Pair of C-5 carbonyl-containing caged ketone compounds serving as enantiomers as well as preparation method and application of C-5 carbonyl-containing caged ketone compounds

Abstract

The invention relates to the technical field of natural medicines, in particular to a C-5 carbonyl group-containing caged ketone compound extracted from yellow leaves of caulis spatholobi and a preparation method and application of the C-5 carbonyl group-containing caged ketone compound. An in-vitro glucose consumption experiment shows that the compound has an obvious promoting effect on glucose consumption of HepG2-IR cells and can improve the insulin resistance state of the HepG2-IR cells to different degrees. An in-vitro anti-proliferative activity experiment is carried out by adopting a CCK-8 method, and the result shows that the compound has obvious anti-proliferative activity on human non-small cell lung cancer cells A549. The invention provides an alternative compound for developing new hypoglycemic and anti-tumor drugs, which is of great significance to comprehensive development and utilization of the yellow leaves of Caulis cephalanopteris.

Description

Technical Field

[0001] The present invention relates to the technical field of natural medicines, and in particular to a pair of mutually enantiomerically caged polysaccharides containing a C-5 carbonyl group extracted from the leaves of Garcinia macrobracta. Ketone compounds, preparation methods and uses thereof. Background Art

[0002] Garcinia bracteata CYWu ex YHLi, also known as flower bark tree and Wannian fruit (Malipo, Yunnan), is a plant of the genus Garcinia in the family Guttiferae. The fruit of Garcinia bracteata is edible and has extremely high nutritional value. It is rich in vitamins, proteins, minerals and other nutrients. In recent years, through phytochemical studies on its stem bark, leaves, branches and fruits, it was found that the plant contains polycyclic polyprenylated acylphloroglucinols (PPAPs), benzophenone derivatives, There are many types of chemical components, including ketones, flavonoids, triterpenes, steroids, biphenyls, etc. Ketones and PPAPs are its characteristic and active ingredients. The research team previously isolated a series of novel PPAPs and Ketone compounds, and exhibit multiple biological activities such as anti-tumor, anti-inflammatory and hypoglycemic activities.

[0003] Therefore, extracting, isolating and discovering new compounds with medicinal value from Garcinia macrobracta leaves is of great significance to the comprehensive development and utilization of Garcinia macrobracta leaves. Summary of the invention

[0004] In view of this, the present invention provides a pair of enantiomers containing a C-5 carbonyl cage obtained by separation and purification from the leaves of Garcinia macrobracta. Ketone compounds, preparation methods and applications thereof.

[0005] The cage-shaped carbonyl containing C-5 provided by the present invention Ketone compounds, the structural formula is shown in formula (I):

[0006]

[0007] The present invention provides a pair of mutually enantiomeric cage-shaped carbonyl-containing C-5 carbonyls. Ketone compounds, the structural formulas are shown in formula A and B:

[0008]

[0009] The present invention also provides the above The preparation method of ketone compounds comprises the following steps:

[0010] S1. Weigh the dried leaves of Garcinia macrobracta, crush them, extract them with ethanol, and then concentrate the extract under reduced pressure to obtain an ethanol extract;

[0011] S2, dissolving the ethanol extract with methanol, then adding petroleum ether for extraction, and concentrating the lower methanol part under reduced pressure to obtain a methanol extract;

[0012] S3, dissolving the methanol extract with a 9:1 (v / v) mixed solvent of water and methanol, extracting with ethyl acetate, and concentrating the ethyl acetate portion under reduced pressure to obtain an ethyl acetate extract;

[0013] S4. The ethyl acetate extract was subjected to normal phase silica gel column chromatography with gradient elution, and similar components were combined by TLC detection to obtain 13 components, which were labeled A to M in order of polarity from small to large;

[0014] S5. The obtained H component is subjected to medium-pressure ODS reverse phase column chromatography and gradient elution. Similar components are combined by TLC detection to obtain 10 components, which are labeled H-01 to H-10 in descending order of polarity;

