Novel hexahydropyrroloindole alkaloid A extracted from fructus lycii as well as preparation method and application of novel hexahydropyrroloindole alkaloid A

Abstract

The invention relates to a novel hexahydropyrroloindole alkaloid extracted from fructus lycii, which can effectively solve the problems of extracting and preparing the novel hexahydropyrroloindole alkaloid A from the fructus lycii and realizing the application of the novel hexahydropyrroloindole alkaloid A in preparation of medicines for preventing and treating type 2 diabetes mellitus. The novel hexahydropyrroloindole alkaloid A is prepared by the following steps: sequentially eluting with water, ethanol and acetone, carrying out reduced pressure concentration on an ethanol part, extracting, stirring a sample with silica gel, carrying out gradient elution, merging the same fractions, eluting a target component, stirring the sample with silica gel, carrying out gradient elution, collecting fractions within retention time, concentrating and drying, thereby obtaining the novel hexahydropyrroloindole alkaloid A in the preparation of the medicine for preventing and treating the type 2 diabetes mellitus and the application of the novel hexahydropyrroloindole alkaloid A in the preparation of the medicine for preventing and treating the type 2 diabetes mellitus. Glucose consumption in insulin-resistant HepG2 cells can be remarkably promoted, glucose uptake capacity is improved, glycogen synthesis is promoted, activity of glycolysis key enzymes hexokinase and pyruvate kinase is enhanced, development and application prospects are wide, and economic and social benefits are remarkable.

Description

Technical Field

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

[0002] With the influence of multiple factors such as changes in people's diet and lifestyle, the incidence of type 2 diabetes mellitus (T2DM) has increased sharply around the world. It has become a chronic disease that seriously endangers human health after tumors and cardiovascular and cerebrovascular diseases. Although a variety of drugs based on different targets have been launched on the market, these drugs have side effects such as weight gain, hypoglycemia and gastrointestinal reactions. Therefore, it is urgent to find and discover new hypoglycemic lead compounds with novel structures, safety and efficiency.

[0003] Lycium barbarum is the dried mature fruit of Lycium barbarum L., a plant of the genus Lycium in the Solanaceae family. It is sweet in taste, neutral in nature, and enters the liver and kidney meridians. It has the effects of nourishing the liver and kidney, improving the essence and improving eyesight. It has important medicinal value in the treatment of diabetes, but the current reports on its hypoglycemic active ingredients are mostly concentrated on Lycium barbarum polysaccharides and total flavonoids. Hexahydropyrroloindole alkaloids have attracted great attention from plant chemists and medicinal chemists due to their unique tricyclic fused ring system and significant biological activity. At present, about 400 hexahydropyrroloindole alkaloids have been discovered in nature. The reports on their biological activities are mainly concentrated on antiviral, antitumor, anti-malarial parasite, antibacterial and acetylcholinesterase inhibition, while there are no reports on their hypoglycemic activity. Lyciumine A is a new type of hexahydropyrroloindole alkaloid first discovered from wolfberry. It has a complex 6 / 5 / 5 / 6 tetracyclic fused ring system connected to a pyrrole ring. Currently, there is no public report 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 novel hexahydropyrroloindole alkaloid extracted from wolfberry fruit and a preparation method and application thereof, which can effectively solve the problem of extracting and preparing a novel hexahydropyrroloindole alkaloid A from wolfberry fruit and realizing its application in the preparation of drugs for the prevention and treatment of type 2 diabetes (T2DM).

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

[0006]

[0007] The preparation method is:

[0008] The dried wolfberry fruit is extracted by heating and refluxing with ethanol 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 part, and the ethanol part is concentrated under reduced pressure to obtain the first extract; the first extract is extracted with ethyl acetate and concentrated to obtain the second extract;

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

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

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

[0012] The target third group 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 (wolfberry alkaloid A, the same below).

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

[0014] The invention discloses a novel hexahydropyrroloindole alkaloid extracted from wolfberry fruit, a preparation method and application thereof. Through experimental research, it is firstly found that wolfberry alkaloid A with a brand-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), thus providing a brand-new chemical structure type for the development of novel diabetes prevention and treatment drugs, having broad development and application prospects, and can be conveniently prepared into a variety of drug dosage forms, convenient for clinical use, and having 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 is a high-resolution mass spectrum of the hexahydropyrroloindole alkaloid A of the present invention;

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

[0018] Figure 4 is a hydrogen nuclear magnetic resonance 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 The results of the glucose consumption test of the hexahydropyrroloindole alkaloid A of the present invention on IR-HepG2 cells;

[0021] Figure 7 The results are the test results 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 modes of the present invention are described in detail below in combination with embodiments and specific situations.

