Polyisopentenyl phloroglucinol compound as well as preparation method and application thereof

Abstract

The invention relates to a polyisopentenyl phloroglucinol compound as well as a preparation method and application thereof, and belongs to the technical field of medicines. The structural formula of the compound is shown in the specification, and the preparation method of the compound comprises the following steps: extracting garcinia macrocarpa fruits in an organic solvent aqueous solution, then extracting with petroleum ether and dichloromethane, and then purifying through silica gel column chromatography, ODS column chromatography, Sephadex LH-20 gel column and semi-preparative high performance liquid chromatography to obtain the compound. The method is simple and easy to implement, and the obtained compound is novel in structure, has a [2, 4, 1] nonane mother nucleus structure, can promote secretion of insulin in pancreas islet beta cells induced by palmitic acid, and has the prospect of preparing drugs for preventing and treating hyperglycemia. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a polyprenylated phloroglucinol compound, a preparation method thereof, and an application thereof, and particularly relates to a novel phloroglucinol compound with a [2,4,1] nonane nucleus having hypoglycemic activity isolated from the fruits of Garcinia pedunculata Roxb. ex Buch.-Ham. of the genus Garcinia in the family Clusiaceae, and a preparation method thereof, belonging to the field of pharmaceutical technology. Background Art

[0002] There are various types of polyprenylated phloroglucinol compounds (PPAP) in plants of the genus Garcinia. According to the type of their parent nucleus, they can be roughly divided into monocyclic polyprenylated acyl phloroglucinol compounds (MPAP), bicyclic isoprenylated acyl phloroglucinol compounds (BPAP), cage-like polyprenylated acyl phloroglucinol compounds (Caged PPAP), and other types of polyprenylated acyl phloroglucinol compounds.

[0003] Compounds of the PPAP type have diverse structures and rich activities, mainly including activities such as anti-tumor, lipid-lowering, anti-inflammatory, antioxidant, antibacterial, antiviral, anti-leishmanial, and hypoglycemic activities. Related scholars isolated garcibractinone C from the fruits of Garcinia bracteata, and detected its hypoglycemic activity through an insulin-resistant hepatocarcinoma cell model. The results showed that this compound could significantly promote glucose uptake. Through the component analysis of the fruits of Garcinia pedunculata Roxb. ex Buch.-Ham., it was found that it contained compounds with other structures, and the compound structure had a great influence on its pharmacological activity. The compound obtained in the present invention has not been reported in other literatures and is a novel compound extracted from the fruits of Garcinia pedunculata. Summary of the Invention

[0004] The present invention provides a polyprenylated phloroglucinol compound, a preparation method thereof, and an application thereof. The present invention extracts a novel phloroglucinol compound from the fruits of Garcinia pedunculata Roxb. ex Buch.-Ham., which has a [2,4,1] nonane nucleus structure and can promote insulin secretion in palmitic acid-induced pancreatic islet β cells.

[0005] A polyprenylated phloroglucinol compound, the structural formula of which is as follows:

[0006]

[0007] Furthermore, the above-mentioned polyprenylated phloroglucinol compounds are extracted and isolated from the fruits of Garcinia pedunculata Roxb.

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

[0009] A method for preparing polyprenylated phloroglucinol compounds includes the following steps:

[0010] S1: Extract the fruits of Garcinia pedunculata Roxb. in an organic solvent aqueous solution, combine the extracts, and concentrate under reduced pressure to obtain an extract.

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

[0012] S3: Further obtain polyprenylated phloroglucinol compounds from the dichloromethane extract by silica gel column chromatography, ODS column chromatography, Sephadex LH-20 gel column, and semi-preparative high performance liquid chromatography methods.

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

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

[0015] Furthermore, it can be extracted by impregnation three times with 50% - 100% acetone aqueous solution for three days each time.

[0016] Furthermore, alternatively, it can be extracted by heating under reflux three times with 50% - 100% ethanol aqueous solution or methanol aqueous solution for three hours each time.

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

[0018] In the above technical solution, in S2, the number of extractions with petroleum ether and dichloromethane is 3 - 5 times each.

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

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

[0021] Further, when using the dichloromethane-methanol solvent system, the eluted fractions with a dichloromethane-methanol volume ratio of 100:0 to 100:3 or 100:1 to 100:5 are collected.

