A polyisoprenyl pyrogallol compound, and a preparation method and application thereof

Abstract

This invention relates to a polyisoprene-based phloroglucinol compound, its preparation method, and its applications, belonging to the field of pharmaceutical technology. The structural formula of the compound is shown below. The preparation method involves extracting the fruit of *Garcinia macrocarpa* in an aqueous organic solvent solution, followed by extraction with petroleum ether and dichloromethane, and then purifying the compound using silica gel column chromatography, ODS column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative high-performance liquid chromatography. The above method is simple and easy to implement, and the resulting compound has a novel structure with a [2,4,1]nonane core structure. It can promote the secretion of insulin in palmitic acid-induced pancreatic β-cells, showing promise for the preparation of drugs for the prevention and treatment of hyperglycemia.

Description

Technical Field

[0001] This invention relates to a polyisoprene-based phloroglucinol compound, its preparation method, and its application. Specifically, it relates to a novel phloroglucinol compound with a [2,4,1]nonane core, extracted and isolated from the fruit of Garcinia pedunculata Roxb. ex Buch.-Ham. (family Clusiaceae), and its preparation method, which has hypoglycemic activity. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Plants in the genus Garcinia contain various types of polyisoprenyl-substituted phloroglucinol compounds (PPAP). Based on their core type, they can be broadly classified into monocyclic polyisoprenyl-substituted acyl-phloroglucinol compounds (MPAP), bicyclic isopentenyl-substituted acyl-phloroglucinol compounds (BPAP), cage-like polyisoprenyl-substituted acyl-phloroglucinol compounds (Caged PPAP), and other types of polyisoprenyl-substituted acyl-phloroglucinol compounds.

[0003] PPAP-type compounds exhibit diverse structures and rich activities, primarily including antitumor, lipid-lowering, anti-inflammatory, antioxidant, antibacterial, antiviral, antileishmaniasis, and hypoglycemic activities. Researchers isolated garcibractinone C from the fruit of *Garcinia bracteata* and tested its hypoglycemic activity using an insulin-resistant liver cancer cell model, showing that this compound significantly promotes glucose uptake. Component analysis of the fruit of *Garcinia pedunculata* Roxb. ex Buch.-Ham. revealed the presence of compounds with other structures, whose structures significantly influence their pharmacological activities. The compounds obtained in this invention have not been reported in other literature and represent a novel compound extracted from the fruit of *Garcinia pedunculata*. Summary of the Invention

[0004] This invention provides a polyisoprene-based phloroglucinol compound, its preparation method, and its application. This invention extracts a novel phloroglucinol compound from the fruit of Garcinia pedunculata Roxb. ex Buch.-Ham., which has a [2,4,1]nonane core structure and can promote insulin secretion in palmitic acid-induced pancreatic β cells.

[0005] A polyisoprene-based phloroglucinol compound has the following structural formula:

[0006]

[0007] Furthermore, the aforementioned polyisoprene-based phloroglucinol compounds were extracted and isolated from the fruit of Garcinia macrocarpa.

[0008] Another object of the present invention is to provide a method for preparing polyisoprene-based phloroglucinol compounds.

[0009] A method for preparing a polyisopentenyl phloroglucinol compound includes the following steps:

[0010] S1: Extract the fruit of Garcinia macrocarpa in an aqueous organic solvent solution, combine the extracts, concentrate under reduced pressure to obtain an extract;

[0011] S2: Disperse the extract obtained in S1 with water, extract with petroleum ether and dichloromethane, concentrate the dichloromethane extract under reduced pressure to obtain dichloromethane extract;

[0012] S3: The dichloromethane extract was further subjected to silica gel column chromatography, ODS column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative high performance liquid chromatography to obtain polyisoprenyl phloroglucinol compounds.

[0013] In the above technical solution, in step S1, the mass-to-volume ratio of the fruit of Garcinia macrocarpa to the aqueous solution of organic solvent is 1:10-20 g / mL.

[0014] In the above technical solution, in step S1, the organic solvent aqueous solution is one of acetone aqueous solution, ethanol aqueous solution, or methanol aqueous solution.

[0015] Furthermore, extraction can be performed by soaking in a 50%–100% acetone aqueous solution three times, for three days each time.

[0016] Alternatively, extraction can be performed three times by heating and refluxing with a 50%–100% aqueous ethanol solution or an aqueous methanol solution, each time for three hours.

[0017] In the above technical solution, in step S2, the volume ratio of water to extract is 2 to 3:1.

