A compound, gynostemma pentaphyllum saponin A, extracted from gynostemma pentaphyllum, a preparation method and application thereof

Abstract

The application discloses a compound, gynostemma pentaphyllum lactone glycoside A, extracted from gynostemma pentaphyllum, a preparation method and application thereof, and effective extraction of the compound gynostemma pentaphyllum lactone glycoside A from gynostemma pentaphyllum, and application in preparation of a medicine with both hypoglycemic and hypolipidemic effects. The preparation method is as follows: drying gynostemma pentaphyllum roots, slicing, heating, reflux extraction, vacuum concentration, sample mixing, gradient elution, chromatography, column loading, separation and purification, and collection of the flow fraction with a retention time, and the compound gynostemma pentaphyllum lactone glycoside A is obtained. The preparation method is easy to operate, the obtained extract has hypoglycemic and hypolipidemic effects, can be used for preparation of a medicine for treating hypoglycemia and hypolipidemia, develops a new medical use of gynostemma pentaphyllum, realizes application in preparation of a medicine with both hypoglycemic and hypolipidemic effects, develops a new medicine with both hypoglycemic and hypolipidemic effects, and has actual popularization and application values.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to a compound extracted from Tongkat Ali, Eurycolactoside A, its preparation method, and its applications. Background Technology

[0002] Tongkat Ali ( Eurycoma longifolia Jack. is an evergreen shrub or small tree belonging to the genus *Codonopsis* in the family Simaroubaceae. Its native habitat is the tropical rainforest of Malaysia, primarily found in the wild. It is also distributed in Indonesia, Vietnam, and Cambodia. The medicinal part of Tongkat Ali mainly comes from its roots, and its active ingredients include quassinolide diterpenes, ferrugin-6-one alkaloids, and... β Tongkat Ali contains primarily carbamate alkaloids, as well as volatile oils, phenylpropanoids, squalene derivatives, triterpenes, amino acids, nucleotides, sterols, saponins, and polysaccharides. Modern pharmacological studies have shown that ketones in Tongkat Ali promote the synthesis of endogenous testosterone, have kidney-tonifying and yang-boosting effects, and strengthen muscles and bones. Its alkaloids, flavonoids, and triterpenoid saponins can regulate hormones, combat fatigue, reduce inflammation, lower uric acid, enhance immunity, and regulate metabolism. To date, there are no publicly available reports of extracting components from Tongkat Ali that simultaneously lower blood sugar and lipids.

[0003] Hyperglycemia and hyperlipidemia are two common metabolic diseases that can be caused by factors such as improper diet, genetics, and aging. Currently, for the treatment of hyperglycemia, medications mainly work by promoting insulin secretion, improving insulin sensitivity, or delaying glucose absorption. For hyperlipidemia, medication selection needs to be individualized according to the type of lipid abnormality. Statins are the first choice for lowering total cholesterol, while fibrates or high-purity fish oil are commonly used to lower triglycerides. If necessary, ezetimibe or PCSK9 inhibitors can be combined, along with appropriate dietary adjustments and exercise. However, whether it is possible to extract active ingredients from Tongkat Ali to prepare drugs with both hypoglycemic and lipid-lowering effects has not yet been publicly reported. Summary of the Invention

[0004] In view of the above situation and to overcome the shortcomings of the prior art, the purpose of this invention is to provide a compound Tongkat Ali extracted from Tongkat Ali, its preparation method and application, which can effectively extract the compound Tongkat Ali and realize its application in the preparation of drugs with both hypoglycemic and lipid-lowering effects.

[0005] Eurycolactoside A, a compound extracted from Tongkat Ali, has the following molecular structure: ; The preparation method is as follows: dried Tongkat Ali root slices are extracted by heating and reflux with ethanol solution, and concentrated under reduced pressure; the sample is loaded, eluted with gradient, and concentrated under reduced pressure to obtain each methanol fraction; the target fraction is precipitated to obtain 6 subfractions; the target subfraction is then chromatographically eluted with gradient, and concentrated under reduced pressure to obtain the second subfraction; the second target subfraction is loaded onto a column, separated and purified, and the fraction with retention time is collected to obtain the compound Tongkat Ali lactone A (Eurycolactoside A).

[0006] The application of the compound Tongkat quassin A, prepared by the above method, in the preparation of drugs with both hypoglycemic and lipid-lowering effects.