[0022] Further, when using the dichloromethane-acetone solvent system, the eluted fractions with a dichloromethane-acetone volume ratio of 100:1 to 100:5 are collected.

[0023] Further, when using the petroleum ether-acetone solvent system, the eluted fractions with a petroleum ether-acetone volume ratio of 100:5 to 100:20 are collected.

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

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

[0026] Further, when performing isocratic elution with dichloromethane-methanol as the mobile phase, the volume ratio of dichloromethane-methanol is 1:8.

[0027] In the above technical solution, in S3, 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 having a volume ratio of 80:100 to 86:100 as the mobile phase to obtain polyprenylated phloroglucinol compounds.

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

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

[0030] Another object of the present invention is to provide the use of the polyprenylated phloroglucinol compounds prepared by the above preparation method in the preparation of hypoglycemic drugs.

[0031] Advantages of the present invention: The compound obtained in the present invention has a novel structure, with a [2,4,1] nonane nuclear structure, and can promote the secretion of insulin in palmitic acid-induced pancreatic islet β cells, having the prospect of preparing drugs for preventing and treating hyperglycemia. Description of the Drawings

[0032] Figure 1 High-resolution ESI-MS spectra of the compounds obtained in Examples 1 to 4.

[0033] Figure 2 For the compounds obtained in Examples 1 to 4 1 1H NMR spectra (solvent CD 3 OD).

[0034] Figure 3 For the compounds obtained in Examples 1 to 4 13 13C NMR spectra (solvent CD 3 OD).

[0035] Figure 4 DEPT135 spectra of the compounds obtained in Examples 1 to 4 (solvent CD 3 OD).

[0036] Figure 5 HMBC spectra of the compounds obtained in Examples 1 to 4 (solvent CD 3 OD).

[0037] Figure 6 HSQC spectra of the compounds obtained in Examples 1 to 4 (solvent CD 3 OD).

[0038] Figure 7 For the compounds obtained in Examples 1 to 4 1 1H- 1 1H COSY spectra (solvent CD 3 OD).

[0039] Figure 8 NOESY spectra of the compounds obtained in Examples 1 to 4 (solvent CD 3 OD).

[0040] Figure 9 For the compounds obtained in Examples 1 to 4 1 1H NMR spectra (solvent DMSO-d 6 ).

[0041] Figure 10 For the compounds obtained in Examples 1 to 4 13 13C NMR spectra (solvent DMSO-d 6 ).

[0042] Figure 11 HMBC spectra of the compounds obtained in Examples 1 to 4 (solvent DMSO-d 6 ).

[0043] Figure 12 HSQC spectra of the compounds obtained in Examples 1 to 4 (solvent DMSO-d 6 ).

[0044] Figure 13 For the compounds obtained in Examples 1 to 4 1 H- 1 H COSY spectra (solvent DMSO-d 6 ).

[0045] Figure 14 NOESY spectra of the compounds obtained in Examples 1 to 4 (solvent DMSO-d 6 ).

[0046] Figure 15 Calculated NMR data analysis diagrams of the compounds obtained in Examples 1 to 4.

[0047] Figure 16 ECD spectra of the compounds obtained in Examples 1 to 4.

[0048] Figure 17 Insulin secretion results of the compounds obtained in Examples 1 to 4 on palmitic acid-induced pancreatic islet β cells. Among them, Figure 17 (A) is the cytotoxicity result of the compound on INS-1 cells, Figure 17 (B) is the cytotoxicity result of palmitic acid on INS-1 cells, Figure 17 (C-D) are immunofluorescence analysis and quantification diagrams of the compound on insulin secretion of palmitic acid-induced INS-1 cells. Detailed implementation manners

[0049] The following non-limiting examples can enable those of ordinary skill in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0050] In the following examples, the test methods are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0051] Example 1

[0052] A preparation method of a polyprenylated phloroglucinol compound, comprising the following steps:

[0053] Take 10 kg of the fruits of Garcinia pedunculata, and impregnate and extract them three times with 100 L of 80% acetone aqueous solution for three days each time. Combine the extracts and concentrate them into an extract; evaporate until there is no acetone smell, disperse them at a volume ratio of 2:1 of the aqueous phase to the extract, and extract with petroleum ether and dichloromethane. Each organic phase is extracted 3 times, and the volume ratio of the aqueous phase to the organic phase is 1:1 each time. Concentrate the dichloromethane extraction layer under reduced pressure; subject the obtained dichloromethane extract to silica gel column chromatography with a mesh size of 200 - 300, and perform gradient elution with a dichloromethane - methanol solvent system with a volume ratio of 100:0 - 0:100. The specific elution ratios are dichloromethane:methanol = 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:50, 100:100, 0:100; subject the elution fractions with a dichloromethane - methanol volume ratio of 100:0 - 100:3 to ODS column chromatography, and perform gradient elution with a methanol - water solvent system with a volume ratio of 30:100 - 100:0. The specific elution ratios are methanol:water = 30:100, 40:100, 50:100, 60:100, 70:100, 80:100, 90:100, 100:0; subject the elution fraction with a methanol - water volume ratio of 60:100 to Sephadex LH - 20 gel column chromatography, and perform isocratic elution with methanol as the mobile phase to obtain fractions A and B. Then, purify fraction B by semi - preparative high - performance liquid chromatography (detection wavelength is 210 nm, the stationary phase of the chromatographic column is ODS, the particle size is 5 μm, the chromatographic column parameters are 250×20 mm, the mobile phase is 80% acetonitrile - water, the flow rate is 2 mL / min, and the retention time is 20 min) to obtain polyprenylated phloroglucinol compounds (6.1 mg, the yield is 0.00061‰).

[0054] Example 2

[0055] A method for preparing polyprenylated phloroglucinol compounds, comprising the following steps:

[0056] Take 15 kg of Garcinia pedunculata fruits, and extract them three times by heating under reflux with 150 L of 95% ethanol for three hours each time. Combine the extracts and concentrate them into an extract; evaporate until there is no ethanol smell, disperse it with a volume ratio of aqueous phase to extract of 3:1, and extract with petroleum ether and dichloromethane. Each organic phase is extracted 3 times, with a volume ratio of aqueous phase to organic phase of 1:2 each time. Concentrate the dichloromethane extraction layer under reduced pressure; separate the obtained dichloromethane extract by silica gel column chromatography with a mesh size of 200 - 300, and perform gradient elution with a solvent system of dichloromethane - acetone with a volume ratio of 100:0 - 0:100. The specific elution ratios are dichloromethane:methanol = 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:50, 100:100, 0:100; separate the elution part with a dichloromethane - acetone volume ratio of 100:1 - 100:5 by ODS column chromatography, and perform gradient elution with a solvent system of methanol - water with a volume ratio of 20:100 - 100:0. The specific elution ratios are methanol:water = 20:100, 30:100, 40:100, 50:100, 60:100, 70:100, 80:100, 90:100, 100:0; separate the elution part with a methanol - water volume ratio of 60:100 by Sephadex LH - 20 gel column chromatography, and perform isocratic elution with methanol as the mobile phase to obtain fractions A and B. Then, purify fraction B by semi - preparative high - performance liquid chromatography (detection wavelength is 210 nm, the stationary phase of the chromatographic column is ODS, particle size is 5 μm, the chromatographic column parameters are 250×20 mm, the mobile phase is 86% methanol - water, the flow rate is 2 mL / min, and the retention time is 26 min) to obtain polyprenylated phloroglucinol compounds (7.0 mg, yield is 0.0007‰).

[0057] Example 3

[0058] Take 20 kg of Garcinia pedunculata fruits, and extract them three times by heating under reflux with 400 L of 75% ethanol for three hours each time. Combine the extracts and concentrate them into an extract; evaporate until there is no ethanol smell, disperse it at a volume ratio of 3:1 of the aqueous phase to the extract, and extract it with petroleum ether and dichloromethane. Each organic phase is extracted 4 times, and the volume ratio of the aqueous phase to the organic phase is 1:2 each time. Concentrate the dichloromethane extraction layer under reduced pressure; separate the obtained dichloromethane extract by silica gel column chromatography with a mesh size of 200 - 300, and perform gradient elution with a petroleum ether - acetone solvent system with a volume ratio of 100:0 - 0:100. The specific elution ratios are dichloromethane:methanol = 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:50, 100:100, 0:100; separate the elution part with a petroleum ether - acetone volume ratio of 100:5 - 100:20 by ODS column chromatography, and perform gradient elution with a methanol - water solvent system with a volume ratio of 30:100 - 100:0. The specific elution ratios are methanol:water = 30:100, 45:100, 55:100, 60:100, 70:100, 80:100, 90:100, 100:0; separate the elution part with a methanol - water volume ratio of 55:100 - 60:100 by Sephadex LH - 20 gel column chromatography, and perform isocratic elution with methanol as the mobile phase to obtain fractions A and B. Then, purify fraction B by semi - preparative high - performance liquid chromatography (detection wavelength is 210 nm, the stationary phase of the chromatographic column is ODS, the particle size is 5 μm, the chromatographic column parameters are 250×20 mm, the mobile phase is 82% acetonitrile - water, the flow rate is 2 mL / min, and the retention time is 22 min) to obtain polyprenylated phloroglucinol compounds (8.3 mg, the yield is 0.00083‰).