[0018] In the above technical solution, in step S2, the extraction is performed 3 to 5 times each using petroleum ether and dichloromethane.

[0019] In the above technical solution, during step S2, the volume ratio of water to petroleum ether and dichloromethane is 1:1 to 2 for each extraction.

[0020] In the above technical solution, the specific process of the silica gel column chromatography method in S3 is as follows: the dichloromethane extract is subjected to gradient elution with a solvent system of dichloromethane-methanol, dichloromethane-acetone or petroleum ether-acetone with a volume ratio of 100:0 to 0:100.

[0021] Furthermore, when using the dichloromethane-methanol solvent system described above, the elution fraction is collected when the volume ratio of dichloromethane to methanol is 100:0 to 100:3 or 100:1 to 100:5.

[0022] Furthermore, when using the dichloromethane-acetone solvent system described above, the elution fraction is collected when the volume ratio of dichloromethane to acetone is 100:1 to 100:5.

[0023] Furthermore, when using the petroleum ether-acetone solvent system described above, the elution fraction is collected when the volume ratio of petroleum ether to acetone is 100:5 to 100:20.

[0024] In the above technical solution, the specific process of the ODS column chromatography method in S3 is as follows: the elution fraction collected by the silica gel column chromatography method is subjected to gradient elution with a methanol-water solvent system with a volume ratio of 20:100 to 100:0 or 30:100 to 100:0, and the elution fraction with a methanol-water volume ratio of 55:100 to 60:100 is collected.

[0025] In the above technical solution, the specific process of the Sephadex LH-20 gel column chromatography method in S3 is as follows: the eluted fraction collected by the ODS column chromatography method is eluted isocratically with methanol or dichloromethane-methanol as the mobile phase to obtain fractions A and B.

[0026] Furthermore, when using dichloromethane-methanol as the mobile phase for isocratic elution, the volume ratio of dichloromethane to methanol is 1:8.

[0027] In the above technical solution, the specific process of the semi-preparative high performance liquid chromatography method in S3 is as follows: the fraction B obtained by the Sephadex LH-20 gel column chromatography method is purified by using acetonitrile-water or methanol-water as the mobile phase with a volume ratio of 80:100 to 86:100 to obtain polyisoprenyl phloroglucinol compounds.

[0028] Furthermore, the chromatographic column used in the semi-preparative high-performance liquid chromatography operation is a COSMOSIL reversed-phase column (ODS, 5μm, 250×20mm), the detection wavelength is 210nm, the flow rate is 2mL / min, and the retention time is 20-30min.

[0029] Another object of the present invention is to provide a pharmaceutical composition comprising the above-described polyisoprene phloroglucinol compound or the polyisoprene phloroglucinol compound prepared by the above-described preparation method.

[0030] Another object of the present invention is to provide the application of the polyisoprene-based phloroglucinol compounds prepared by the above-described method in the preparation of hypoglycemic drugs.

[0031] The beneficial effects of the present invention are as follows: The compound obtained by the present invention has a novel structure with a [2,4,1]nonane core structure, and can promote the secretion of insulin in pancreatic β cells induced by palmitic acid, and has the prospect of preparing drugs for the prevention and treatment of hyperglycemia. Attached Figure Description

[0032] Figure 1 The high-resolution ESI-MS spectra of the compounds obtained in Examples 1-4 are shown.

[0033] Figure 2 The compounds obtained in Examples 1-4 1 1H NMR spectrum (solvent CD3OD).

[0034] Figure 3 The compounds obtained in Examples 1-4 13 C10 NMR spectrum (solvent CD3OD).

[0035] Figure 4 The DEPT135 spectra (solvent CD3OD) of the compounds obtained in Examples 1-4 are shown.

[0036] Figure 5 The HMBC spectra (solvent CD3OD) of the compounds obtained in Examples 1-4 are shown.

[0037] Figure 6 The HSQC spectra (solvent CD3OD) of the compounds obtained in Examples 1-4 are shown.

[0038] Figure 7 The compounds obtained in Examples 1-4 1 H- 1 H COSY spectrum (solvent CD3OD).

[0039] Figure 8 The NOESY spectra (solvent CD3OD) of the compounds obtained in Examples 1-4 are shown.

[0040] Figure 9 The compounds obtained in Examples 1-4 1 1H NMR spectrum (solvent DMSO-d6).

[0041] Figure 10 The compounds obtained in Examples 1-4 13 C10 NMR spectrum (solvent DMSO-d6).

[0042] Figure 11 The HMBC spectra (solvent DMSO-d6) of the compounds obtained in Examples 1-4 are shown.