[0024] Embodiment 1:

[0025] A novel hexahydropyrroloindole alkaloid extracted from wolfberry fruit, the chemical molecular structure of which is:

[0026]

[0027] The preparation method comprises the following steps:

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

[0029] (2) adding the concentrated solution to a macroporous adsorption resin column of 2 times the volume, adsorbing at a flow rate of 4 BV / h, first eluting with 8 times the volume of distilled water, then with 4 BV of 95% ethanol, and finally with 3 BV of acetone, collecting the 95% ethanol eluate, and concentrating under reduced pressure to a first extract (i.e., a 95% ethanol eluate concentrate) of 1 / 10-12 the volume 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, and the extraction was repeated 8 times. The extracts were combined and concentrated under reduced pressure to a second extract whose volume was 1 / 9-12 of the 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 plate, using petroleum ether-ethyl acetate with a volume ratio of 2:1 and dichloromethane-methanol with a volume ratio of 10:1 as developing solvents, respectively, and analyzing under ultraviolet light at 254 nm. The same fractions were merged according to the 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 gradient of methanol:water in a volume ratio of 10:90, 30:70, 50:50, 70:30, and 90:10, 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, respectively. 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 a GF254 thin layer plate and dichloromethane:methanol with a volume ratio of 10:1 as the developing solvent. The analysis was detected at 254 nm of an ultraviolet lamp. The same fractions were merged according to the thin layer spots to obtain four third subgroups Fr.D3.1-D3.4);

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

[0035] The invention discloses an application of the novel hexahydropyrroloindole alkaloid extracted from wolfberry fruit prepared by the above method in preparing 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 medicine for preventing and treating type 2 diabetes (T2DM) with a pharmaceutically acceptable salt or halide.

[0037] The medicine is a tablet, capsule, injection, powder injection, granule, powder, pill, fat emulsion, microcapsule, dripping pill, ointment, sustained release or controlled rapid release preparation prepared from hexahydropyrroloindole alkaloid A (wolfberry alkaloid A) and a pharmaceutically acceptable carrier.

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

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

[0040] 1. Instruments and test drugs

[0041] Bruker AVANCEⅢ500 nuclear magnetic resonance instrument (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, WatersAlliance series 2695 high-performance liquid phase system equipped with 2998 diode array detector, Empower3 chromatography data workstation, LC50 high-pressure preparative liquid chromatograph, UV200 ultraviolet detector [Syplex (Beijing) Technology Co., Ltd.], YMC-Pack ODS-A chromatographic column (250×10mm.DS-5mm,12mm) (YMC Co., Ltd.), the rest include N-1100 rotary evaporator (Shanghai Ailang Instrument Co., Ltd.), A-1000S water flow vacuum pump (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.), AB204-N one-ten-thousandth precision analytical balance (METTLER TOLEDO).