[0059] Example 4

[0060] 15 kg of Garcinia pedunculata fruits were taken and extracted three times by heating under reflux with 300 L of 75% methanol for 3 hours each time. The extracts were combined and concentrated into an extract; it was volatilized until there was no methanol smell, dispersed at a volume ratio of aqueous phase to extract of 2:1, and extracted with petroleum ether and dichloromethane. Each organic phase was extracted 5 times, with a volume ratio of aqueous phase to organic phase of 1:2 each time. The dichloromethane extraction layer was concentrated under reduced pressure; the obtained dichloromethane extract was separated by silica gel column chromatography with a mesh size of 200 - 300, and gradient elution was carried out with a solvent system of dichloromethane - methanol with a volume ratio of 100:0 - 0:100. The specific elution ratios were dichloromethane:methanol = 100:0, 100:1, 100:3, 100:5, 100:7, 100:10, 100:20, 100:50, 100:100, 0:100; the elution fractions with a dichloromethane - methanol volume ratio of 100:1 - 100:5 were separated by ODS column chromatography, and gradient elution was carried out with a methanol - water solvent system with a volume ratio of 30:100 - 100:0. The specific elution ratios were methanol:water = 30:100, 45:100, 55:100, 60:100, 70:100, 80:100, 90:100, 100:0; the elution fraction with a methanol - water volume ratio of 60:100 was separated by Sephadex LH - 20 gel column chromatography, and isocratic elution was carried out with a dichloromethane - methanol solvent system with a volume ratio of 1:8 as the mobile phase to obtain fractions A and B. Then, fraction B was purified by semi - preparative high - performance liquid chromatography (detection wavelength was 210 nm, the stationary phase of the chromatographic column was ODS, particle size was 5 μm, the chromatographic column parameters were 250×20 mm, the mobile phase was 85% methanol - water, flow rate was 2 mL / min, retention time was 30 min) to obtain polyprenylated phloroglucinols (7.3 mg, yield was 0.00073‰).

[0061] The polyprenylated phloroglucinols obtained in Examples 1 - 4 were identified for their structures by high - resolution mass spectrometry, nuclear magnetic resonance spectroscopy ( 1 HNMR, 13 C NMR, 2D - NMR), etc. The results were as follows:

[0062] The above - obtained compound was dissolved in methanol and then precipitated, showing as a yellow powder, and the specific rotation [α]20D was - 86.64 (c 0.30 MeOH). The HR - ESI - MS spectrum ( Figure 1 ) gave a quasi - molecular ion peak of m / z 641.3442 [M + Na] + (calcd for C 38 H 50 O 7 Na, 641.3454), determining the molecular formula of this compound to be C 38 H 50 O7 , with an unsaturation degree of 14. 1 HNMR (600 MHz, CD 3 OD, Table 1) spectrum ( Figure 2 ) and HSQC spectrum ( Figure 6 ) show: a set of ABX-coupled aromatic proton signals at 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 at δ 5.16 (1H, m, H-25), 2.03 (1H, m, H-24), 1.68 (3H, s, CH 3 -28), 1.57 (3H, s, CH 3 -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, CH 3 -38), 1.70 (1H, m, H-34) and 1.63 (3H, s, CH 3 -37); an olefinic proton signal at δ 6.15 (1H, d, J = 1.9 Hz, H-17); two sets of methylene proton signals at 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 at δ 5.07 (1H, d, J = 1.9 Hz, H-18), 1.67 (1H, m, H-30) and 1.46 (1H, m, H-6); and six methyl proton signals at δ 1.32 (3H, s, CH 3 -22), 1.25 (3H, s, CH 3 -32), 1.21 (3H, s, CH 3 -33), 1.17 (3H, s, CH 3 -20), 1.16 (3H, s, CH 3 -23) and 1.00 (3H, s, CH 3 -21). 13 CNMR (150 MHz, CD 3 OD, Table 1) spectrum ( Figure 3 ) and DEPT135 spectrum ( Figure 4 ) give 38 carbon signals, including 16 sp 2 -hybridized carbon signals and 22 sp 3 -hybridized carbon signals. Among them, 16 sp 2Among the hybridized carbon signals, there are two carbonyl carbon signals at δ 203.86 (C-9) and 190.75 (C-10); 22 sp 3 Among the hybridized carbon signals, there are four methylene carbon signals at δ 36.40 (C-29), 32.02 (C-7), 30.91 (C-34), 29.20 (C-24), three methine carbon signals at δ 87.63 (C-18), 45.97 (C-6), 42.80 (C-30), and ten methyl carbon signals at δ 30.61 (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), 18.01 (C-37). Based on the above information, it is speculated that the compound is a polyisoprenyl-substituted acylphloroglucinol compound.

