Novel triterpenoid glycoside compound extracted from rosa roxburghii tratt fruits as well as preparation method and application of novel triterpenoid glycoside compound
By performing multi-step chromatographic separation and purification on prickly pear fruit, a new triterpenoid glycoside compound, Roxbuterpene E, with significant PTP1B enzyme inhibitory activity was obtained. This solves the problem of insufficient research on triterpenoid components in prickly pear fruit for the treatment of diabetes and realizes the potential application of diabetes treatment drugs.
Patent Information
- Application Number
- CN202511490072.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-17
- Publication Date
- 2026-02-06
AI Technical Summary
In the current technology, there is limited research on the use of triterpenoids in the treatment of diabetes in prickly pear fruit, and there is a lack of effective active ingredients to be discovered and applied.
The methanol extract of Roxbuterpene fruit was separated and purified by normal-phase silica gel column chromatography, reversed-phase ODS column chromatography and HPLC to obtain a new triterpenoid glycoside compound, Roxbuterpene E. Its potential activity in treating diabetes was verified by PTP1B enzyme inhibition activity.
A novel triterpenoid glycoside, Roxbuterpene E, was successfully isolated and identified, exhibiting significant PTP1B enzyme inhibition, confirming its potential application value in diabetes treatment and providing technical support for novel diabetes treatment drugs.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of medicine, in particular to a new triterpenoid glycoside compound extracted from Rosa roxburghii fruit and its preparation method and application. BACKGROUND
[0002] Rosa roxburghii fruit is the mature fruit of Rosaceae Rosaceae plant Rosa roxburghii, also known as Wenxian fruit and Chunghui fruit, mainly distributed in Guizhou, Hunan, Yunnan, Sichuan and other provinces, among which Guizhou has the most varieties of Rosa roxburghii, which is an economic plant resource with large storage capacity.
[0003] Rosa roxburghii fruit, as a medicinal and edible plant, is listed in the Quality Standard of Guizhou Province Traditional Chinese Medicine and Ethnic Medicine (2003 edition) and is a traditional Chinese medicine in Guizhou area. Previous studies have found that Rosa roxburghii fruit contains rich triterpenoid components and has significant therapeutic effect on diabetes. At present, most of the studies on the treatment of diabetes by Rosa roxburghii fruit are focused on the extract level and polysaccharide and flavonoid components, while there are few reports on the treatment of diabetes by triterpenoid components and individual compounds in Rosa roxburghii fruit.
[0004] In order to further discover active ingredients with potential therapeutic effect on diabetes in Rosa roxburghii fruit, the present application adopts normal phase silica gel column chromatography, reverse phase ODS column chromatography and HPLC high performance liquid chromatography to separate and purify the methanol extract of Rosa roxburghii fruit, and obtains a new triterpenoid glycoside component. The PTP1B enzyme inhibition activity experiment proves that it has potential therapeutic effect on diabetes. SUMMARY
[0005] The present application aims to provide a new triterpenoid glycoside compound extracted from Rosa roxburghii fruit and a preparation method thereof.
[0006] Another object of the present application is to provide the application of the new triterpenoid glycoside compound in the preparation of diabetes treatment drugs.