[0015] S6. Component H-06 was subjected to normal phase silica gel column chromatography with gradient elution, and similar components were combined by TLC detection to obtain 13 components, which were labeled H-06-01 to H-06-13 in order of polarity from small to large;

[0016] S7, component H-06-10 was segmented by semi-preparative HPLC to obtain 8 components, which were labeled as (H-06-10-01 to H-06-10-08) in order of peak appearance;

[0017] S8 and component H-06-10-08 were separated by semi-preparative high performance liquid chromatography to obtain a racemate of the compound represented by formula (I);

[0018] S9, performing chiral separation on the racemate obtained in step S8 to obtain compound A and compound B.

[0019] Furthermore, in step S4, the conditions for gradient elution of the ethyl acetate extract are: gradient elution using petroleum ether and ethyl acetate in a volume ratio of 10:1, 8:2, 7:3, 6:4, 1:1, 3:7, and 0:1.

[0020] Furthermore, in step S5, the conditions for gradient elution of component H are: gradient elution using water and methanol in a volume ratio of 7:3, 5:5, 4:6, 3:7, 2:8, and 0:1.

[0021] Furthermore, in step S6, the conditions for gradient elution of component H-06 are: gradient elution using petroleum ether and ethyl acetate in a volume ratio of 15:1, 10:1, 6:1, 8:2, 7:3, 6:4, and 1:1.

[0022] Furthermore, in step S7, the chromatographic conditions for component H-06-10 by high performance liquid chromatography segmentation are: using a mixed solvent of acetonitrile and water as the eluent, isocratic elution is performed according to a volume ratio of acetonitrile: water of 51:49, a flow rate of 3 mL / min, and a detection wavelength of 254 nm.

[0023] Furthermore, in step S8, the conditions for high performance liquid chromatography separation of component H-06-10-08 are: using a mixed solvent of acetonitrile and water as the eluent, gradient elution is performed according to the volume ratio of acetonitrile: water of 8:92 (0-45min), 75:25 (45-60min), and 95:5 (60-70min), the flow rate is 3mL / min, and the detection wavelength is 254nm.

[0024] Furthermore, in step S9, the chromatographic conditions for chiral separation are: a detection wavelength of 254 nm, a mixed solvent of n-hexane and isopropanol as the eluent, isocratic elution is performed according to a volume ratio of n-hexane: isopropanol of 95:5, and a flow rate of 2 mL / min to obtain compound A and compound B.

[0025] The above cage Ketone compounds can serve as lead compounds for hypoglycemic and anti-tumor drugs and have further research and development value.

[0026] The advantages and beneficial effects of the technical solution provided by the present invention are as follows: the present invention separates and purifies the ethanol extract of Garcinia macrobracta leaves to obtain a pair of new compounds that are enantiomers of each other, and comprehensively applies a variety of spectral techniques and quantum chemical calculation methods to determine that the new cage-like compound contains a C-5 carbonyl group. Ketone compounds; by conducting in vitro anti-proliferation and glucose consumption experiments on the isolated compounds, it was found that they had significant anti-proliferation activity on A549 and a significant promoting effect on glucose consumption of HepG2-IR cells; the present invention provides a lead compound for the development of new anti-tumor and hypoglycemic drugs, and is of great significance for the comprehensive development and utilization of Garcinia macrobracte leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Preparation of cage-shaped Flow chart of extraction and separation of ketone compounds;

[0028] Figure 2 The product obtained in step S8 of Example 1 1H-NMR (500MHz, CD3OD) spectrum;

[0029] Figure 3 The product obtained in step S8 of Example 1 13 C-NMR (125MHz, CD3OD) spectrum;

[0030] Figure 4 DEPT (Distortionless Enhancement by Polarization Transfer) (θ=90°) spectrum of the product obtained in step S8 of Example 1;