[0063] The planar structure of the compound was determined by HMBC spectrum ( Figure 5 ) and HSQC spectrum ( Figure 6 ), and the signals of hydrogen and carbon spectra were assigned according to these spectra. In the HMBC spectrum ( Figure 5) Among them, δ7.29 (1H, d, J = 2.1 Hz, H-12) has a long-range correlation with δ190.75 (C-10), 153.07 (C-14), 146.67 (C-13), 125.08 (C-16), δ7.16 (1H, dd, J = 8.3, 2.1 Hz, H-16) has a long-range correlation with δ190.75 (C-10), 153.07 (C-14), 116.57 (C-12), and δ6.76 (1H, d, J = 8.3 Hz, H-15) has a long-range correlation with δ153.07 (C-14), 146.67 (C-13), 130.98 (C-11), indicating 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) have a long-range correlation with δ203.86 (C-9), 73.19 (C-3), 45.97 (C-6), 39.94 (C-5), 36.40 (C-29), 29.20 (C-24), δ2.03 (1H, m, H-24) and 1.29 (1H, m, H-24) have a long-range correlation with δ45.97 (C-6), 39.94 (C-5), δ6.15 (1H, d, J = 1.9 Hz, H-17) has a long-range correlation with δ150.45 (C-4), 73.19 (C-3), 39.94 (C-5), δ1.76 (1H, m, H-29) and 1.65 (1H, m, H-29) have a long-range correlation with δ73.19 (C-3), 45.93 (C-8), 32.02 (C-7). Combining the two angular methyls δ1.32 (3H, s, CH 3 -22) and 1.16 (3H, s, CH 3 -23) have a long-range correlation with δ150.45 (C-4), 39.94 (C-5), 45.97 (C-6). Moreover, when measuring the spectrum with deuterated dimethyl sulfoxide ( Figures 9 to 14 ), it was found that δ1.46 (1H, m, H-6) has a correlation with δ150.45 (C-4), 125.00 (C-25), 45.93 (C-8), 39.94 (C-5), 32.02 (C-7), 29.20 (C-24), indicating the presence of the C3-C4-C5-C6-C7-C8-C9 cycloheptane nucleus; δ2.03 (1H, m, H-24) and 1.29 (1H, m, H-24) have a long-range correlation with δ133.01 (C-26), 125.00 (C-25), 45.97 (C-6), 39.94 (C-5), and 1 H- 1 H COSY spectrum ( Figure 7 and Figure 13)Show H-25, H 2 -24, H-6 and H 2 -7 are related and belong to the same spin coupling system. It is speculated that one isopentenyl group is attached to the 6th position of the cycloheptane nucleus; δ6.15(1H, d, J = 1.9Hz, H-17) has a long-range correlation with δ150.45(C-4), 87.63(C-18), 73.19(C-3), 39.94(C-5), and δ5.07(1H, d, J = 1.9Hz, H-18) has a long-range correlation with δ150.45(C-4), 125.06(C-17), 26.02(C-20), 23.56(C-21). Combining the two methyl groups δ1.17(3H, s, CH 3 -20) and 1.00(3H, s, CH 3 -21) has a long-range correlation with δ87.63(C-18), 72.06(C-19), and based on the carbon spectrum data δ125.06(C-17), 87.63(C-18), 72.06(C-19), it is inferred that there is a 2-(1-hydroxy-1-methylethyl)-2,5-dihydrofuran ring fragment, which is fused to the 3rd and 4th positions of the cycloheptane nucleus; δ1.76(1H, m, H-29) and 1.65(1H, m, H-29) have a long-range correlation with δ78.19(C-31), 73.19(C-3), 45.93(C-8), 42.80(C-30), 32.02(C-7). The two methyl groups δ1.25(3H, s, CH 3 -32) and 1.21(3H, s, CH 3 -33) have a long-range correlation with δ78.19(C-31), 42.80(C-30), and δ2.18(1H, m, H-34) and 1.70(1H, m, H-34) have a long-range correlation with δ39.94(C-30). Combining 1 H- 1 H COSY spectrum, H 2 -29, H-30, H 2 -34 and H-35 are related and belong to the same spin coupling system. It is inferred that a 2,2-dimethyltetrahydropyran fragment is fused to the 1st and 8th positions of the cycloheptane nucleus, and this isopentenyl group is attached to the 30th position. According to the molecular formula, there are still two degrees of unsaturation and two carbon atoms left. The signals are δ186.85 and 117.36 respectively. It is speculated that there is a carbon-carbon double bond forming a ring. δ186.85 is the signal of the sp 2 hybridized carbon atom with oxygen at the 1st position, and δ117.36 is the signal of the carbon atom connected to the benzoyl group at the 2nd position. Therefore, this compound is a phloroglucinol compound with a [2,4,1]nonane nucleus.