[0007] To achieve the above object, the technical scheme adopted by the present application is as follows: The new triterpenoid glycoside compound extracted from Rosa roxburghii fruit according to the present application is Roxbuterpene E, with a molecular formula of C 41 H 66 O 13 , and its structural formula is as follows: 。
[0008] The preparation method of the new triterpenoid glycoside compound according to the present application comprises the following steps: S1, taking dried Rosa roxburghii fruit, crushing and adding 6-10 times amount of methanol, heating and refluxing for 1-3 times, each time for 1-3 hours, filtering and combining the filtrate, reducing pressure and concentrating, recovering the solvent, and obtaining the methanol extract; S2, the methanol extract obtained in step S1 is suspended and dispersed with water in equal quality, and then extracted with equal volume of petroleum ether, ethyl acetate and n-butanol, respectively, each solvent is extracted for 1-4 times, the extraction liquid is combined respectively, and the solvent is recovered by rotary evaporation to obtain petroleum ether extraction part, ethyl acetate extraction part, n-butanol extraction part and water part extract, respectively; S3, taking the n-butanol part extract obtained in step S2, column chromatography separation is carried out through 5 times amount of AB-8 type macroporous adsorption resin column, gradient elution is carried out with methanol-water and pure water as mobile phase with volume ratio of 3:7-7:3, to obtain A, B, C, D four parts; S4, taking component C obtained in step S3, dissolving after adding methanol, the obtained sample is mixed with 80-100 mesh silica gel with mass ratio of 1:1.5, wet column is adopted, gradient elution is carried out through silica gel column chromatography with dichloromethane-methanol as mobile phase with volume ratio of 50:1-0:1, TLC detection is carried out, and the same parts are combined to obtain 9 components Fr.1~Fr.9; S5, taking component Fr.5 obtained in step S4, ODS column chromatography separation is carried out, gradient elution is carried out with methanol-water as mobile phase with volume ratio of 1:9-10:0, to obtain 8 components Fr5.1~Fr5.8; S6, taking component Fr5.7 obtained in step S5, HPLC chromatography separation is carried out, to obtain compound Roxbuterpene E; wherein, the HPLC chromatography separation conditions are as follows: The chromatographic column is COSMOSIL 5C18-MS-II C 18 , the specification is 250mm×10mm, 5μm; the mobile phase is methanol: water = 85:15, the flow rate is 3mL / min; the detector is differential detector.
[0009] Preferably, in the preparation method of the new triterpenoid glycosides, step S1 is specifically: taking dried Rosa roxburghii fruit, crushing, adding 7-9 times amount of methanol, heating reflux extraction for 2-3 times, each time for 1.5-2.5h, filtering and combining the filtrate, reducing pressure concentration, recovering the solvent to obtain the methanol extract.
[0010] Further preferably, in the preparation method of the new triterpenoid glycosides, step S1 is specifically: taking dried Rosa roxburghii fruit, crushing, adding 8 times amount of methanol, heating reflux extraction for 3 times, each time for 2h, filtering and combining the filtrate, reducing pressure concentration, recovering the solvent to obtain the methanol extract.
[0011] Preferably, in the preparation method of the new triterpenoid glycosides, the extraction times of each solvent in step S2 is 2-4 times.
[0012] In a further preferred embodiment, in the method for preparing the new triterpenoid glycosides of the present invention, the extraction of each solvent in step S2 is performed three times.
[0013] Preferably, in the method for preparing the new triterpenoid glycosides of the present invention, the gradient elution procedure in step S3 is as follows: gradient elution is performed using methanol-water with volume ratios of 3:7, 5:5, 7:3, and 0:1 as the mobile phase, with each gradient elution consisting of 4 column volumes.
[0014] Preferably, in the method for preparing the new triterpenoid glycosides of the present invention, the gradient elution procedure in step S4 is as follows: gradient elution is performed using dichloromethane-methanol with volume ratios of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, and 0:1, with each gradient elution consisting of 4 column volumes.
[0015] Preferably, in the method for preparing the new triterpenoid glycosides of the present invention, the gradient elution procedure in step S5 is as follows: gradient elution is performed using methanol-water with volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 10:0 as the mobile phase, with each gradient elution consisting of 3 column volumes.
[0016] The application of the novel triterpenoid glycosides described in this invention in the preparation of drugs for treating diabetes.
[0017] The beneficial effects of this invention are: This invention employs normal-phase silica gel column chromatography, reversed-phase ODS column chromatography, and HPLC to separate and purify the methanol extract of *Rhizophora stylosa* fruit, yielding a novel triterpenoid glycoside, Roxbuterpene E. PTP1B enzyme inhibition activity assays confirmed that Roxbuterpene E significantly inhibits PTP1B, demonstrating its potential activity in treating diabetes. This invention provides technical support for the development of novel diabetes treatment drugs. Attached Figure Description
[0018] Figure 1 For the compound RoxbuterpeneE 1 H-NMR spectrum; Figure 2 For the compound RoxbuterpeneE 1 C-NMR spectrum; Figure 3 Graph of DEPT-135 for compound Roxbuterpene E; Figure 4 Here is the HSQC diagram of compound Roxbuterpene E; Figure 5The HR-ESI-MS spectrum of compound Roxbuterpene E is as follows: 1 H- 1 The COSY spectrum of compound Roxbuterpene E is as follows: Figure 6 The HMBC spectrum of compound Roxbuterpene E is as follows: Figure 7 The NOESY spectrum of compound Roxbuterpene E is as follows: Figure 8 The HR-ESI-MS spectrum of compound Roxbuterpene E is as follows: Figure 9 The optical rotation value of compound Roxbuterpene E is as follows: Figure 10 The IR spectrum of compound Roxbuterpene E is as follows. DETAILED DESCRIPTION
[0019] The technical solutions of the present application are described in detail below in combination with specific examples. The following examples are only used for explanation and illustration, and do not constitute a limitation on the technical solutions of the present application.