[0007] This invention discloses a compound, Tongkat quassin A, extracted from Tongkat Ali. The preparation method is easy to operate, and the resulting extract has hypoglycemic and lipid-lowering effects. It can be used to prepare drugs for treating hypoglycemic and lipid-lowering diseases, opening up new pharmaceutical applications for Tongkat Ali and realizing its application in the preparation of drugs with both hypoglycemic and lipid-lowering effects. It has developed new drugs with both hypoglycemic and lipid-lowering effects and has practical application value. Attached Figure Description

[0008] Figure 1 This is a molecular structural diagram of Tongkat quassin A, the present invention. Figure 2 This invention relates to Tongkat lactone A. 1 H- 1 Dynamic planar structure diagrams of H COSY, HMBC (A) and ROESY (B); Figure 3 The experimental and calculated ECD curves for Tongkat quassin A of this invention are shown. Figure 4 This diagram illustrates the establishment of a HepG2 insulin resistance (IR-HepG2) model induced by high concentrations of D-(+)-glucosamine hydrochloride (GlcN) of tongkat lactone A and the screening of positive drug dosages. (a) The effect of different concentrations of GlcN on HepG2 cell survival rate was detected using the MTT assay (`x±s, n=6). (b) The effect of GlcN on glucose consumption in HepG2 cells (`x±s, n=4). (c) The effect of different concentrations of metformin (Met) on HepG2 cell survival rate was detected using the MTT assay (`x±s, n=6). (d) The effect of Met on glucose consumption in HepG2 cells (`x±s, n=4). Compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01; Figure 5This invention investigates the effects of tongkat lactone A on the viability (x±s, n=6) and glucose consumption (x±s, n=4) of GlcN-induced IR-HepG2 model cells. Compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01; Figure 6 The effect of 0.1 μM tongkat lactone A of this invention on GlcN-induced IR-HepG2 model glycogen synthesis (`x±s, n=4). Compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01; Figure 7 The effect of 0.1 μM tongkat lactone A of this invention on Oil Red O-stained positive lipid droplets in a HepG2 lipid deposition model induced by 0.25 mM palmitic acid and 0.5 mM oleic acid (PA+OA) (x±s, n=3). Compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01; Figure 8 This invention investigates the effect of tongkat lactone A on intracellular TC and TG levels in a PA+OA-induced HepG2 lipid deposition model (x±s, n=4). Compared with the normal group, ** P<0.01; compared with the model group, ## P<0.01. Detailed Implementation

[0009] The specific implementation of the present invention will be described in detail below with reference to examples and specific circumstances.

[0010] The present invention is illustrated by the following embodiments: Eurycolactoside A, a compound extracted from Tongkat Ali, has the following molecular structure: ; The preparation method is as follows: 1.5 kg of dried Tongkat Ali root slices were added to 10 times their weight (volume) of 95% ethanol solution (volume concentration). The weight (volume) refers to kg for solids and L for liquids. The system was heated to a slight boiling reflux state under normal pressure, maintaining the internal liquid phase temperature at 78 °C. Extraction was performed three times, 2 hours each time. The extracts were combined and concentrated under reduced pressure using a rotary evaporator to obtain 69.68 g of extract with a density of 1.13. The extract was loaded onto an MCI column with polyamide at a 1:1 mass ratio. A gradient elution system of 1%, 5%, 30%, 60%, 90%, and 100% methanol / water was used in a medium-pressure preparation system at a flow rate of 15 mL / min. Elution continued until the chromatographic elution curve returned to the baseline and no obvious chromatographic peak was detected. The next gradient elution solvent was then switched. All eluents from each gradient were collected in fractions and concentrated under reduced pressure using a rotary evaporator to obtain six fractions with a relative density of 1.15, designated as M1 in the 1% methanol fraction. The sample contained 36.9430g of methanol, 2.0854g of 5% methanol fraction M2, 6.5247g of 30% methanol fraction M3, 9.0056g of 60% methanol fraction M4, 4.4758g of 90% methanol fraction M5, and 0.7355g of 100% methanol fraction M6. Fraction M3 (6.5247g) was subjected to Sephadex LH-20 chromatography to obtain six subfractions: M3N1 (267.92mg), M3N2 (5.1310g), M3N3 (513.72mg), M3N4 (58.77mg), M3N5 (31.91mg), and M3N6 (0.7355g). 8.51 mg; then the subfractions M3N1, M3N2, and M3N3 were combined and subjected to ODS gradient chromatography in a medium-pressure preparation system. Gradient elution was performed using a methanol / water gradient system at volume ratios of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 70%, 90%, and 100%, with a flow rate of 15 mL / min. Elution was continued at each gradient until the chromatographic elution curve returned to the baseline and no obvious chromatographic peak was detected. The next gradient elution solvent was then switched, and all eluents from each gradient were collected in fractions. The fractions were concentrated under reduced pressure using a rotary evaporator to obtain 19 fractions with a relative density of 1.15, designated as M3N1-g1…M3N1-g9…M3N1-g19. 251.39 mg of fraction M3N1-g9 was taken and analyzed using a Cosmosil 5C18-MS-II column with a mobile phase of CH3CN:H2O at a volume ratio of 10:90–30:70, at a flow rate of 3... The flow rate was measured at mL / min, and the solution was purified by semi-preparative liquid chromatography, with the retention time t collected. R After a fractionation time of 25.20 min, 6.07 mg of the compound eurycolactoside A was obtained.