[0064] In the NOESY spectrum( Figure 8and Figure 14 ) in, CH 3 -22 is related to H-6 and H-7β. It is speculated that the above hydrogen atoms and groups are coplanar with the β orientation. Since the [2,4,1]nonane nucleus is a rigid structure, the carbonyl group at the 9th position is specified above the C1-C2-C3-C4-C5-C6-C7-C8 cyclooctane nucleus, and the 29th carbon and the 3rd oxygen atom are located below the cyclooctane nucleus; CH 3 -23 and the hydrogen signal of H-24 are related, indicating that the isopentenyl group attached to the 6th position is located below the cyclooctane nucleus. According to the biogenetic relationship, the [2,4,1]nonane nucleus of this compound is formed by the isomerization of the [3,3,1]nonane nucleus of the known compound cambogin isolated. Therefore, it is inferred that the absolute configurations of C-3, C-6, C-8, and C-30 are the same. 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 density functional theory was used to calculate the nuclear magnetic resonance method at the mPW1PW91 / 6-311+G(d,p) level to determine the configuration of this compound. By calculating the linear regression coefficient (R 2 ) between the measured and calculated carbon spectrum data and the DP4+ probability evaluation of the measured and calculated NMR data ( Figure 15 ), the possibility of the absolute configuration being 1b is 100.00%. Thus, it is confirmed 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 at the B3LYP / 6-31G(d,p) theoretical level ( Figure 16 ), and the absolute configuration of the compound was determined to be (3R,6R,8S,18S,30S) by comparing the measured and calculated ECD spectra.

[0065] Table 1 Compounds 1 H NMR and 13 C NMR chemical shift values (the solvent is CD 3 OD and DMSO-d 6 )

[0066]

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

[0068]

[0069] Experimental study on the compounds obtained in Examples 1 to 4 for promoting insulin secretion activity in palmitic acid-induced pancreatic islet β cells in vitro:

[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, the INS-1 cells, an islet β-cell line, were incubated in the above-prepared medium in an incubator at 37 °C and 5% CO 2 2 with saturated humidity.

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

[0072]

[0073] Immunofluorescence confocal microscopy was used to quantify insulin secretion. Clean coverslips were placed in a 6-well plate, and INS-1 cells were added for culture. After the cells grew on the coverslips, the cells were treated normally according to the grouping. 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 to fix the cells for 20 min. The cells were washed three times with PBS to remove residual paraformaldehyde, and care was taken to add the PBS along the wall of the 6-well plate to prevent the cells from being washed off the glass slide. The washing liquid was discarded. Then, 1 mL / well of 0.15% Triton solution was used to punch holes for 15 min, and the cells were washed three times with PBS. The glass slides were taken out, the cells at the edges were wiped off, and a circle was drawn using an immunohistochemistry pen. After the glass slides were placed back in the 6-well plate, blocking solution (PBS containing 10% serum) was added, with a dosage of 200 μL per well and a blocking time of 30 min. The cells were incubated with primary antibody insulin B (1:200) prepared with PBS overnight at 4 °C.