[0031] Figure 5 The DEPT (θ=135°) spectrum of the product obtained in step S8 of Example 1;

[0032] Figure 6 HSQC of the product obtained in step S8 of Example 1 ( 1 H-detected Heteronuclear SingleQuantum Coherence, heteronuclear single quantum coherence correlation spectrum) spectrum;

[0033] Figure 7 is the HMBC of the product obtained in step S8 of Example 1 ( 1 H-detected Heteronuclear MultipleBond Correlation (H-detected Heteronuclear MultipleBond Correlation) spectrum;

[0034] Figure 8 The product obtained in step S8 of Example 1 1 H- 1 H COSY (Correlation spectroscopy) spectrum;

[0035] Fig. 9 HR-ESI-MS spectrum of the product obtained in step S8 of Example 1;

[0036] Fig.10 DP4+ analysis of two possible relative configurations of the compound prepared in Example 1;

[0037] Fig.11 This is the chiral separation chromatogram of step S9 in Example 1;

[0038] Fig.12 CD spectra of compounds A and B prepared in Example 1;

[0039] Fig.13The experimental ECD and calculated ECD spectra of compounds A and B prepared in Example 1;

[0040] Fig.14 The antiproliferative activities of compounds A, B, oridonin and paclitaxel on A549 cells in experiment (I) of Example 2;

[0041] Fig.15 The effect of compounds A and B on the viability of HepG2 cells in experiment (II) of Example 2;

[0042] Fig.16 The effect of the compound of Example 1 on glucose consumption in HepG2 / IR cells, wherein NC: control group; MC: model group; Met: metformin. Statistical data are expressed as mean ± SEM. ### p<0.001, # indicates control group vs model group; ***p<0.001, **p<0.01, *p<0.05, * indicates model group vs drug-treated group. DETAILED DESCRIPTION

[0043] In order to make the objectives, technical solutions and advantages of the present invention more clear, the implementation modes of the present invention are further described below in conjunction with specific embodiments.

[0044] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0045] Example 1

[0046] This example provides a method for extracting The preparation process of ketone compounds includes the following specific steps:

[0047] Step S1. Weigh 20 kg of dried Garcinia bracteata leaves, crush them and extract them three times with 95% (v / v) ethanol, soak them at room temperature (25°C, the same below) for 24 hours each time, filter them after each soaking, collect the extract, combine the extracts after 3 filtrations, and concentrate them under reduced pressure to obtain a total extract (2.29 kg); The Garcinia bracteata leaves used in this embodiment were collected in Xichou County, Wenshan Prefecture, Yunnan Province in September 2021, and were identified by Professor Liu Xinqiao of the School of Pharmacy of South-Central University for Nationalities as Garcinia bracteata CYWu ex YHLi of the Garcinia family. The plant specimens are preserved in the medicinal plant specimen room of the School of Pharmacy of South-Central University for Nationalities, and the specimen number is 20210901.

[0048] Step S2, dissolving the total extract with methanol, then adding petroleum ether for extraction, concentrating the upper petroleum ether portion under reduced pressure to obtain a petroleum ether extract (100 g), and concentrating the lower methanol portion under reduced pressure to obtain a methanol extract;

[0049] Step S3, dissolving the methanol extract again with a 9:1 (v / v) mixed solvent of water and methanol, then adding ethyl acetate for extraction, and concentrating the ethyl acetate portion under reduced pressure to obtain an ethyl acetate extract (430 g).

[0050] Step S4, subjecting the ethyl acetate extract to normal phase silica gel column chromatography, petroleum ether-ethyl acetate gradient elution (petroleum ether: ethyl acetate = 10: 1, 8: 2, 7: 3, 6: 4, 1: 1, 3: 7, 0: 1, v / v), using TLC (developing solvent is dichloromethane: methanol = 20: 1, v / v) to detect and combine similar components to obtain 13 components, which are labeled AM in order from small to large in polarity, and the eluents with a petroleum ether-ethyl acetate volume ratio of 8: 2, 7: 3, and 6: 4 are collected and combined, numbered H, and concentrated to dryness under reduced pressure for standby use.