[0011] The application of the compound Tongkat quassin A, prepared by the above method, in the preparation of drugs with both hypoglycemic and lipid-lowering effects.

[0012] The compounds given in the above embodiments can be prepared in any amount as needed. The given embodiments are only used to illustrate specific implementation methods of the present invention, and are not intended to limit the scope of protection of the present invention. Any technical solutions that are essentially the same as those made by using equivalent substitutions or transformations are within the scope of protection of the present invention.

[0013] This invention discloses a compound, tongkatali lactone A, extracted from tongkat Ali. The preparation method is easy to operate, and the resulting extract has both hypoglycemic and lipid-lowering effects. Experiments have shown very good and beneficial technical results, and the relevant experimental data are as follows: 1. Instruments and reagents: 1.1 Instruments: Triple TOF 6600 high-performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry system (ABSCIEX, USA); Bruker AM-500MHz, TMS internal standard, superconducting nuclear magnetic resonance spectrometer (Bruker, Germany); MCP5100 fully automated polarimeter (Antonpaght, Austria); EVO300 ultraviolet spectrophotometer and Nicolet IS10 infrared spectrometer (Thermo Scientific, USA); LC-52 semi-preparative liquid chromatograph (Saipu Ruisi Beijing Technology Co., Ltd.); N-1100 rotary evaporator and EYELACA-3310 cryogenic condenser circulating pump (Tokyo RIKEN Equipment Co., Ltd.); Atomic 1820C pure water system (Chongqing Moore Water Treatment Equipment Co., Ltd.).

[0014] NuAire NU-5810 cell culture incubator (NuAire, USA); micropipette (Eppendorf, Germany); SpectraMax M2 multi-functional microplate reader (Molecular Devices, Sunnyvale, USA); DL-CJ-2NDI clean bench (Beijing Donglian Haer Instrument Manufacturing Co., Ltd.); fully automated cell analyzer (Shanghai Ruiyu Biotechnology Co., Ltd.); AE2000 inverted microscope (Motic, USA); AB series 0.0001 ppm electronic balance (Shanghai Shangtian Precision Instrument Co., Ltd.); Multifuge X1 Pro general-purpose benchtop centrifuge (Thermo Fisher Scientific, USA); CytoFLEX flow cytometer (Beckman Coulter, USA).

[0015] 1.2. Packing materials, chromatographic columns, drugs, and reagents: MCI Gel CHP-20 column packing material (Mitsubishi Chemical Corporation, Japan, catalog number: 02000-095030, lot number: CP08M01MX); Toyopearl HW-40 packing material (TOSOH Corporation, Japan, catalog number: 0014682, lot number: 40HWFB01Y); ODS gel packing material (TOSOH Corporation, Japan, catalog number: 13371, lot number: AAG12S50); Sephadex LH-20 gel column packing material (GE Healthcare, USA, catalog number: 17-0090-02, lot number: 10276077); preparative Cosmosil 5C18-MS-II column (S-5 μm; 250×20 mm, Nacalai, Japan). Tesque Corporation), normal-phase silica gel column chromatography material (200-300 mesh, Qingdao Ocean Chemical Plant, China, batch number: 0250506) and GF254 silica gel thin-layer plate (Qingdao Ocean Chemical Plant, China, batch number: 2020112), analytical methanol (Tianjin Siyou Fine Chemicals Co., Ltd., China, batch number: 20260113), ethyl acetate (Tianjin Siyou Fine Chemicals Co., Ltd., China, batch number: 20251125), petroleum ether (Tianjin Siyou Fine Chemicals Co., Ltd., China, batch number: 20250421), ethanol (Tianjin Siyou Fine Chemicals Co., Ltd., China, batch number: 20251101), dichloromethane (Tianjin Siyou Fine Chemicals Co., Ltd., China, batch number: 20250402) and chromatographic methanol (Tianjin Siyou Fine Chemicals Co., Ltd., China, batch number: 20260129), and acetonitrile reagent (Tianjin Siyou Fine Chemicals Co., Ltd., China, batch number: 20251110).