[0043] Figure 12 The HSQC spectra (solvent DMSO-d6) of the compounds obtained in Examples 1-4 are shown.

[0044] Figure 13 The compounds obtained in Examples 1-4 1 H- 1 H COSY spectrum (solvent DMSO-d6).

[0045] Figure 14 The NOESY spectra (solvent DMSO-d6) of the compounds obtained in Examples 1-4 are shown.

[0046] Figure 15 The graphs show the calculated NMR data analysis of the compounds obtained in Examples 1-4.

[0047] Figure 16 The ECD spectra of the compounds obtained in Examples 1-4 are shown.

[0048] Figure 17 The results of the compounds obtained in Examples 1-4 on palmitic acid-induced insulin secretion from pancreatic β cells are shown, wherein, Figure 17 (A) shows the toxicity results of the compound on INS-1 cells. Figure 17 (B) shows the toxicity of palmitic acid to INS-1 cells. Figure 17 (CD) is an immunofluorescence analysis and quantification diagram of the effect of the compound on palmitic acid-induced insulin secretion in INS-1 cells. Detailed Implementation

[0049] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0050] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0051] Example 1

[0052] A method for preparing a polyisopentenyl phloroglucinol compound includes the following steps:

[0053] 10 kg of Garcinia pedunculata fruit was extracted three times with 100 L of 80% acetone aqueous solution for three days each time. The extracts were combined and concentrated into a paste. The paste was then dispersed at a volume ratio of 2:1 (aqueous phase to paste) and extracted with petroleum ether and dichloromethane. Each organic phase was extracted three times, with a volume ratio of 1:1 (aqueous phase to organic phase). The dichloromethane extract was concentrated under reduced pressure. The resulting dichloromethane extract was separated by silica gel column chromatography at a mesh size of 200–300, using a gradient elution system of dichloromethane-methanol at a volume ratio of 100:0 to 0:100 (dichloromethane:methanol = 100:0, 100:1, 100:3, 1...). 00:5, 100:7, 100:10, 100:20, 100:50, 100:100, 0:100; the eluent fractions with a dichloromethane-methanol volume ratio of 100:0 to 100:3 were separated by ODS column chromatography and eluted with a methanol-water solvent system with a volume ratio of 30:100 to 100:0, specifically methanol:water = 30:100, 40:100, 50:100, 60:100, 70:100, 80:100, 90:100, 100:0; the eluent fraction with a methanol-water volume ratio of 60:100 was separated by Sephadex chromatography. LH-20 gel column chromatography was used for isocratic elution with methanol as the mobile phase to obtain fractions A and B. Fraction B was then purified by semi-preparative high performance liquid chromatography (detection wavelength 210 nm, stationary phase ODS, particle size 5 μm, column parameters 250 × 20 mm, mobile phase 80% acetonitrile-water, flow rate 2 mL / min, retention time 20 min) to obtain polyisoprenyl phloroglucinol compounds (6.1 mg, yield 0.00061‰).

[0054] Example 2

[0055] A method for preparing a polyisopentenyl phloroglucinol compound includes the following steps:

[0056] 15 kg of Gamboge fruit was extracted three times by heating and refluxing with 150 L of 95% ethanol for three hours each time. The extracts were combined and concentrated into a paste. The paste was then dispersed at a volume ratio of 3:1 (aqueous phase to paste) and extracted with petroleum ether and dichloromethane. Each organic phase was extracted three times, with a volume ratio of 1:2 (aqueous phase to organic phase) each time. The dichloromethane extract was concentrated under reduced pressure. The resulting dichloromethane extract was separated by silica gel column chromatography using a 200–300 mesh filter. Gradient elution was performed using a dichloromethane-acetone solvent system with a volume ratio of 100:0 to 0:100 (dichloromethane:methanol = 100:0, 100:1, 100:3, 100:5). The eluent fractions with a dichloromethane-acetone volume ratio of 100:1 to 100:5 were separated by ODS column chromatography and eluted with a methanol-water solvent system at a volume ratio of 20:100 to 100:0, specifically methanol:water = 20:100, 30:100, 40:100, 50:100, 60:100, 70:100, 80:100, 90:100, 100:0. The eluent fraction with a methanol-water volume ratio of 60:100 was separated by Sephadex chromatography. LH-20 gel column chromatography was used for isocratic elution with methanol as the mobile phase to obtain fractions A and B. Fraction B was then purified by semi-preparative high performance liquid chromatography (detection wavelength 210 nm, stationary phase ODS, particle size 5 μm, column parameters 250 × 20 mm, mobile phase 86% methanol-water, flow rate 2 mL / min, retention time 26 min) to obtain polyisoprenyl phloroglucinol compounds (7.0 mg, yield 0.0007‰).