[0051] Step S5, dry-load the H component (14.27 g), use medium-pressure ODS reverse-phase column chromatography, and perform gradient elution with methanol and water (water: methanol = 7:3, 5:5, 4:6, 3:7, 2:8, 0:1, v / v), and detect and combine similar components by TLC (developing solvent is dichloromethane: methanol = 20:1, v / v), and finally obtain 10 components, which are marked as H-01 to H-10 in descending order of polarity, and the eluate with a water: methanol volume ratio of 3:7 is collected, numbered H-06, and concentrated to dryness under reduced pressure for use.

[0052] Step S6, subjecting component H-06 to normal phase silica gel column chromatography, petroleum ether-ethyl acetate gradient elution (petroleum ether: ethyl acetate = 15: 1, 10: 1, 6: 1, 8: 2, 7: 3, 6: 4, 1: 1, v / v), and using TLC (developing solvent is dichloromethane: methanol = 20: 1, v / v) to detect and combine similar components to obtain 13 components, which are labeled in order from small to large in polarity (H-06-01 to H-06-13), and the eluate with a petroleum ether-ethyl acetate volume ratio of 6:4 is collected, numbered H-06-10, and concentrated to dryness under reduced pressure for later use.

[0053] Step S7, component H-06-10 was segmented by HPLC using a semi-preparative column YMC-Pack ODS-A (250×10 mm, 5 μm), a mixed solution of acetonitrile and water (acetonitrile: water = 51:49, v / v) was used as the elution solvent, the detection wavelength was set to 254 nm, and isocratic elution was performed at a flow rate of 3 mL / min to obtain 8 components, which were labeled (H-06-10-01 to H-06-10-08) in order of peak elution, wherein the retention time of H-06-10-08 was t R =128.258min.

[0054] Step S8, component H-06-10-08 was purified by HPLC using a semi-preparative column YMC-Pack ODS-A (250×10 mm, 5 μm), with acetonitrile and water in a volume ratio of 8:92 (0-45 min), 75:25 (45-60 min), and 95:5 (60-70 min) as elution solvents, the detection wavelength was set to 254 nm, and gradient elution was performed at a flow rate of 3 mL / min to obtain a cage-shaped The racemate of ketone compounds (t R =55.0min).

[0055] Step S9, the racemate obtained in step S8 was separated using a Daicel chiral preparative column CHIRALPAK AD-H (250 mm×10 mm, 5 μm), the detection wavelength was set to 254 nm, n-hexane and isopropanol were used as the mobile phase, and isocratic elution was performed at a volume ratio of n-hexane: isopropanol of 95:5 at a flow rate of 2 mL / min to obtain compound A (t R :13.5min) and compound B (t R :17.6min), see Fig.11 .

[0056] The flowchart of the above steps is shown in Figure 1 .

[0057] The product obtained in this example was subjected to high-resolution mass spectrometry, ultraviolet spectroscopy, optical rotation, and nuclear magnetic resonance analysis to determine the structure of the compound. The physicochemical data and spectral data are as follows:

[0058] Racemate: white amorphous powder, molecular formula is C 30 H 38 O7. HR-ESI-MS m / z 511.2690[M+H] + (calcd for C 30 H 39 O7 + ,511.2690).