[0016] DMEM culture medium (Wuhan Procell Life Sciences Co., Ltd., catalog number: FM150210, batch number: WHB825F281); PBS (Wuhan Servicebio Biotechnology Co., Ltd., catalog number: C4202, batch number: GA25060044722); South American premium fetal bovine serum (LONSA SCIENCE SRL, Uruguay, catalog number: bs711-001S, batch number: VE06899), 0.25% Trypsin-EDTA (Thermo Fisher Scientific, USA, Catalog No.: 25200-072, Lot No.: 2068002), D-(+)-glucosamine hydrochloride (Beyotime Biotechnology Co., Ltd., Catalog No.: ST1222, Lot No.: Z153251020), Metformin Hydrochloride (Shanghai Yuanye Co., Ltd., Catalog No.: B25331, Lot No.: D16GB171362), MTT (Beijing Solarbio Co., Ltd., Catalog No.: M8180, Lot No.: 5550516009), Dimethyl sulfoxide (Shanghai Macklin Co., Ltd., Catalog No.: D806645, Lot No.: :C17413072), Oleic acid + Sodium palmitate high-fat inducer (Xi'an Kunchuang Technology Development Co., Ltd., item number: KC006), Tween-20 (Beijing Solarbio Co., Ltd., item number: T8220, batch number: 1121S0113), Trilatengine X-100 (Beijing Solarbio Co., Ltd., item number: IT9100, batch number: 1111J021), Bovine serum albumin (BSA) (Beijing Solarbio Co., Ltd., item number: A8010, batch number: 5550407004), Simvastatin (Merck, Germany, item number: P HR1438, batch number: LRAD7005), Glucose Assay Kit (Shanghai Rongsheng, catalog number: F006-II, batch number: 20250814101), Glycogen Content Assay Kit (Abbkine, USA, catalog number: KTB1340, batch number: ATYJ13101), Omni-Easy™ Ready-to-Use BCA Protein Quantitative Kit (Shanghai Yamei Biomedical Technology Co., Ltd., catalog number: ZJ102, batch number: 03A21470), Enzymatic Assay Kit for Triglyceride Content (Abbkine, USA, catalog number: KTB2) 200, batch number: ATYL29101), Total Cholesterol (TC) assay kit (Abbkine, USA, catalog number: KTB2220, batch number: ATYG15101), 96-well cell culture plate (Corning, USA, catalog number: CCP-96H, batch number: 04025601), cell culture dish (LABSELECT, catalog number: 12310, batch number: 31525038E), Oil Red O working solution (Solarbio, Beijing, catalog number: G1262, batch number: 2500030003).

[0017] 1.3. Cells: The human hepatocellular carcinoma HepG2 cell line was purchased from Procell Life Sciences Co., Ltd. (Wuhan Procell Life Sciences Co., Ltd.; batch number: CL-0103). 1.4 Medicinal Materials: Tongkat Ali medicinal material was purchased from Malaysia in March 2025 and identified by Henan University of Traditional Chinese Medicine as... Eurycoma longifolia The dried root of Jack. Specimen (No. 202503-EN) is deposited in the Herbarium of Traditional Chinese Medicine and Natural Product Chemistry, School of Pharmacy, Henan University of Traditional Chinese Medicine.

[0018] 2. Method: 2.1 ECD Calculation: Conformation search was conducted using the MMFF94s force field to screen conformations with relative internal energy differences within 3 kcal / mol. Using Gaussian 09 software, based on density functional theory (DFT), these conformations were geometrically optimized at the B3LYP / 6-31G (d, p) calculation level. Based on the Boltzmann distribution, dominant conformations were selected, and combined with the polarized continuum model (PCM), time-dependent density functional theory (TDDFT) was used in the gas phase to perform circular dichroism (ECD) calculations at the B3LYP / 6-31G (d, p) level. After UV correction, the ECD chromatograms were fitted using SpecDis software, and weighted according to the Boltzmann distribution.

[0019] 2.2 Acid hydrolysis and derivatization of sugars: Weigh 1.0 mg of tongkadsori lactone A and add it to 3 mL of a 2 mol·L⁻¹ solution. -1 The mixture was sealed with dilute hydrochloric acid and heated in a 90°C water bath for 2.5 h to allow for complete reaction. After drying, it was extracted three times with 1 mL of distilled water and 1 mL of ethyl acetate, respectively. The aqueous layer was concentrated and dried. 1 mg of L-cysteine ​​methyl ester hydrochloride and 1 mL of pyridine were added. The mixture was sealed and heated in a 60°C water bath for 1.5 h. 20 μL of o-toluene isothiocyanate was added. The mixture was sealed and heated in a 60°C water bath for 1.5 h. The mixture was analyzed by LC-MS. Chromatographic conditions: Phenomenex Kinete C18 (2.6 μm, 2.1 mm × 100 mm); isocratic elution: water (A)-methanol (B), 0–28 min, 28% B; column temperature 40°C, flow rate 0.3 mL·min⁻¹; mass spectrometry conditions: positive ion mode. Tongkat quassin A (t R = 9.33 min) and D-glucose standard derivative (t R The retention times of the glucose in Tongkat quassin A (9.16 min) were basically consistent, and the absolute configuration of glucose in Tongkat quassin A was identified as type D.