[0057] Example 3

[0058] Take 20 kg of Gamboge fruit and extract three times with 400 L of 75% ethanol under reflux for three hours each time. Combine the extracts and concentrate them into a paste. Evaporate until no ethanol odor remains. Disperse the paste at a volume ratio of 3:1 (aqueous phase to paste). Extract with petroleum ether and dichloromethane, four times for each organic phase, with a volume ratio of 1:2 (aqueous phase to organic phase each time). Concentrate the dichloromethane extract under reduced pressure. Separate the obtained dichloromethane extract by silica gel column chromatography (200-300 mesh) at a volume ratio of 1:1. A gradient elution was performed using a petroleum ether-acetone solvent system with a volume ratio of 0:0 to 0:100, specifically at dichloromethane:methanol ratios of 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:50, 100:100, and 0:100. The eluted fractions with a petroleum ether-acetone volume ratio of 100:5 to 100:20 were then separated by ODS column chromatography. Gradient elution was performed using a methanol:water solvent system with a volume ratio of 30:100 to 100:0, specifically methanol:water = 30:100, 45:100, 55:100, 60:100, 70:100, 80:100, 90:100, and 100:0. The eluent fractions with a methanol:water volume ratio of 55:100 to 60:100 were then separated by Sephadex LH-20 gel column chromatography, using methanol... Isocratic elution was performed on the mobile phase to obtain fractions A and B. Fraction B was then purified by semi-preparative high-performance liquid chromatography (210 nm detection wavelength, ODS stationary phase with a particle size of 5 μm, column parameters of 250 × 20 mm, 82% acetonitrile-water mobile phase, flow rate of 2 mL / min, and retention time of 22 min) to obtain polyisoprenyl phloroglucinol compounds (8.3 mg, yield of 0.00083‰).

[0059] Example 4

[0060] 15 kg of Gamboge fruit was extracted three times by heating and reflux with 300 L of 75% methanol for three hours each time. The extracts were combined and concentrated into a paste. The paste was then dispersed at a volume ratio of 2:1 (aqueous phase to paste) and extracted with petroleum ether and dichloromethane, five times per organic phase, with a volume ratio of 1:2 (aqueous phase to organic phase each time). The dichloromethane extract was concentrated under reduced pressure. The resulting dichloromethane extract was separated by silica gel column chromatography at a mesh size of 200–300. The extracts were then mixed at a volume ratio of 100:1. A gradient elution was performed using a dichloromethane-methanol solvent system with a volume ratio of 0 to 100:100, specifically dichloromethane:methanol = 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:50, 100:100, and 0:100. The eluted fractions with a dichloromethane-methanol volume ratio of 100:1 to 100:5 were then separated by ODS column chromatography at a volume ratio of 3... Gradient elution was performed using a methanol-water solvent system with a ratio of 0:100 to 100:0, specifically methanol:water = 30:100, 45:100, 55:100, 60:100, 70:100, 80:100, 90:100, and 100:0. The eluent fraction with a methanol-water volume ratio of 60:100 was separated by Sephadex LH-20 gel column chromatography, using a dichloromethane-methanol mixture with a volume ratio of 1:8. The solvent system was used as the mobile phase for isocratic elution to obtain fractions A and B. Then, fraction B was purified by semi-preparative high performance liquid chromatography (detection wavelength 210 nm, stationary phase ODS, particle size 5 μm, column parameters 250 × 20 mm, mobile phase 85% methanol-water, flow rate 2 mL / min, retention time 30 min) to obtain polyisoprenyl phloroglucinol compounds (7.3 mg, yield 0.00073‰).

