30% acetonitrile at a flow rate of 3 mL / min and a volume of 1 L. The elution time was collected. t R The fraction at 12.8 min was concentrated and dried to obtain hexahydropyrroloindole alkaloid A, namely, wolfberry alkaloid A.

3. Use of the hexahydropyrroloindole alkaloid extracted from wolfberry fruit according to claim 1 in the preparation of drugs for preventing and treating type 2 diabetes mellitus (T2DM).

4. The use of the hexahydropyrroloindole alkaloid extracted from wolfberry fruit in the preparation of a drug for preventing and treating type 2 diabetes mellitus (T2DM) according to claim 3, wherein: The medicine is a medicine containing hexahydropyrroloindole alkaloid A, or a medicine for preventing and treating type 2 diabetes mellitus (T2DM) in a pharmaceutically acceptable salt thereof.

5. The use of the hexahydropyrroloindole alkaloid extracted from wolfberry fruit in the preparation of a drug for preventing and treating type 2 diabetes mellitus (T2DM) according to claim 3, characterized in that: The medicine is a tablet, capsule, injection, powder injection, granule, powder, pill, fat emulsion, microcapsule, dripping pill, ointment, sustained-release agent or controlled-release dosage form prepared from hexahydropyrroloindole alkaloid A and a pharmaceutically acceptable carrier.

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