[0020] 2.3 Establishment and grouping of a high-concentration D-(+)-glucosamine hydrochloride (GlcN)-induced HepG2 insulin resistance (IR-HepG2) model: Human hepatocellular carcinoma HepG2 cells were cultured under the following parameters: 37°C, 5% CO2, DMEM complete culture medium, and 10% heat-inactivated fetal bovine serum. HepG2 cells from passage 3 to 8 were used in this study.

[0021] This experiment used a high concentration of D-(+)-glucosamine hydrochloride (GlcN) to establish an in vitro cultured human hepatocellular carcinoma cell line IR-HepG2. The stability of the obtained model was then evaluated. After HepG2 cells were cultured in 96-well plates for 24 hours, different concentrations of GlcN (1mM, 5mM, 10mM, 20mM, 40mM, 60mM, 80mM, 100mM) were added to the complete culture medium in the other groups (except for the blank control group). After 24 hours of culture, the medium was replaced with complete culture medium, and the cells were cultured for another 24 hours. The supernatant was collected to detect glucose consumption, and 10mM of GlcN was determined as the model concentration. Then, the optimal dose of metformin hydrochloride (Met) was screened. Similarly, after HepG2 cells were cultured in 96-well plates for 24 hours, different concentrations of Met (0.01mM, 0.1mM, 1mM, 5mM, 10mM, 20mM) were added to the model group in the other groups (except for the blank control group and the model group), and 0.1mM of Met was determined as the positive drug concentration. The cells were subsequently divided into eight groups: a blank control group, a model group [10 mM GlcN], a positive control group [10 mM GlcN + 0.1 mM Met], and groups with different concentrations of the compound [10 mM GlcN + (0.01, 0.1, 1, 5, 10) μM eurycolactoside A]. After 24 hours of treatment, the culture medium was replaced and the cells were cultured for another 24 hours. Cells and supernatant were then collected for MTT and glucose consumption measurements.

[0022] 2.4 Detection of cell viability and glucose consumption: MTT assay was used to determine cell viability. Cell viability was determined using 6 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 μL in 96-well plates and cultured for 24 h. After modeling and drug administration, the culture medium was replaced and cultured for another 24 h. The culture medium was collected, and glucose consumption was measured using a glucose assay kit according to the kit instructions to determine the optimal concentration of eurycolactoside A. After collecting the culture medium, 200 μL of blank culture medium and 20 μL of 5% MTT solution were added to each well. The cells were incubated at 37°C for 4 h, the supernatant was discarded, and 150 μL of dimethyl sulfoxide solution was added to each well. The absorbance was measured at 490 nm using a SpectraMax M2 multimicroplate reader, and the cell viability (%) was calculated. Six parallel wells were set up for each group.

[0023] 2.5 Glycogen content detection: HepG2 cells (2 × 10⁻⁶) were digested with 0.25% Trypsin-EDTA. 6 Cells were prepared by mixing cells (number per well) to form a single-cell suspension. The cell pellet was washed with PBS, and an alkaline solution was added. The cells were then sonicated to disrupt the cell structure, boiled in a water bath for 20 minutes, and cooled with tap water to prepare the glycogen assay solution. Glycogen content was measured using the glycogen content detection kit according to the manufacturer's instructions.

[0024] 2.6 Establishment and grouping of a HepG2 cell lipid deposition model induced by 0.25 mM palmitic acid and 0.5 mM oleic acid (PA+OA): HepG2 cells were fed at a rate of 1×10 6 HepG2 cells were evenly seeded in 6-well plates at a density of cells / well. After 24 h of culture, an in vitro lipid deposition model was established, divided into four groups: control group, model group [0.25 mM palmitic acid (PA) and 0.5 mM moleic acid (OA)], positive control group [0.25 mM PA and 0.5 mM OA + 5 μM simvastatin], and monomer compound group [0.25 mM PA and 0.5 mM OA + eurycolactoside A]. Intracellular TC and TG levels were measured and Oil Red O staining was performed 24 h after each group treatment.