[0061] The polyisopentenyl phloroglucinol compounds obtained in Examples 1-4 were analyzed by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. 1 HNMR, 13 The structure was identified using methods such as C NMR and 2D-NMR, and the results are as follows:

[0062] The compound obtained above was dissolved in methanol and then precipitated as a yellow powder with a specific optical rotation [α]20D of -86.64 (c 0.30MeOH). HR-ESI-MS spectrum ( Figure 1 The quasi-molecular ion peak is given as m / z 641.3442 [M+Na]. + (calcdfor C 38 H 50 The molecular formula of this compound was determined to be C7Na, 641.3454. 38 H 50 O7 has an unsaturation degree of 14.1 HNMR (600MHz, CD3OD, Table 1) spectrum Figure 2 ) and HSQC spectrum ( Figure 6 The results show: one set of ABX-coupled aromatic proton signals δ 7.29 (1H, d, J = 2.1 Hz, H-12), 7.16 (1H, dd, J = 8.3, 2.1 Hz, H-16), and 6.76 (1H, d, J = 8.3 Hz, H-15); two sets of isopentenyl proton signals δ 5.16 (1H, m, H-25), 2.03 (1H, m, H-24), and 1.68 (3H, s, ... CH3-28), 1.57 (3H,s,CH3-27) and 1.29 (1H,m,H-24); δ 5.14 (1H,m,H-35), 2.18 (1H,m,H-34), 1.74 (3H,s,CH3-38), 1.70 (1H,m,H-34) and 1.63 (3H,s,CH3-37); one olefin proton signal δ 6.15 (1H,d,J= 1.9 Hz, H-17); two sets of methylene proton signals δ 2.06 (1H,dd,J=14.4,4.2 Hz, H-7) and 1.93 (1H,dd,J=14.4,4.2 Hz, H-7), 1.76 (1H,m,H-29) and 1.65 (1H,m,H-29); three methine proton signals δ 5.07 (1H,d,J=1.9 Hz, H-18), 1. 67 (1H,m,H-30) and 1.46 (1H,m,H-6); there are also six methyl proton signals δ 1.32 (3H,s,CH3-22), 1.25 (3H,s,CH3-32), 1.21 (3H,s,CH3-33), 1.17 (3H,s,CH3-20), 1.16 (3H,s,CH3-23) and 1.00 (3H,s,CH3-21). 13 CNMR (150MHz, CD3OD, Table 1) spectrum Figure 3 ) and DEPT135 spectrum ( Figure 4 The paper provides 38 carbon signals, including 16 sp signals. 2 Hybridized carbon signal and 22 sp 3 Hybridized carbon signals, of which 16 sp... 2 The hybrid carbon signals have two carbonyl carbon signals at δ 203.86 (C-9) and 190.75 (C-10); and 22 sp... 3Among the hybrid carbon signals, the values ​​of the four methylene carbon signals are δ36.40 (C-29), 32.02 (C-7), 30.91 (C-34), and 29.20 (C-24); the values ​​of the three methine carbon signals are δ87.63 (C-18), 45.97 (C-6), and 42.80 (C-30); and the values ​​of the ten methyl carbon signals are δ30.61 (C-22), 30.19 (C-32), 26.02 (C-20), 26.28 (C-38), 26.84 (C-23), 25.97 (C-28), 23.56 (C-21), 20.85 (C-33), 18.04 (C-27), and 18.01 (C-37). Based on the above information, it is speculated that the compound is a polyisopentenyl-substituted acyl phloroglucinol compound.