[0059] A: (c 0.05, MeOH), ECD (9.80×10 -4 M,MeOH)λ(θ)205(-8.23),323(+5.41)nm;

[0060] B: (c 0.05, MeOH), ECD (9.80×10 -4 M,MeOH)λ(θ)204(+6.39),323(-3.42)nm;

[0061] The NMR data of the compounds are shown in Table (2). 1 H NMR (500MHz, CD3OD) spectrum see Figure 2 , 13 C-NMR (125MHz, CD3OD) spectrum Figure 3 , DEPT (θ=90°) spectrum is shown in Figure 4 , DEPT (θ=135°) spectrum see Figure 5 , HSQC spectrum see Figure 6 , HMBC spectrum see Figure 7 , 1 H- 1 H COSY spectrum see Figure 8 , HR-ESI-MS spectrum see Fig. 9 Based on the above high-resolution mass spectrometry and various NMR data, it can be determined that the planar structure of the compound is 1-O-methyl-8-methoxy-8,8a-dihydroneobractiatin.

[0062] At the same time, it was also found that the C-8 signal in compound (I) was C70.6(d), which is -5ppm higher than 8-methoxy-8,8a-dihydroneobractiatin (J.Nat.Prod.2018,81,749-757), indicating that the configuration of C-8 in compound (I) is inconsistent with 8-methoxy-8,8a-dihydroneobractiatin. The coupling constant between H-8 and H-8a is J=2.5Hz, which further confirms that H-8 and H-8a are in trans. The relative configuration of C-6, C-7, C-17, and C-10a can be determined as rel-6S, 7S, 10aS, and 17S according to the origin and the rigid structure of the ring. Based on the above analysis, compound (I) may have two relative configurations: rel-(6S,7S,8S,8aR,10aS,17S)-Ia and rel-(6S,7S,8R,8aS,10aS,17S)-Ib. 1 H, 13 DP4+ analysis of C-NMR calculation data showed that the possibility of compound Ib reached 100%. In summary, the relative configuration of compound (I) was determined to be rel-(6S, 7S, 8R, 8aS, 10aS, 17S). Using the chiral column separation method, (I) was separated under the chromatographic conditions of 95% n-hexane-5% isopropanol, and finally compound and The absolute configurations of A and B were further determined using the ECD calculation method. The results showed that the experimental ECD curves of A and B had a good fit with the calculated ECD curves of (6S,7S,8R,8aS,10aS,17S)-I and (6R,7R,8S,8aR,10aR,17R)-I, respectively. Therefore, the absolute configurations of compounds A and B were determined to be (6S,7S,8R,8aS,10aS,17S) and (6R,7R,8S,8aR,10aR,17R), respectively.

[0063] Table 1: Chinese and English names of compounds represented by formula (I)

[0064]

[0065] Table 2: Compounds 1 H-NMR and 13 C-NMR data

[0066]

[0067]

[0068] Measured at CD3OD

[0069] Example 2: In vitro antiproliferative activity test

[0070] The compound prepared in Example 1 was subjected to an in vitro antiproliferative activity test.

[0071] Materials and reagents: DMEM high-glucose medium was purchased from Hyclone, USA; penicillin-streptomycin solution was purchased from Hyclone, USA; fetal bovine serum was purchased from Hangzhou Sijiqing Bioengineering Materials Co., Ltd.; CCK-8 kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO, BIOSHARP, USA); glucose solution was purchased from Hyclone, USA; 6mmol / L ready-to-use palmitic acid solution was purchased from Xi'an Kunchuang Technology Development Co., Ltd.; metformin hydrochloride tablets were purchased from Shanghai Bristol-Myers Squibb Pharmaceuticals Co., Ltd.; glucose detection kit was purchased from Nanjing Jiancheng Bioengineering Institute; paclitaxel (PTX, product number: B21695, Shanghai Yuanye Biotechnology Co., Ltd.); oridonin (homemade, purity greater than 98%); Multiskan GO multifunctional microplate reader (Thermo Fisher, USA).

[0072] Test tumor cell lines: human non-small cell lung cancer cell A549, purchased from Nanjing Senbeijia Biotechnology Co., Ltd. (Cat NO: BC-C-HU-009); human liver cancer cell HepG2, purchased from Wuhan Pronocell Life Science Co., Ltd. (Cat NO: CL-0103).