[0025] 2.7 Detection of intracellular TC and TG levels: Twenty-four hours after cell drug intervention, the culture medium was aspirated, washed twice with PBS, and 500 μL of 0.25% Trypsin-EDTA was added to each well for digestion. The cells were then collected by gentle pipetting with cold PBS at 1200 r·min⁻¹. −1 Centrifuge for 3 min. Discard the supernatant, add 500 μL of cell lysis buffer, vortex for 20 s, incubate at room temperature for 10 min until all lipids are dissolved, then centrifuge to collect the protein. Quantitatively determine the total protein concentration of each sample using the BCA protein quantification kit, and perform protein concentration correction. Then, determine the total cholesterol and triglyceride levels according to the kit instructions.

[0026] 2.8 Oil Red O staining: Adipocytes were obtained from 6-well plates induced by 2.7 μm differentiation. The culture medium in the wells was discarded, and the cells were washed twice with PBS. Oil Red O fixative was added for 30 min, followed by two washes with distilled water. The cells were then immersed in 60% isopropanol for 20 s, stained with Oil Red O working solution for 20 min, and the staining solution was discarded. The cells were rinsed with 60% isopropanol until the stroma was clear, and Mayer hematoxylin staining solution was added for nucleus counterstaining for 2 min. The cells were then incubated with Oil Red O buffer for 1 min to allow them to regain their blue color. The buffer was discarded, and the cells were covered with distilled water for microscopic observation of intracellular lipid droplet staining. After dissolving the stained lipid droplets with 60% isopropanol, the A value at 492 nm was measured using a microplate reader. The A value reflects the lipid content.

[0027] 2.9 Statistical Analysis: Statistical analysis was performed on all data using SPSS 22.0 and GraphPad Prism 8 software. Data are expressed as mean ± standard deviation (Means ± SD). One-way ANOVA was used when all groups followed a normal distribution and homogeneous variances, followed by the least significant difference (LSD) test. Nonparametric tests were used if the data did not follow a normal distribution or had unequal variances. A p-value < 0.05 was considered statistically significant.

[0028] 3. Results: 3.1 Structural assessment: Eurycolactoside A, its molecular structure is as follows:

[0029] Brown oily, exhibiting positive optical rotation. α ]20 D+89.73 (c 0.067, MeOH); HR-ESI-MS results confirmed its molecular formula as C 25 H 36 O 10 (m / z 519.2201 [M+Na] + calcd for C 25 H 36 O 10 Na, 519.2206). UV(MeOH) λmax (log ε): 209.0 (2.326), 223.0 (2.150), 229.0 (2.098) nm. Infrared spectroscopy showed that compound 1 contains hydroxyl (3364 cm⁻¹), lactone carbonyl (1658 cm⁻¹), and CO bond (1023, 992 cm⁻¹) groups. The compound was hydrolyzed with 2 mol / L HCl, and the hydrolysis products were analyzed by LC-MS, which detected the presence of D-glucose in the structure.1 H, 13 The CNMR spectrum (Table 1) indicates the presence of one [structure / component]. β -D-glucopyranosyl[ δ 4.37 (1H, d, J = 7.7 Hz, H-1'). 1 The 1H NMR spectrum indicates the presence of three methyl groups in its structure. δ 0.87 (3H, s, H-17), 1.27 (3H, s, H-18), 0.79 (3H, d, J = 7.4 Hz, H-19)], 3 hydroxymethyl groups δ 4.17 (1H, s, H-1), 3.38 (1H, s, H-7), 5.03 (1H, s, H-11), 1 terminal olefin hydrogen δ 4.76 (1H, s, H-16a), 4.85 (1H, s, H-16b), 1 cis double bond 5.66 (1H, d, J =10.1 Hz, H-2), 6.17 (1H, d, J = 10.1 Hz, H-3); 13 C10 NMR and HSQC data indicate the presence of 25 carbon signals, including one ester carbonyl group. δ C 175.0, 4 olefin carbons δ C 128.0, 130.3, 144.8, 110.7 and 4 carbon atoms δ C 81.5, 83.0, 71.7 and 75.1 and a group of glycosyl carbons δ C 99.9, 76.5, 76.9, 73.4, 70.0, 61.2. These observations indicate that the compound is a C14 compound with a glycosyl group and a lactone ring. 19 -Quercusin compounds, at the same time δ H 2.49 (1H, m, H-14) and δ C The HMBC correlation signals of 175.0 (C-15), 26.8 (C-13), 14.7 (C-19), 41.4 (C-8), 21.7 (C-18), and 75.3 (C-12) are combined with those of H-9 / H-10 / H-11 / H-12 / H-13 / H-19. 1 H 1The presence of the lactone ring was confirmed in the H COSY-related coupled spin system. δ H 4.17 (1H,s, H-1) and δ C The association of 99.9 (C-1')HMBC determines the position of the glycosyl linkage ( Figure 2 In addition, the presence of double bonds was further confirmed by H-2 with C-10 and C-4, and H-3 with C-1, C-4 and C-16; the HMBC signals of H-1 with C-10 and C-17, H-6 with C-10, H-7 with C-9, C-14 and C-18, H-11 with C-8 and C-12, H-13 with C-12 and C-19, and H-17 with C-10 and C-5 further confirmed the structure of eurycolactoside A.