[0063] The planar structure of the compound was determined by HMBC spectroscopy. Figure 5 ) and HSQC spectrum ( Figure 6 The hydrogen and carbon spectral signals were determined and assigned based on these spectra. In the HMBC spectrum ( Figure 5In the above, δ7.29 (1H, d, J = 2.1Hz, H-12) shows long-range correlation with δ190.75 (C-10), 153.07 (C-14), 146.67 (C-13), and 125.08 (C-16); δ7.16 (1H, dd, J = 8.3, 2.1Hz, H-16) shows long-range correlation with δ190.75 (C-10), 153.07 (C-14), and 116.57 (C-12); and δ6.76 (1H... The long-range correlation between δ1H, dd, J = 8.3 Hz, H-15 and δ153.07 (C-14), 146.67 (C-13), 130.98 (C-11) indicates the presence of 3,4-dihydroxybenzoyl; δ2.06 (1H, dd, J = 14.4, 4.2 Hz, H-7) and 1.93 (1H, dd, J = 14.4, 4.2 Hz, H-7) and δ203.86 (C-9), 73.19 (C-3), 45.97 (C-6) indicates the presence of 3,4-dihydroxybenzoyl; Long-range correlations exist at δ1H, d, J = 1.9 Hz, H-17, 39.94 (C-5), 36.40 (C-29), and 29.20 (C-24). Long-range correlations also exist between δ2.03 (1H, m, H-24) and 1.29 (1H, m, H-24) and δ45.97 (C-6), 39.94 (C-5). Long-range correlations also exist between δ6.15 (1H, d, J = 1.9 Hz, H-17) and δ150.45 (C-4), 73.19 (C-3), and 39.94 (C-5). Long-range correlations were found between δ1.76 (1H,m,H-29) and 1.65 (1H,m,H-29) and δ73.19 (C-3), 45.93 (C-8), 32.02 (C-7). Furthermore, long-range correlations were found between δ1.32 (3H,s,CH3-22) and 1.16 (3H,s,CH3-23) and δ150.45 (C-4), 39.94 (C-5), 45.97 (C-6) when using deuterated dimethyl sulfoxide (DMSO) spectra. Figures 9-14 It was found that δ1.46 (1H,m,H-6) was correlated with δ150.45 (C-4), 125.00 (C-25), 45.93 (C-8), 39.94 (C-5), 32.02 (C-7), and 29.20 (C-24), indicating the presence of a C3-C4-C5-C6-C7-C8-C9 cycloheptane core; δ2.03 (1H,m,H-24) and 1.29 (1H,m,H-24) were long-range correlated with δ133.01 (C-26), 125.00 (C-25), 45.97 (C-6), and 39.94 (C-5), and... 1 H- 1 H COSY spectrum ( Figure 7 and Figure 13The results show correlations between H-25, H2-24, H-6, and H2-7, indicating a single spin-coupled system, suggesting a single isopentenyl group attached to the 6-position of the cycloheptane core. Long-range correlations are observed between δ6.15 (1H, d, J = 1.9 Hz, H-17) and δ150.45 (C-4), 87.63 (C-18), 73.19 (C-3), and 39.94 (C-5), with a correlation between δ5.07 (1H, d, J = 1.9 Hz, H-17). Long-range correlations were found between δ150.45 (C-4), 125.06 (C-17), 26.02 (C-20), and 23.56 (C-21) for z, H-18, and δ150.45 (C-4), 125.06 (C-17), 26.02 (C-20), and 23.56 (C-21) for the two methyl groups. Long-range correlations were also found between δ1.17 (3H,s,CH3-20) and 1.00 (3H,s,CH3-21) for z, H-18, and δ87.63 (C-18) and 72.06 (C-19) for δ125.06 (C-21). (C-17), 87.63 (C-18), 72.06 (C-19), suggesting the presence of a 2-(1-hydroxy-1-methylethyl)-2,5-dihydrofuran ring fragment, fused at positions 3 and 4 of the cycloheptane core; δ 1.76 (1H,m,H-29) and 1.65 (1H,m,H-29) and δ 78.19 (C-31), 73.19 (C-3), 45.93 (C-8), 42.8 Long-range correlations exist between 0 (C-30) and 32.02 (C-7); long-range correlations exist between the two methyl groups at δ 1.25 (3H,s,CH3-32) and 1.21 (3H,s,CH3-33) and δ 78.19 (C-31) and 42.80 (C-30); long-range correlations exist between δ 2.18 (1H,m,H-34) and 1.70 (1H,m,H-34) and δ 39.94 (C-30). 1 H- 1 In the 1H COSY spectrum, H2-29, H-30, H2-34, and H-35 show correlation, indicating they belong to the same spin-coupled system. This suggests that the 2,2-dimethyltetrahydropyran fragment is coupled at positions 1 and 8 of the cycloheptane core, with this isopentenyl group attached at position 30. Based on the molecular formula, two unsaturations and two carbon atoms remain, with signals of δ186.85 and 117.36, respectively. This suggests the presence of a carbon-carbon double bond forming a ring, with δ186.85 representing the sp bond of an oxygen atom at position 1. 2 The hybrid carbon signal, δ117.36, is the carbon signal at position 2 where the benzoyl group is attached. Therefore, this compound is a phloroglucinol compound with a [2,4,1]nonane core.