[0073] (I) CCK-8 assay to detect the antiproliferative activity of compounds A and B on A549 cells

[0074] Experimental methods:

[0075] Experimental grouping and dosage setting: Compounds A and B (50 μM) were used as the experimental group, Rubescensine A (50 μM) and Paclitaxel (20 μM) were used as positive drugs, and a control group (an equal volume of serum-free culture medium containing cells but not compounds) and a blank control group (an equal volume of serum-free culture medium without compounds and cells) were set up.

[0076] A549 cells in the logarithmic growth phase were cultured in DMEM high-glucose medium (containing penicillin and streptomycin) containing 10% fetal bovine serum until they grew to about 80% and then A549 cells (1×10 596-well plates were inoculated with 100 μL of PBS (1×, pH=7.6, the same below) and inoculated into 96-well plates with a final volume of 100 μL, and cultured in a 37°C cell culture incubator for 6 hours until the cells adhered. Compounds A and B of Example 1 were prepared with DMSO, with a mother solution concentration of 10 mM, and then diluted with DMEM culture medium to different concentrations. After the cells adhered, the culture medium in the 96-well plate was discarded, and 100 μL of the test compound with a concentration of 50 μmol / L was added to each well after rinsing with PBS (1×, pH=7.6, the same below), and cultured in an incubator at 37°C and 5% CO2 for 48 hours. The supernatant of each culture well was then discarded, and 100 μL of culture medium containing 10% (volume percentage) CCK-8 working solution was added to each well, and cultured for 1 hour at 37°C and 5% CO2, and the absorbance (A) value of each well at 450 nm was measured with an enzyme marker. Six replicate wells were set for each group. The inhibition rate was calculated based on the measured A value. For compounds with an inhibition rate of 50% or more, a gradient concentration (1, 3.125, 6.25, 12.5, 25, 50 μM) was set and the corresponding IC 50 Value (see Fig.14 ).

[0077] Inhibition rate = [(A 对照 -A 实验 ) / (A 对照 -A 空白 )]×100%

[0078] Experimental conclusion:

[0079] from Fig.14 It can be seen that the results of the A549 cell toxicity screening showed that the positive drugs Rubescensine A (50 μM) and paclitaxel (20 μM) and compounds A and B had an inhibition rate of 85-97% on A549 cells at a concentration of 50 μM, showing obvious anti-proliferation activity. The IC 50 The values ​​were 9.20±1.51μM, 1.37±1.11μM, 7.53±2.83μM, and 6.81±1.92μM, respectively.

[0080] (II) Determination of glucose consumption activity

[0081] 1. Effects of Compounds A and B on HepG2 Cell Viability

[0082] Experimental methods:

[0083] Experimental grouping and dosage setting: Compounds A and B (5 and 10 μM) were used as the experimental group, metformin (10 μM) was used as the positive control group, and a control group (an equal volume of serum-free culture medium containing cells but no compounds) and a blank control group (serum-free culture medium without compounds and cells) were set up at the same time.

[0084] The CCK-8 method was used to detect the effect of the compounds on the viability of HepG2 cells. The cells in the logarithmic growth phase were digested with trypsin and prepared with serum-free DMEM medium at a concentration of 1×10 5 100 μL of cell suspension of 5 μM / mL was inoculated in each well of a 96-well plate and cultured at 37°C and 5% CO2. When the cell monolayer adhered to 80%-90%, the supernatant was discarded and 100 μL of serum-free DMEM medium containing different concentrations (5 μM, 10 μM) of compounds was added respectively (the compounds were pre-dissolved in DMSO to prepare a 10 mM stock solution). A control group (an equal volume of serum-free DMEM medium containing cells but not compounds) and a blank control group (serum-free medium without compounds and cells) were set up at the same time. After incubation for 24 hours, the supernatant was discarded and 100 μL of medium containing 10% (volume percentage) CCK-8 working solution was added to each well. The plates were cultured at 37°C and 5% CO2 for 1 hour. Finally, the absorbance (A value) at 450 nm was measured with an enzyme reader and the cell survival rate was calculated. The results are shown in Fig.15 .