[0020] Example 1 The new triterpene glycoside compound Roxbuterpene E has a molecular formula of C 41 H 66 O 13 , and its structural formula is as follows: 。
[0021] Example 2 The preparation method of the new triterpene glycoside compound Roxbuterpene E is as follows: S1, take dried Rosa roxburghii fruit, crush and add 8 times the amount of methanol, heat reflux extraction 3 times, 2 h each time, filter and combine the filtrate, reduce pressure concentration, recover the solvent, and obtain the methanol extract.
[0022] S2, after the methanol extract obtained in step S1 is suspended and dispersed with water in equal quality, it is extracted with equal volume of petroleum ether, ethyl acetate and n-butanol in turn, and each solvent is extracted 3 times. After recovering the solvent by rotary evaporation, the petroleum ether extract, the ethyl acetate extract, the n-butanol extract and the water extract are prepared respectively.
[0023] S3, take the n-butanol extract obtained in step S2, and separate it by AB-8 type macroporous adsorption resin column chromatography, and gradient elute with methanol-water with a volume ratio of 3:7, 5:5, 7:3 and 0:1 as the mobile phase, 4 column volumes for each gradient elution, to obtain four parts A, B, C and D.
[0024] S4, taking the component C obtained in step S3, dissolving after adding methanol, mixing the obtained sample with 80-100 mesh silica gel at a mass ratio of 1:1.5, adopting wet column packing, gradient elution with dichloromethane-methanol at a volume ratio of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:1 as the mobile phase, 4 column volumes for each gradient elution, TLC detection, and combining the same parts to obtain 9 components Fr.1-Fr.9.
[0025] S5, taking the component Fr.5 obtained in step S4, adopting ODS column chromatography separation, gradient elution with methanol-water at a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 10:0 as the mobile phase, 3 column volumes for each gradient elution, to obtain 8 components Fr5.1-Fr5.8.
[0026] S6, taking the component Fr5.7 obtained in step S5, HPLC chromatography separation to obtain compound Roxbuterpene E; wherein the HPLC chromatography separation conditions are as follows: The chromatographic column is COSMOSIL 5C18-MS-II C 18 , the specification is 250 mm x 10 mm, 5 μm; the mobile phase is methanol: water = 85:15, the flow rate is 3 mL / min; and the detector is a differential detector.
[0027] Example 3 The preparation method of the new triterpenoid glycoside compound Roxbuterpene E is as follows: S1, taking dried Malus hallasis fruit, crushing, adding 10 times the amount of methanol, heating reflux extraction for 3 h, filtering and combining the filtrate, reducing pressure concentration, recovering the solvent to obtain methanol extract.
[0028] S2, dispersing the methanol extract obtained in step S1 with water in equal mass, sequentially extracting with equal volume of petroleum ether, ethyl acetate, n-butanol, 4 times for each solvent, recovering the solvent by rotary evaporation, and then preparing petroleum ether extract, ethyl acetate extract, n-butanol extract and water extract, respectively.
[0029] S3, taking the n-butanol extract obtained in step S2, adopting 5 times the amount of AB-8 type macroporous adsorption resin column chromatography separation, gradient elution with methanol-water at a volume ratio of 3:7, 5:5, 7:3, 0:1 as the mobile phase, 4 column volumes for each gradient elution, to obtain four parts A, B, C, D.
[0030] S4, taking the component C obtained in step S3, dissolving after adding methanol, mixing the obtained sample with 80-100 mesh silica gel at a mass ratio of 1:1.5, adopting wet column packing, gradient elution is carried out by using dichloromethane-methanol at a volume ratio of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:1 as the mobile phase, 4 column volumes for each gradient elution, TLC detection is carried out, and 9 components Fr.1-Fr.9 are obtained by combining the same parts.