[0074] Equilibrate the glass slides at room temperature for 30 min. First, wash them 3 times with TBST to remove unbound primary antibody, and then wash 3 times with PBS to remove the remaining Tween. Next, all steps related to incubating the fluorescent secondary antibody are carried out in the darkroom. Prepare a fluorescent secondary antibody with a concentration of 1:500 using PBS, 100 μL per slide. After incubating in the dark for 2 h, wash 3 times with PBS to remove unbound fluorescent secondary antibody, and then add 100 μL of DAPI (1:10,000) prepared with PBS to each well for incubation. After 10 min, wash 3 times with PBS to remove excess DAPI. Drop a fluorescent quencher on the glass slide, remove the coverslip, invert the cell side onto the glass slide, and seal the slide with nail polish. Place it in a dark box for further measurement. Operate according to the laser confocal microscope operation procedure, obtain fluorescent images through a fluorescence microscope, and perform fluorescence quantification using Image J software.

[0075] The main function of pancreatic islet β cells is to synthesize and release insulin. When pancreatic islet cells are damaged, it will lead to dysfunction. Figure 17 (A) shows the cytotoxicity results of the compound on INS-1 cells. Figure 17 (B) shows the cytotoxicity results of palmitic acid on INS-1 cells. It can be seen that the compound can protect pancreatic islet β cells under the condition of 10 μM. With the help of immunofluorescence experiments, the content of insulin in INS-1 cells was examined using a fluorescence microscope. The results are shown in Figure 17 (C) and (D). It can be seen that under the condition of 10 μM, compared with the control group and the blank group, the compound showed a certain promoting effect on insulin secretion in pancreatic islet β cells induced by palmitic acid, and has the prospect of preparing clinical drugs for the prevention and treatment of hyperglycemia.

Claims

1. A polyisopentenyl phloroglucinol compound, characterized in that: The structural formula of the compound is shown below:

2. A method for preparing a polyisopentenyl phloroglucinol compound, characterized in that: The polyisopentenyl phloroglucinol compounds are extracted and separated from Garcinia macrocarpa fruit.

3. A method for preparing a polyisopentenyl phloroglucinol compound, characterized in that: The following steps are involved: S1: extracting the fruit of Garcinia macrocarpa in an organic solvent aqueous solution, combining the extracts, 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 the dichloromethane extract layer under reduced pressure to obtain a dichloromethane extract; S3: The dichloromethane extract was further subjected to silica gel column chromatography, ODS column chromatography, Sephadex LH-20 gel column and semi-preparative high performance liquid chromatography to obtain the compound.

4. The preparation method according to claim 3, characterized in that: In S1, 50% to 100% acetone aqueous solution can be used for immersion extraction three times, each time for three days; or 50% to 100% ethanol aqueous solution or methanol aqueous solution can be used for heating reflux extraction three times, each time for three hours.

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

6. The preparation method according to claim 3, characterized in that: In S3, the specific process of the ODS column chromatography method is: the eluted portion collected by the silica gel column chromatography method is gradient eluted with a methanol-water solvent system with a volume ratio of 20:100 to 100:0 or 30:100 to 100:0, and the eluted portion with a methanol-water volume ratio of 55:100 to 60:100 is collected.

7. The preparation method according to claim 3, characterized in that: The specific process of the Sephadex LH-20 gel column chromatography method is: the eluted portion 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 the volume ratio of dichloromethane to methanol when isocratic elution is performed with dichloromethane-methanol as the mobile phase is 1:

8.

8. The preparation method according to claim 3, characterized in that: The specific process of the semi-preparative high performance liquid chromatography method is: the fraction B obtained by the Sephadex LH-20 gel column chromatography method is purified using acetonitrile-water or methanol-water with a volume ratio of 80:100 to 86:100 as the mobile phase to obtain polyisopentenyl phloroglucinol compounds, wherein the chromatographic column is a COSMOSIL reverse chromatographic column (ODS, 5μm, 250×20mm), the detection wavelength is 210nm, the flow rate is 2mL / min, and the retention time is 20 to 30min.

9. A pharmaceutical composition, characterized in that: Contains the polyisopentenyl phloroglucinol compound according to claim 1.

10. Use of the polyisopentenyl phloroglucinol compound according to claim 1 in the preparation of hypoglycemic drugs.

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