[0030] In the ROESY spectrum, H-1 is associated with H-5, H-7, and H-11; H-7 with H-14 and H-18; H-12 with H-10 and H-19; and H-19 with H-14. H-1, H-5, H-7, H-10, H-11, H-14, and H-19 are defined as... β Orientation was determined to clarify the relative configuration of eurycolactoside A. ECD quantum chemical calculations at the B3LYP / 6-31G(d, p) level were performed using the TD-SCF method in the Gaussian 09 package to determine the absolute configuration of eurycolactoside A. The calculated ECD spectra of (1S, 5R, 7R, 8R, 9R, 10S, 11S, 12R, 13R, 14S)⁻¹ are consistent with experimental data, thus defining its absolute configuration, as shown below. Figure 3 As shown. Therefore, according to the IUPAC nomenclature system, this compound was identified as (1S,2R,5S,5aR,6R,7aR,11S,11aS,11bR,12R)-1,6-dihydroxy-5a,11a,12-trimethyl-8-methylene-11-(((2R,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1,5,5a,6,7,7a,8,11,11a,11b-decahydro-2,5-methanonaphtho[1,2-d]oxepin-4(2H)-one, and named Eurycolactoside A.

[0031] Table 1. Eurycolactoside A (Tongkat) 1 H NMR (600 MHz, DMSO) and 13 CNMR (151 MHz, DMSO) data

[0032] 3.2 Activity test: 3.2.1 Establishment of a high-concentration D-(+)-glucosamine hydrochloride (GlcN)-induced HepG2 insulin resistance (IR-HepG2) model: like Figure 4 As shown in Figure a, GlcN concentrations below 10 mM had no significant effect on HepG2 cell viability, while GlcN concentrations above 10 mM significantly reduced cell survival (P < 0.01). Figure 4 As shown in b, compared with the control group, the cellular glucose consumption after treatment with 10 mM GlcN for 24 h was significantly reduced (P < 0.01); therefore, this experiment used 10 mM GlcN treatment for 24 h to establish the IR-HepG2 model. Figure 4 As shown in c, treatment with 0.01 mM, 0.1 mM, and 1 mM metformin (Met) did not significantly reduce cell viability compared to the model group; Figure 4 As shown in d, 0.1 mM Met significantly increased glucose consumption in HepG2 cells (P<0.01); therefore, 0.1 mM Met was selected as the positive control concentration in this experiment.

[0033] 3.2.2 Effect of Eurycolactoside A on glucose consumption in the GlcN-induced IR-HepG2 model: Cell viability was determined using the MTT assay, and cellular glucose uptake was measured using the glucose oxidase method to determine the effective concentration of eurycolactoside A. Figure 5As shown, compared with the control group, the cell viability and glucose consumption in the model group were significantly reduced (p<0.01). 0.01μM-0.1μM eurycolactoside A significantly increased cell viability (p<0.01), and 0.01μM-10μM eurycolactoside A significantly increased glucose consumption (p<0.01). The optimal effective concentration of eurycolactoside A was 0.1μM. Therefore, 0.1μM eurycolactoside A was selected for subsequent experiments.

[0034] 3.2.3 Effect of eurycolactoside A on GlcN-induced glycogen synthesis in the IR-HepG2 model: This experiment investigated the effect of eurycolactoside A on liver glycogen synthesis. Figure 6 As shown, compared with the model group, 0.1 μM eurycolactoside A significantly promoted glycogen synthesis in IR-HepG2 (p<0.01), suggesting that it may improve glucose levels in the insulin resistance model by promoting liver glycogen synthesis.

[0035] 3.2.4 Effect of eurycolactoside A on Oil Red O-stained positive lipid droplets in a HepG2 cell lipid deposition model induced by 0.25 mM palmitic acid and 0.5 mM oleic acid (PA+OA): like Figure 7 As shown, Oil Red O staining was performed 24 h after PA+OA induction. No obvious lipid droplets were observed in the cells of the normal group, while lipid droplets of varying sizes were observed in the cells of the model group. After treatment with 0.1 μM eurycolactoside A, the number of lipid droplets observed under the microscope was significantly less than that in the model group, indicating that eurycolactoside A can inhibit lipid synthesis in hepatocytes and reduce lipid accumulation in hepatocytes.