[0031] S5, taking the component Fr.5 obtained in step S4, ODS column chromatography is adopted for separation, gradient elution is carried out by using methanol-water at a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 10:0 as the mobile phase, 3 column volumes for each gradient elution, and 8 components Fr5.1-Fr5.8 are obtained.
[0032] S6, taking the component Fr5.7 obtained in step S5, HPLC chromatography separation is carried out, and compound Roxbuterpene E is obtained; wherein, the HPLC chromatography separation conditions are as follows: The chromatographic column is COSMOSIL 5C18-MS-II C 18 , the specification is 250 mm*10 mm, 5 μm; the mobile phase is methanol: water = 85:15, the flow rate is 3 mL / min; and the detector is a differential detector.
[0033] Example 4 The preparation method of the new triterpenoid glycoside compound Roxbuterpene E is as follows: S1, taking dried Malus hallasis fruit, crushing, adding 6 times the amount of methanol, heating reflux extraction 3 times, 1 h each time, filtering and combining the filtrate, reducing pressure concentration, recovering the solvent, and obtaining methanol extract.
[0034] S2, dispersing the methanol extract obtained in step S1 with water in equal mass, sequentially extracting with equal volume of petroleum ether, ethyl acetate and n-butanol, 1 time for each solvent, recovering the solvent by rotary evaporation after extracting the extract, and respectively preparing petroleum ether extract part, ethyl acetate extract part, n-butanol extract part and water part extract.
[0035] S3, taking the n-butanol extract part extract obtained in step S2, 5 times the amount of AB-8 type macroporous adsorption resin column chromatography separation is carried out, gradient elution is carried out by using methanol-water at a volume ratio of 3:7, 5:5, 7:3, 0:1 as the mobile phase, 4 column volumes for each gradient elution, and A, B, C, D four parts are obtained.
[0036] S4, taking the component C obtained in step S3, dissolving after adding methanol, mixing and sample mixing the obtained sample with 80-100 mesh silica gel at a mass ratio of 1:1.5, adopting wet column packing, gradient elution with dichloromethane-methanol at a volume ratio of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:1 as the mobile phase, 4 column volumes for each gradient elution, TLC detection, and combining the same parts to obtain 9 components Fr.1-Fr.9.
[0037] S5, taking the component Fr.5 obtained in step S4, separating by ODS column chromatography, gradient elution with methanol-water at a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 10:0 as the mobile phase, 3 column volumes for each gradient elution, to obtain 8 components Fr5.1-Fr5.8.
[0038] S6, taking the component Fr5.7 obtained in step S5, separating by HPLC chromatography to obtain compound Roxbuterpene E; wherein the HPLC chromatography separation conditions are as follows: The chromatographic column is COSMOSIL 5C18-MS-II C 18 , with a specification of 250 mm x 10 mm, 5 μm; the mobile phase is methanol: water = 85:15, and the flow rate is 3 mL / min; and the detector is a differential detector.
[0039] Example 5 The preparation method of the new triterpenoid glycoside compound Roxbuterpene E is as follows: S1, taking dried Malus hallasis fruit, crushing, adding 7 times the amount of methanol, heating to reflux and extracting 3 times, 1.5 h each time, filtering and combining the filtrates, recovering the solvent under reduced pressure to obtain a methanol extract.
[0040] S2, dispersing the methanol extract obtained in step S1 with water in equal mass, successively extracting with equal volumes of petroleum ether, ethyl acetate, and n-butanol, 4 times for each solvent, recovering the solvent by rotary evaporation, and then preparing petroleum ether extract, ethyl acetate extract, n-butanol extract, and water extract, respectively.
[0041] S3, taking the n-butanol extract obtained in step S2, separating by 5 times the amount of AB-8 type macroporous adsorption resin column chromatography, gradient elution with methanol-water at a volume ratio of 3:7, 5:5, 7:3, 0:1 as the mobile phase, 4 column volumes for each gradient elution, to obtain four parts A, B, C, and D.