[0042] Column chromatography fillers Diaion HP-20, MCI Gel CHP-20 (Mitsubishi Chemical Company, Japan), Toyopearl HW-40 (TOSOH Company, Japan), Sephadex LH-20 (Parmacia Biotech Company), silica gel H (100-200 mesh, 200-300 mesh) used for column chromatography, thin layer chromatography silica gel (GF254) produced by Qingdao Ocean Chemical Plant, chromatographic reagents used were produced by Tianjin Siyou Fine Chemicals Co., Ltd., analytical reagents used were produced by Beijing Chemical Plant and Tianjin Third Chemical Reagent Factory. Lycium barbarum was collected in Ningxia and identified as the dried mature fruit of Ningxia 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 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 N 3 O 5 The calculated unsaturation degree is 14. The infrared spectrum shows that the compound has free hydroxyl groups (3358 cm -1 ) and associated carbonyl (1702 and 1662 cm -1 ). 1 H-NMR (600MHz, CD 3 CN) spectrum, 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)]. According to 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-OCH 3 )]. 13 C-NMR (150MHz, CD 3 CN) and HSQC spectra show a total of 21 carbon signals, including 3 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 determine the structure of the compound, its 2D-NMR spectrum was further analyzed, H-2 and C-3, C-4, C-9 and C-11, H-5 and C-3, H-8 and C-9, H-11 and C-3, H 2 -10 is associated with the HMBC of C-2 and C-3, as well as H-5 / H-6 / H-7 / H-8 and H 2 -10 / H-11 1 H- 1 HCOSY correlation indicated the presence of a hexahydropyrrolo[2,3-b]indole fragment. In addition, H-2 to N-1 and N-13, H-8 to N-1, H 2 -10 to N-13 1 H- 15 N HMBC correlation further confirmed the existence of this fragment. H-3′ and C-2′ and C-5′, H-4′ and C-2′ and C-5′, H 2 -6′ and C-3, C-4, C-10, C-5′ and N-1′, and H-7′ and C-2′ further HMBC correlation, combined with H-3′ / H-4′ 1 H- 1 H COSY correlation confirmed the existence of 5-methyl-2-formylpyrrole fragment, which is connected to hexahydropyrrolo[2,3-b]indole fragment via C-3. According to HMBC correlation of H-10 and H-11 with C-12, ester carbonyl (C-12) is connected to ring C via C-11. The remaining structure is a pyrimidine-4,6-dione fragment composed of some atoms of ring B and ring C. 2 The HMBC correlation from C-15 to C-14, C-16, N-1 and N-13 confirmed the existence of the fragment. Therefore, 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. The NOE correlations of H-2 / H-6′b, H-2 / H-1′ and H-2 / H-10a showed that H-2 and H-6′b were located on the same face of the cis-fused rings B and C, which is also consistent with the rigid fusion mode of the two five-membered rings. In addition, there was no obvious NOE effect on H-2 / H-11, indicating that they were on opposite sides. At the same time, the relative configuration of the compound was further confirmed by calculating NMR and DP4+ probability analysis. Finally, the absolute configuration of the compound's 2R, 3R, and 11S was determined by calculating ECD. The structure of the compound was finally determined and named Lycium barbarum alkaloid A, and the molecular structure is:

[0046]

[0047] Table 1. 1 H, 13 C,and15 N NMR spectroscopic data of compounds 1

[0048]

[0049] a Recorded in CD 3 CN,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 with good growth status were cultured at 2×10 5 The cells were seeded at a density of 100 μL / mL in a 96-well plate, and each well was incubated at 37°C with 5% CO 2 In the incubator, the cells were attached to the wall and the culture medium was discarded and washed twice with PBS. Serum-free medium was added for starvation for 24 hours. The culture medium was discarded and 100 μL of serum-free medium containing 1 μmol / L insulin was added to induce the HepG2 cell insulin resistance model. The model was successfully established. At the same time, a blank group, a model group, a positive drug metformin group (1mmol / L) and groups of various compounds at different concentrations were set up. A blank group containing only culture medium without cells was set up. After 36 hours of drug intervention, the supernatant of each well was aspirated for glucose content detection. The glucose content was detected using a glucose determination kit, the cell number was detected using a CCK-8 kit, and the relative glucose consumption was calculated. Six replicate wells were set up in each group of the experiment, and repeated 3 times.

[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 could 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 was better than that of the positive drug metformin (1 mmol / L). As the concentration of wolfberry alkaloid A gradually increased, the glucose consumption of wolfberry alkaloid A gradually increased, and the effect was best at a concentration of 10 μmol / L, and the difference was 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 with good growth status were cultured at 1×10 5 The cells were inoculated at a density of 100 μL / mL in a black transparent bottom 96-well plate. Each well contained 100 μL of cell suspension. After the cells adhered to the wall, an insulin resistance model was established and the drug was treated for 36 h. 2 μL of 5 mmol / L 2-NBDG solution was directly added to the original culture medium and incubated at 37°C and 5% CO 2 After incubation in the incubator for 60 min, the 2-NBDG solution was discarded, the plate was washed twice with pre-cooled PBS, 100 μL PBS was added, fluorescence images were obtained using a fluorescence microscope, and 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 can significantly increase the ability to absorb glucose at 10 μmol / L (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 inoculated in 12-well plates at a density of 100 / mL, with 1 mL of cell suspension per well. After cell adhesion, the insulin resistance model was established and the drug treatment was carried out for 24 hours. The cells were washed with PBS twice, centrifuged at 1000 rpm / min for 5 minutes, and the cell pellets were collected in 1.5 mL centrifuge tubes. 500 μL of pre-cooled PBS was added to each EP tube, and the cells were ultrasonically disrupted. The protein concentration was measured by the BCA protein concentration assay kit, and the glycogen content, HK and PK activity were measured by the glycogen content, PK and HK activity assay kits, respectively.