[0036] 3.2.5. Eurycolactoside A reduces PA+OA-induced intracellular TC and TG accumulation in HepG2 cells: like Figure 8As shown, compared with the normal group, the intracellular TC and TG contents of HepG2 cells in the model group were significantly increased (P<0.01); compared with the model group, the intracellular TC and TG contents of the 0.1 μM eurycolactoside A group were significantly decreased (P<0.01).

[0037] 4. Conclusion: A new compound was extracted, isolated, and purified from Tongkat Ali. The structure of this compound, identified according to the IUPAC nomenclature system, is (1R,2R,5S,5aR,6R,7aR,11S,11aS,11bR,12R)-1,6-dihydroxy-5a,11a,12-trimethyl-8-methylene-11-(((2R,3R,4R,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-1,5,5a,6,7,7a,8,11,11a,11b-decahydro-2,5-methanonaphtho[1,2-d]oxepin-4(2H)-one, and named Eurycolactoside A. (0.1 μM Eurycolactoside A...) A) It can promote glucose consumption in HepG2 hepatocytes, promote liver glycogen synthesis, and inhibit lipid accumulation in hepatocytes. It can be effectively used to prepare hypoglycemic and lipid-lowering drugs, opening up new pharmaceutical applications for Tongkat Ali, realizing its application in the preparation of drugs with both hypoglycemic and lipid-lowering effects, providing technical support for the development of new drugs with both hypoglycemic and lipid-lowering effects, and has practical application value.

Claims

1. A compound extracted from Tongkat Ali, Tongkat lactone A, has the following molecular structural formula: 。 2. The method for preparing the compound Tongkat quassin A according to claim 1, characterized in that: 1.5 kg of dried Tongkat Ali root was sliced ​​and added to 10 times its weight (volume) of 95% ethanol solution. The weight (volume) refers to kg for solids and L for liquids. The system was heated to a near-boiling reflux state under normal pressure, with the internal liquid phase temperature maintained at 78°C. Extraction was performed three times, 2 hours each time. The extracts were combined and concentrated under reduced pressure using a rotary evaporator to obtain 69.68 g of extract with a density of 1.

13. The extract was loaded onto an MCI column with polyamide at a 1:1 mass ratio. A gradient elution system of 1%, 5%, 30%, 60%, 90%, and 100% methanol / water was used in a medium-pressure preparation system at a flow rate of 15 mL / min. Elution continued until the chromatographic elution curve returned to baseline and no obvious peak was detected. The next gradient elution solvent was then switched. All eluents from each gradient were collected and concentrated under reduced pressure using a rotary evaporator to obtain six fractions with a relative density of 1.15: M1 (1% methanol) 36.9430 g, M2 (5% methanol) 2, and M3 (5% methanol). The sample contained 2.0854 g of methanol, 6.5247 g of 30% methanol fraction M3, 9.0056 g of 60% methanol fraction M4, 4.4758 g of 90% methanol fraction M5, and 0.7355 g of 100% methanol fraction M6. Fraction M3 (6.5247 g) was subjected to Sephadex LH-20 chromatography to obtain six subfractions: M3N1 (267.92 mg), M3N2 (5.1310 g), M3N3 (513.72 mg), M3N4 (58.77 mg), M3N5 (31.91 mg), and M3N6 (0.7355 g). 8.51 mg; then the subfractions M3N1, M3N2, and M3N3 were combined and subjected to ODS gradient chromatography in a medium-pressure preparation system. Gradient elution was performed using a methanol / water gradient system at volume ratios of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 70%, 90%, and 100%, with a flow rate of 15 mL / min. Elution was continued at each gradient until the chromatographic elution curve returned to the baseline and no obvious chromatographic peak was detected. The next gradient elution solvent was then switched, and all eluents from each gradient were collected in fractions. The fractions were concentrated under reduced pressure using a rotary evaporator to obtain 19 fractions with a relative density of 1.15, designated as M3N1-g1…M3N1-g9…M3N1-g19. 251.39 mg of fraction M3N1-g9 was taken and analyzed using a Cosmosil 5C18-MS-II column with a mobile phase of CH3CN:H2O at a volume ratio of 10:90–30:70, at a flow rate of 3... The flow rate was measured at mL / min, and the solution was purified by semi-preparative liquid chromatography, with the retention time t collected. R After a fractionation time of 25.20 min, 6.07 mg of the compound Tongkat lactone A was obtained.

3. The use of the compound Tongkat lactone A according to claim 1 in the preparation of a drug with both hypoglycemic and lipid-lowering effects.

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