[0042] S4, taking the component C obtained in step S3, dissolving after adding methanol, mixing the obtained sample with 80-100 mesh silica gel at a mass ratio of 1:1.5, adopting wet column packing, gradient elution with dichloromethane-methanol at a volume ratio of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:1 as the mobile phase, 4 column volumes for each gradient elution, TLC detection, and combining the same parts to obtain 9 components Fr.1-Fr.9.
[0043] S5, taking the component Fr.5 obtained in step S4, adopting ODS column chromatography separation, gradient elution with methanol-water at a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 10:0 as the mobile phase, 3 column volumes for each gradient elution, to obtain 8 components Fr5.1-Fr5.8.
[0044] S6, taking the component Fr5.7 obtained in step S5, HPLC chromatography separation to obtain compound Roxbuterpene E; wherein the HPLC chromatography separation conditions are as follows: The chromatographic column is COSMOSIL 5C18-MS-II C 18 , the specification is 250 mm x 10 mm, 5 μm; the mobile phase is methanol: water = 85:15, the flow rate is 3 mL / min; and the detector is a differential detector.
[0045] Example 6 The preparation method of the new triterpenoid glycoside compound Roxbuterpene E is as follows: S1, taking dried Malus hallasis fruit, crushing, adding 9 times the amount of methanol, heating to reflux extraction 2 times, 2.5 h each time, filtering and combining the filtrate, reducing pressure concentration, recovering the solvent to obtain the methanol extract.
[0046] S2, dispersing the methanol extract obtained in step S1 with water in equal mass, sequentially extracting with equal volume of petroleum ether, ethyl acetate, n-butanol, 2 times for each solvent, combining the extraction liquid, recovering the solvent by rotary evaporation, and respectively preparing petroleum ether extraction part, ethyl acetate extraction part, n-butanol extraction part and water part extract.
[0047] S3, taking the n-butanol part extract obtained in step S2, adopting 5 times the amount of AB-8 type macroporous adsorption resin column chromatography separation, gradient elution with methanol-water at a volume ratio of 3:7, 5:5, 7:3, 0:1 as the mobile phase, 4 column volumes for each gradient elution, to obtain four parts A, B, C, D.
[0048] S4, taking the component C obtained in step S3, dissolving after adding methanol, mixing the obtained sample with 80-100 mesh silica gel at a mass ratio of 1:1.5, adopting wet column packing, performing gradient elution with dichloromethane-methanol at a volume ratio of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:1 as the mobile phase, 4 column volumes for each gradient elution, identifying by TLC, and combining the same parts to obtain 9 components Fr.1-Fr.9.
[0049] S5, taking the component Fr.5 obtained in step S4, separating by ODS column chromatography, performing gradient elution with methanol-water at a volume ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 10:0 as the mobile phase, 3 column volumes for each gradient elution, to obtain 8 components Fr5.1-Fr5.8.
[0050] S6, taking the component Fr5.7 obtained in step S5, separating by HPLC chromatography to obtain compound Roxbuterpene E; wherein the HPLC chromatography separation conditions are as follows: The chromatographic column is COSMOSIL 5C18-MS-II C 18 with a specification of 250 mm x 10 mm, 5 μm; the mobile phase is methanol: water = 85:15, and the flow rate is 3 mL / min; and the detector is a differential detector.
[0051] In order to further verify the reliability of the present application, the inventors have carried out a series of tests, as follows: 1. Separation and extraction of new triterpenoid glycosides Take 15.0 kg of dried Malus hallasis fruit, crush, add 8 times the amount of pure methanol, heat reflux extraction 3 times, 2 h each time, filter and combine the filtrate, reduce pressure concentration, recover the solvent, obtain 2655 g of alcohol extract; disperse it in water according to 1:1, extract with petroleum ether, ethyl acetate, n-butanol (volume ratio 1:1) in turn, 3 times for each polar solvent, combine the extract, recover the solvent by rotary evaporator, respectively obtain 212 g of petroleum ether extract, 562 g of ethyl acetate extract, 1172 g of n-butanol extract and water part.