[0064] In NOESY spectrum ( Figure 8 and Figure 14In the [2,4,1]nonane core, CH3-22 is correlated with H-6 and H-7β, suggesting that the hydrogen atoms and groups are coplanar with the β orientation. Since the [2,4,1]nonane core is a rigid structure, the carbonyl group at position 9 is designated as being above the C1-C2-C3-C4-C5-C6-C7-C8 cyclooctane core, while the carbon atom at position 29 and the oxygen atom at position 3 are located below the cyclooctane core. The hydrogen signals of CH3-23 and H-24 are correlated, indicating that the isopentenyl group at position 6 is located below the cyclooctane core. Based on the etymological relationship, it is inferred that the [2,4,1]nonane core of this compound is formed by the isomerization of the [3,3,1]nonane core of the isolated known compound cambogin. Therefore, it is inferred that the absolute configurations of C-3, C-6, C-8, and C-30 are the same. Thus, the possible absolute configurations of this compound are (3R,6R,8S,18R,30S) and (3R,6R,8S,18S,30S), named 1a and 1b, respectively. The configuration of this compound was determined by calculated NMR at the mPW1PW91 / 6-311+G(d,p) level using density functional theory. The linear regression coefficient (R²) between the measured and calculated carbon spectra was calculated. 2 ) and DP4+ probability assessment of measured and calculated NMR data ( Figure 15 The probability of the absolute configuration being 1b is 100.00%, thus confirming that (3R, 6R, 8S, 18S, 30S) is the most likely configuration of this compound. To further determine the absolute configuration, the ECD spectrum of 1b was calculated using the B3LYP / 6-31G(d,p) theoretical level. Figure 16 The absolute configuration of the compound was determined to be (3R, 6R, 8S, 18S, 30S) by comparing measured and calculated ECD spectra.

[0065] Table 1 Compounds 1 H NMR and 13 Chemical shift values ​​of 10⁻⁶ C NMR (solvents: CD₃OD and DMSO-d₆)

[0066]

[0067] In summary, the structural formula of this compound is shown below. It is a new compound not reported in the literature and is named (-)garpedvinin A.

[0068]

[0069] Experiments on the in vitro activity of the compounds obtained in Examples 1-4 in promoting insulin secretion in palmitic acid-induced pancreatic β-cells:

[0070] 100 U / mL penicillin, 100 μg / mL streptomycin, 50 μM β-mercaptoethanol, 1 mM pyruvate, 10 mM HEPES, 2 mM L-glutamine and 2 g / L sodium bicarbonate were added to RPMI-1640 medium containing 10% (V / V) heat-inactivated fetal bovine serum. Then, pancreatic β-cell line INS-1 cells were incubated in the above-prepared medium in an incubator at 37°C and 5% CO2 saturated humidity.

[0071] The effect of the test compound and palmitic acid on the viability of INS-1 cells was detected by the MTT assay: INS-1 cells in logarithmic growth phase were seeded at a rate of 20,000 per well in a 96-well cell culture plate and cultured at 37°C with 5% CO2 for 48 h. Then, different concentrations of the test compound (0.1, 1, 10, 20, 30 μM) and palmitic acid (0.1, 1, 5, 10, 20, 30 μM) were added according to the grouping. After 24 h of treatment, MTT solution (0.5 mg / mL) diluted with serum-free culture medium was added, and the reaction was continued in the incubator for 2–4 h. The cell culture plate was then removed, and the unreacted supernatant mixture was discarded. 150 μL of 1640 culture medium was added to each well, and the mixture was shaken thoroughly to completely dissolve the MTT. Absorbance reflects cell viability. The absorbance of the solution was measured at a wavelength of 492 nm. Data processing was performed using a microplate reader and corresponding software. The formula is as follows:

[0072]

[0073] Insulin secretion was quantified using immunofluorescence confocal microscopy. Clean coverslips were placed in 6-well plates, and INS-1 cells were cultured. After cell growth on the slides, cells were processed normally according to their assigned groups. The supernatant was discarded, and the cells were washed twice with PBS to remove residual culture medium. Then, 500 μL of paraformaldehyde solution was added to each well for fixation for 20 min. The cells were then washed three times with PBS to remove any remaining paraformaldehyde, being careful to add the solution along the wall of the 6-well plate to prevent cells from being washed off the slides. The washing liquid was discarded. Next, 1 mL of 0.15% Triton solution was used to punch up the wells, and the cells were incubated for 15 min. The cells were then washed three times with PBS. The slides were removed, the edges were wiped clean, and immunohistochemical circles were drawn. The slides were then returned to the 6-well plates, and blocking buffer (PBS containing 10% serum) was added at 200 μL per well for 30 min. Primary antibody insulin B (1:200) was prepared in PBS and incubated overnight at 4°C.

[0074] The slides were equilibrated at room temperature for 30 min. They were then washed three times with TBST to remove unbound primary antibody, followed by three washes with PBS to remove Tween residue. Subsequent steps involving the incubation of the fluorescent secondary antibody were performed in a dark room. A 1:500 concentration of fluorescent secondary antibody was prepared in PBS, 100 μL per slide. After incubation in the dark for 2 h, the slides were washed three times with PBS to remove unbound fluorescent secondary antibody. Then, 100 μL of DAPI (1:10000) prepared in PBS was added to each well for incubation. After 10 min, the slides were washed three times with PBS to remove excess DAPI. A fluorescence quencher was placed on the slide, the coverslip was removed, the cell side was inverted onto the slide, and the slide was sealed with nail polish and placed in a dark cassette for analysis. Following the laser confocal microscope operating procedure, fluorescence images were obtained using a fluorescence microscope, and quantitative fluorescence analysis was performed using ImageJ software.