[0085] Cell survival rate = [(A s -A b ) / (A c -A b )]×100%

[0086] A s : Experimental group; A c : Control group; A b : Blank control group

[0087] 2. Effects of compounds on glucose consumption in HepG2 / IR cell model

[0088] Experimental grouping and dosage setting: the serum-free DMEM group without compounds and cells was the blank control group; the serum-free DMEM group without compounds but with cells was the control group (NC); the serum-free DMEM group without compounds but with palmitic acid (0.25 mM) and glucose solution (30 mM) was the model group (MC); the serum-free DMEM group containing palmitic acid (0.25 mM), glucose solution (30 mM) and metformin (10 μM) was the positive control group (Met); the serum-free DMEM group containing palmitic acid (0.25 mM), glucose solution (30 mM) and the test compound (10 μM) was the experimental drug group.

[0089] According to the cytotoxicity results of the compounds in Experiment 1, the glucose consumption experiment was conducted on the compounds with a cell survival rate of 90% at a concentration of 10 μM, and the glucose oxidase method was used to determine the effect of the compounds on the glucose consumption of HepG2 / IR model cells induced by palmitic acid (PA).5 The cell suspension of 100 μL / mL was inoculated into a 96-well plate, and each well was incubated at 37°C and 5% CO2 until 80%-90% of the cells were attached to the wall. After the supernatant was discarded, the cells were washed once with PBS, and then 100 μL of serum-free medium was added to each well for starvation for 12 hours. The old culture medium was discarded, and a blank control group, a control group (NC), a model group (MC), a positive drug group (Met) and an experimental drug group were set up respectively, with a final volume of 100 μL. After incubation at 37°C and 5% CO2 for 24 h, 2 μL of the supernatant was aspirated, and 2 μL of glucose calibration solution was aspirated as the calibration group to eliminate the influence of the glucose kit on the absorbance. The supernatant aspirated from each group was added to the well containing 200 μL of glucose detection reagent. After incubation at 37°C and 5% CO2 for 0.5 h, the absorbance was measured at 505 nm using an enzyme reader, and the glucose consumption (GC) of each group was calculated. At the same time, 100 μL of culture medium containing 10% (volume percentage) CCK-8 working solution was added to each well of the original 96-well plate from which 2 μL of supernatant had been aspirated, and the cells were incubated at 37°C and 5% CO2 for 1 h. Finally, the absorbance at 450 nm was measured using an enzyme reader, and the cell survival rate CV of each group was determined (the calculation method was the same as that of Experiment 1). The GC / CV value was used to evaluate the effect of the actual monomer compound on the glucose consumption of HepG2 / IR cells. The experimental results are shown in Fig.16 .

[0090] Experimental conclusion:

[0091] After 24h of co-incubation with palmitic acid and high glucose, the glucose consumption of the MC group decreased significantly compared with the NC group, and the GC / CV value was 2.62±0.23mM, indicating that HepG2 cells produced IR and the model was successfully established. Compared with the MC group, metformin significantly increased glucose consumption. After treatment with 10μM compounds A and B, the glucose consumption of HepG2 / IR cells was promoted to varying degrees, the insulin resistance of HepG2 cells was improved, and good hypoglycemic activity was shown. The GC / CV values ​​were 8.21±0.05mM and 8.31±0.04mM, respectively, showing an effect comparable to that of metformin, which could restore the cell's ability to utilize glucose to the level of the NC group.