[0052] The n-butanol fraction (1000 g) was taken and subjected to AB-8 macroporous adsorption resin column chromatography (5 times the amount), gradient elution (4 column volumes for each gradient elution) was performed with methanol-water (3:7, 5:5, 7:3, 0:1), to obtain 4 fractions (A-D). Fraction C (120 g) was dissolved in an appropriate amount of methanol and then mixed with silica gel (80-100 mesh) (sample: silica gel = 1:1.5), and the mixture was subjected to wet column chromatography. Gradient elution (4 column volumes for each gradient elution) was performed with dichloromethane-methanol (50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 0:1), and TLC detection was performed. Fractions with the same properties were combined to obtain 9 fractions Fr.1-Fr.9. Fraction Fr.5 (3.5 g) was subjected to ODS column chromatography, gradient elution (3 column volumes for each gradient elution) was performed with methanol-water (1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 10:0), and 8 fractions Fr5.1-Fr5.8 were obtained. Fraction Fr5.7 (180 mg) was subjected to HPLC high performance liquid chromatography separation (the chromatographic column was COSMOSIL 5C18-MS-II C 18 , the specifications were 250 mm x 10 mm, 5 μm; the mobile phase was methanol: water = 85:15, the flow rate was 3 mL / min; the detector was a differential detector), the retention time was 21.3 min, and compound Roxbuterpene E (1.3 mg) was obtained.
[0053] 2. Structure identification of compound Roxbuterpene E of compound Roxbuterpene E 1 The H-NMR chart is shown in Figure 1 , 1 The C-NMR chart is shown in Figure 2 , Figure 3 The HSQC chart is shown in Figure 4 , 1 H- 1 The H Figure 5 COSY spectrum is shown in Figure 6 The HMBC spectrum is shown in Figure 7 The NOESY spectrum is shown in Figure 8 The HR-ESI-MS spectrum is shown in Figure 9 The optical rotation value is shown in Figure 10 The IR chart is shown in
[0054] Roxbuterpene E is a colorless crystal, and its HR-ESI-MS (m / z: 789.4381 [M+Na] + , C 41 H 66 O 13Na, 789.4395) Figure 8 Its molecular formula is determined to be C 41 H 66 O 13 Characteristic absorption peaks in the infrared spectrum (3431, 1733, and 1634 cm⁻¹) -1 This indicates the presence of hydroxyl, carbonyl, and olefin functional groups. Figure 10 Roxbuterpene E 1 H NMR data (Table 1) show that the signal contains six methyl protons: δ H 1.17 (3H, s, H3-23), 0.81 (3H, s, H3-25), 0.95 (3H, s, H3-26), 0.99 (3H, s, H3-27), 1.01 (3H, d, J=6.6 Hz, H3-29), 1.60 (3H, s, H3-30); Three oxygen-bonded proton signals: δ H 3.35 (overlapped, H-3), 4.02 (d, J=11.4 Hz, H-24a), 3.27 (d, J=11.4 Hz, H-24b); a proton signal for an olefin: δ H 5.22 (brd, J=6.6 Hz, H-22); two glycan proton signals: δ H 5.41 (d, J=8.1 Hz, H-1'), 4.35 (d, J=6.6 Hz, H-1〞). Roxbuterpene E 13 C NMR spectra (Table 1) and DEPT spectra ( Figure 3 ) and HSQC spectrum ( Figure 4 The display shows 41 carbon signals, including six methyl carbon signals: δ C 23.1 (C-23), 16.7 (C-25), 16.6 (C-26), 15.3 (C-27), 23.8 (C-29), 22.0 (C-30); Ten methylene carbon signals: δ C 39.7 (C-1), 27.2 (C-2), 19.5 (C-6), 35.5 (C-7), 28.7 (C-11), 30.8 (C-12), 30.2 (C-15), 33.7 (C-16), 38.3 (C-22), 64.1 (C-24); Six methylene carbon signals: δ C 90.7 (C-3), 57.3 (C-5), 51.9 (C-9), 40.4 (C-13), 50.3 (C-18), 38.5 (C-19); a carbonyl carbon signal: δ C 175.8 (C-28); Two olefin carbon signals: δ C144.1 (C-20), 118.2 (C-21), six quaternary carbon signals: δ C 44.8 (C-4), 42.2 (C-8), 37.8 (C-10), 43.0 (C-14), 50.4 (C-17); and two sets of glycosyl carbon signals: δ C 95.3 (C-1'), 74.3 (C-2'), 78.6 (C-3'), 71.2 (C-4'), 78.4 (C-5'), 62.5 (C-6') and δ C 106.1 (C-1"), 72.8 (C-2"), 74.0 (C-3"), 69.1 (C-4"), 65.9 (C-5"). The above spectral data confirmed that this compound was a pentacyclic triterpene skeleton, combined with 2D-NMR techniques ( 1 H- 1 H COSY and HMBC, Figure 5 and Figure 6 ) further confirmed that Roxbuterpene E had a typical skeleton of Euphane type [1] .