[0075] The main function of pancreatic β cells is to synthesize and release insulin. When pancreatic β cells are damaged, it can lead to dysfunction. Figure 17 (A) shows the toxicity results of the compound on INS-1 cells. Figure 17 (B) shows the toxicity of palmitic acid to INS-1 cells, indicating that this compound can protect pancreatic β cells at 10 μM. Insulin levels in INS-1 cells were examined using immunofluorescence microscopy, and the results are shown below. Figure 17 As shown in (C) and (D), under 10 μM conditions, compared with the control group and the blank group, this compound showed a certain promoting effect on insulin secretion in palmitic acid-induced pancreatic β cells, and has the potential to be used to prepare clinical drugs for the prevention and treatment of hyperglycemia.

Claims

1. A polyisoprenyl depsidoid compound, characterized by: The structural formula of the compound is as follows: 。 2. A method for preparing the polyisoprene-based phloroglucinol compound as described in claim 1, characterized in that: The polyisoprenyl phloroglucinol compound is extracted and separated from Garcinia xanthochymus fruit, including the following steps: S1: Extracting the fruit of Garcinia xanthochymus in an organic solvent aqueous solution, combining the extract, and concentrating under reduced pressure to obtain an extract; S2: Dispersing the extract obtained in S1 with water, extracting with petroleum ether and dichloromethane, and concentrating under reduced pressure to obtain a dichloromethane extract; S3: The dichloromethane extract is further subjected to silica gel column chromatography, ODS column chromatography, Sephadex LH-20 gel column chromatography, and semi-preparative high performance liquid chromatography to obtain the compound.

3. The method of claim 2, wherein: In S1, the fruit can be soaked and extracted three times with 50% to 100% acetone aqueous solution, each time for three days; or the fruit can be heated and refluxed three times with 50% to 100% ethanol aqueous solution or methanol aqueous solution, each time for three hours.

4. The preparation method of claim 2, characterized in that: In S3, the specific process of the silica gel column chromatography method is as follows: the dichloromethane extract is gradient eluted with a solvent system of dichloromethane-methanol, dichloromethane-acetone, or petroleum ether-acetone at a volume ratio of 100:0 to 0:100; When using the dichloromethane-methanol solvent system, the elution part at a volume ratio of 100:0 to 100:3 or 100:1 to 100:5 of dichloromethane-methanol is collected; When using the dichloromethane-acetone solvent system, the elution part at a volume ratio of 100:1 to 100:5 of dichloromethane-acetone is collected; When using the petroleum ether-acetone solvent system, the elution part at a volume ratio of 100:5 to 100:20 of petroleum ether-acetone is collected.

5. The method of claim 2, wherein: In S3, the specific process of the ODS column chromatography method is as follows: the elution part collected by the silica gel column chromatography method is gradient eluted with a methanol-water solvent system at a volume ratio of 20:100 to 100:0 or 30:100 to 100:0, and the elution part at a volume ratio of 55:100 to 60:100 of methanol-water is collected.

6. The method of claim 2, wherein: The specific process of the Sephadex LH-20 gel column chromatography method is as follows: the elution part collected by the ODS column chromatography method is eluted with methanol or dichloromethane-methanol as the mobile phase to obtain fractions A and B, wherein, when eluted with dichloromethane-methanol as the mobile phase, the volume ratio of dichloromethane-methanol is 1:

8.

7. The method of claim 2, wherein: The specific process of the semi-preparative high performance liquid chromatography method is as follows: fraction B obtained by the Sephadex LH-20 gel column chromatography method is purified with acetonitrile-water or methanol-water at a volume ratio of 80:100 to 86:100 as the mobile phase to obtain the polyisoprenyl phloroglucinol compound, wherein, the chromatographic column is a COSMOSIL reverse chromatographic column, the filler is ODS, the particle size is 5 µm, the specification is 250×20 mm, the detection wavelength is 210 nm, the flow rate is 2 mL / min, and the retention time is 20-30 min.

8. A pharmaceutical composition, characterized by: The polyisoprenyl phloroglucinol compound of claim 1.

9. Use of the polyisoprenyl depsidate compound according to claim 1 for the preparation of a hypoglycemic medicament.

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