Claims

1. A cage-like structure containing a C-5 carbonyl group Ketone compounds, the structural formula of which is:

2. The cage-shaped A ketone compound, characterized in that The structural formula of the compound is:

3. A method for preparing the compound according to claim 2, characterized in that: The following steps are involved: S1. Weigh the dried leaves of Garcinia macrobracta, crush them, extract them with ethanol, and then concentrate the extract under reduced pressure to obtain an ethanol extract; S2, dissolving the ethanol extract with methanol, then adding petroleum ether for extraction, and concentrating the lower methanol part under reduced pressure to obtain a methanol extract; S3, dissolving the methanol extract with a mixed solvent of water and methanol, then adding ethyl acetate for extraction, and concentrating the ethyl acetate portion under reduced pressure to obtain an ethyl acetate extract; S4, subjecting the ethyl acetate extract to normal phase silica gel column chromatography and gradient elution to obtain 13 components A to M; S5. Subject the obtained H component to medium-pressure ODS reverse phase column chromatography and gradient elution to obtain 10 components H-01 to H-10; S6. Perform normal phase silica gel column chromatography on component H-06 and gradient elution to obtain 1313 components from H-06-01 to H-06-1; S7, component H-06-10 was segmented by semi-preparative HPLC to obtain 8 components H-06-10-01 to H-06-10-08; S8 and component H-06-10-08 were separated by semi-preparative high performance liquid chromatography to obtain the racemate of the compound represented by formula (I); S9. The racemate obtained in step S8 is subjected to chiral resolution to obtain compound A and compound B.

4. The preparation method according to claim 3, characterized in that: In step S4, the conditions for gradient elution of the ethyl acetate extract are: using petroleum ether-ethyl acetate as the eluent, gradient elution is performed according to the volume ratio of 10:1, 8:2, 7:3, 6:4, 1:1, 3:7, and 0:1, and similar components are combined by TLC detection, wherein the developing solvent is a mixed solvent of dichloromethane-methanol with a volume ratio of 20:

1.

5. The preparation method according to claim 3, characterized in that: In step S5, the conditions for gradient elution of component H are: using water-methanol as the eluent, gradient elution is performed according to the volume ratio of 7:3, 5:5, 4:6, 3:7, 2:8, and 0:1, and similar components are combined by TLC detection, wherein the developing solvent is a mixed solvent of dichloromethane-methanol with a volume ratio of 20:

1.

6. The preparation method according to claim 3, characterized in that: In step S6, the conditions for gradient elution of component H-06 are: using petroleum ether-ethyl acetate as the eluent, gradient elution is performed according to the volume ratio of 15:1, 10:1, 6:1, 8:2, 7:3, 6:4, and 1:1, and similar components are combined by TLC detection, wherein the developing solvent is a mixed solvent of dichloromethane-methanol with a volume ratio of 20:

1.

7. The preparation method according to claim 3, characterized in that: In step S7, the chromatographic conditions for component H-06-10 by semi-preparative HPLC segmentation are: detection wavelength is 254nm, a mixed solvent of acetonitrile and water is used as the eluent, isocratic elution is performed according to the volume ratio of acetonitrile: water of 51:49, and the flow rate is 3mL / min.

8. The preparation method according to claim 3, characterized in that: In step S8, the conditions for semi-preparative high performance liquid chromatography separation of component H-06-10-08 are as follows: the detection wavelength is 254 nm, a mixed solution of acetonitrile and water is used as the eluent, and gradient elution is performed according to the acetonitrile-water volume ratio of 8:92 from 0 to 45 min, the acetonitrile-water volume ratio of 75:25 from 45 to 60 min, and the acetonitrile-water volume ratio of 95:5 from 60 to 70 min, and the flow rate is 3 mL / min.

9. The preparation method according to claim 3, characterized in that: In step S9, the chromatographic conditions for chiral separation are: a detection wavelength of 254 nm, a mixed solvent of n-hexane and isopropanol as an eluent, isocratic elution according to a volume ratio of n-hexane:isopropanol of 95:5, and a flow rate of 2 mL / min.

10. The cage-shaped Application of ketone compounds in the preparation of anti-tumor drugs; The cage-shaped Application of ketone compounds in the preparation of hypoglycemic drugs.

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