[0055] The aglycone NMR data of Roxbuterpene E were similar to those of the aglycone of Nayabin C, but the structural difference between them was obvious [2] . In Nayabin C, the C-23 position was connected with a glucopyranosyl group, while in Roxbuterpene E, the C-23 position was a methyl group, and the C-3 position was connected with an arabinopyranosyl group, and there was a correlation between δ H 4.35 (d, J = 6.6 Hz, H-1") and δ C 90.7 (C-3) in the HMBC spectrum, further confirming this conclusion. In addition, δ H 5.41 (d, J = 8.1 Hz, H-1') and δ C 175.8 (C-28) had a correlation signal, confirming that the C-28 position was connected with a glucopyranosyl group. By comparing the chemical shift signals and coupling constants of the glycosyl part in the published literature [3] , the relative configurations of the anomeric carbons of the arabinosyl and glucosyl groups in Roxbuterpene E were determined to be α-type and β-type, respectively. After hydrolysis of Roxbuterpene E, the sugar derivative was analyzed by thin layer chromatography, and by comparing the Rf value with the standard, the absolute configuration of the arabinose was further determined to be L, and the absolute configuration of the glucose was D.
[0056] ; ; Structural analysis of Roxbuterpene E aglycone revealed that it possesses a taraxane-type skeleton; NOESY spectra ( Figure 7 The stereoconfiguration of the aglycone was confirmed to be consistent with that of Nayabin C. Furthermore, single crystals of Roxbuterpene E were obtained by culturing with methanol and analyzed by CuKα X-ray single crystallography. The absolute configuration parameter was -0.2 (5), and the relative configuration of Roxbuterpene E was determined to be 3S*, 4S*, 5R*, 8R*, 9R*, 10R*, 13R*, 14R*, 17R*, 18R*, 19S*, 1'S*, 2'R*, 3'S*, 4'S*, 5'R*, 1''S*, 2''R*, 3''S*, 4''S*. Based on this, the structure of Roxbuterpene E was finally determined to be: 3. PTP1B enzyme inhibitory activity experiment 3.1 Experimental Methods This study investigated the inhibitory effect of Roxbuterpene E on PTP1B using an in vitro enzyme inhibition assay. The specific method was as follows: Roxbuterpene E was dissolved in DMSO to prepare different concentrations (1-50 μM). PTP1B enzyme was diluted with Tris-HCl buffer (buffer formulation: 25 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 mM DTT, and 0.1 M NaCl), and then treated with different concentrations of Roxbuterpene E (1-50 μM). The substrate (p-NPP) was reacted in Tris-HCl buffer (pH=7.5) in 96-well plates. The reaction mixture was incubated at 37°C for 20 min. Ursolic acid was used as a positive control, with three replicates per group. Finally, the amount of p-nitrophenol released by p-NPP was estimated by measuring the absorbance at 405 nm using a microplate spectrophotometer, and the IC50 was calculated. 50 value.
[0057] 3.2 Conclusions and Analysis The results of the inhibitory activity of compound Roxbuterpene E on PTP1B enzyme are shown in Table 2.
[0058] ; It was found that compound Roxbuterpene E had a significant inhibitory effect on PTP1B through PTP1B inhibition. Protein tyrosine phosphatase 1B (PTP1B) belongs to the PTP superfamily and is an important signal transduction enzyme that negatively regulates the insulin signaling pathway. PTP1B inhibits insulin signaling by dephosphorylating the insulin receptor and insulin receptor substrate (IRS), thereby reducing the activation of PI3K. The decrease in PI3K activity leads to a decrease in the production of PIP3, a downstream molecule, which in turn inhibits the phosphorylation and activation of AKT. AKT is a key molecule in the insulin signaling pathway, and its activity reduction inhibits the expression of GLUT4 (glucose transporter 4) and GSK-3β (glycogen synthase kinase-3β), thereby inducing and aggravating the occurrence of diabetes. Therefore, the discovery of PTP1B inhibitors is an important means to treat diabetes.