[0062] The experimental results are as follows Figure 8 As shown, 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] It can be seen from the above that the beneficial effects of the present invention are:

[0064] 1. The present invention explores new medical uses for Lycium barbarum alkaloid A and opens up a new application field for Lycium barbarum alkaloid A. 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 experimental research, it was found that the product discovered this time, 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 the ability to absorb glucose, 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] The experiment fully shows that Lycium barbarum alkaloid A has potential hypoglycemic activity and can regulate sugar metabolism, and thus can be used to prevent and treat diabetes. Therefore, the present invention provides a new chemical structure type for the development of new diabetes prevention and treatment drugs, expands the medicinal value of Lycium barbarum, and opens up a new way for the preparation of drugs for the treatment and prevention of type 2 diabetes (T2DM), which has a good development and application prospect and significant economic and social benefits.

Claims

1. A novel hexahydropyrroloindole alkaloid extracted from wolfberry fruit, the chemical molecular structure of which is: 。 2. A method for preparing a novel hexahydropyrroloindole alkaloid extracted from wolfberry fruit, characterized in that: The following steps are involved: (1) 50 kg of dried wolfberry was extracted with 95% ethanol by volume for 3 times under heating and reflux, with the amount of ethanol used each time being 3-5 times the weight volume of the wolfberry, and each time for 2 hours. The weight volume refers to the solid in kg and the liquid in L. The 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 2-fold volume of macroporous adsorption resin column, adsorb at a flow rate of 4 BV / h, first elute with 8-fold volume of distilled water, then with 4 BV volume concentration of 95% ethanol, and finally with 3 BV of acetone, collect the 95% ethanol eluate, and concentrate under reduced pressure to a first extract (i.e., 95% ethanol eluate concentrate) of 1 / 10-12 the volume of the 95% ethanol eluate; (3) suspending the first extract in 2.5 L of distilled water, extracting with ethyl acetate, 2.5 L each time for 1 h, repeating the extraction 8 times, combining the extracts, and concentrating under reduced pressure to obtain the second extract with a volume of 1 / 9-12 of the extract; (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, 1:1 and a dichloromethane-methanol mixed solvent with a volume ratio of 30:1, 20:1, 10:1, 5:1, 1:1, 0:1, each gradient dosage was 5-10L, the flow rate was 10-15ml / min, each 500mL was a fraction, and each fraction was concentrated under reduced pressure. According to silica gel thin layer chromatography, a GF254 thin layer plate was used, and petroleum ether-ethyl acetate with a volume ratio of 2:1 and dichloromethane-methanol with a volume ratio of 10:1 were used as the developing solvents, respectively. The analysis was detected at 254nm of an ultraviolet lamp. 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 component Fr.D was passed through an ODS column and eluted with a gradient of methanol:water in a volume ratio of 10:90, 30:70, 50:50, 70:30, and 90:10, with each gradient volume of 5 L and a flow rate of 10-15 ml / min to obtain five second group components Fr.D1, Fr.D2, Fr.D3, Fr.D4, and Fr.D5; (6) 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, respectively. 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 a GF254 thin layer plate and a dichloromethane:methanol volume ratio of 10:1 as the developing solvent. The analysis was detected at 254 nm by ultraviolet lamp. The same fractions were merged according to the thin layer spots to obtain four third subgroups Fr.D3.1-D3.4); (7) The third group fraction 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 (wolfberry alkaloid A).

3. Use of the novel 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 novel 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 medicine containing hexahydropyrroloindole alkaloid A, or a medicine for preventing and treating type 2 diabetes (T2DM) with a pharmaceutically acceptable salt or halide.

5. The use of the novel 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 or controlled rapid release preparation prepared from hexahydropyrroloindole alkaloid A and a pharmaceutically acceptable carrier.

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