[0059] Although the present application has been described in detail above with general description, specific embodiments and experiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application.
[0060] References: [1] Chiang, Y. M., & Kuo, Y. H. (2000). Taraxastane-type triterpenesfrom the aerial roots of Ficus microcarpa. Journal of natural products, 63(7), 898-901. [2] Kanwal, Nayab, et al. "Isolation and characterization of non-sulfated and sulfated triterpenoid saponins from Fagonia indica."Phytochemistry 143 (2017): 151-159. [3] Yin X, Liu Y et al. "Melongenaterpenes A–L, Vetispirane-Type Sesquiterpenoids from the Roots of Solanum melongena." Journal of Natural Products 82.12 (2019): 3242-3248.
Claims
1. A novel triterpenoid glycoside compound extracted from prickly pear fruit, characterized in that, The novel triterpenoid glycoside compound is Roxbuterpene E, with the molecular formula C0. 41 H 66 O 13 Its structural formula is as follows: 。 2. The method for preparing the novel triterpenoid glycoside compound as described in claim 1, characterized in that, Includes the following steps: S1. Take dried prickly pear fruit, crush it, add 6-10 times the amount of methanol, heat and reflux to extract 1-3 times, each time for 1-3 hours, filter and combine the filtrates, concentrate under reduced pressure, recover the solvent, and obtain the methanol extract. S2. After suspending and dispersing the methanol extract obtained in step S1 with water of equal mass, extract it sequentially with equal volumes of petroleum ether, ethyl acetate and n-butanol, each solvent being extracted 1-4 times. The extracts are combined and the solvents are recovered by rotary evaporation to obtain petroleum ether extract, ethyl acetate extract, n-butanol extract and water extract, respectively. S3. Take the n-butanol extract obtained in step S2 and separate it by chromatographic chromatography using 5 times the amount of AB-8 macroporous adsorption resin column. Use methanol-water and pure water as mobile phases with a volume ratio of 3:7-7:3 to perform gradient elution to obtain four fractions: A, B, C and D. S4. Take component C obtained in step S3, dissolve it in methanol, mix the resulting sample with 80-100 mesh silica gel at a mass ratio of 1:1.5, pack the sample into a column using a wet method, and perform gradient elution using silica gel column chromatography with a mobile phase of dichloromethane-methanol at a volume ratio of 50:1-0:
1. After TLC detection, merge the same parts to obtain 9 components Fr.1~Fr.
9. S5. Take the component Fr.5 obtained in step S4 and separate it by ODS column chromatography. Use methanol-water with a volume ratio of 1:9-10:0 as the mobile phase for gradient elution to obtain 8 components Fr5.1~Fr5.
8. S6. Take the component Fr5.7 obtained in step S5 and separate it by HPLC to obtain the compound Roxbuterpene E; wherein the HPLC separation conditions are as follows: The chromatographic column was COSMOSIL. 5C18-MS-II C 18 The device has a size of 250 mm × 10 mm and a diameter of 5 μm; the mobile phase is methanol:water = 85:15, the flow rate is 3 mL / min; and the detector is a differential detector.
3. The method for preparing the new triterpenoid glycoside compound according to claim 2, characterized in that, The specific steps of step S1 are as follows: take dried prickly pear fruit, crush it, add 7-9 times the amount of methanol, heat and reflux to extract 2-3 times, each time for 1.5-2.5 h, filter and combine the filtrates, concentrate under reduced pressure, recover the solvent, and obtain methanol extract.
4. The method for preparing the new triterpenoid glycoside compound according to claim 3, characterized in that, Step S1 specifically involves: taking dried prickly pear fruit, crushing it, adding 8 times the amount of methanol, heating and refluxing to extract 3 times, 2 hours each time, filtering and combining the filtrates, concentrating under reduced pressure, recovering the solvent, and obtaining the methanol extract.
5. The method for preparing the new triterpenoid glycoside compound according to claim 2, characterized in that, In step S2, each solvent is extracted 2-4 times.
6. The method for preparing the new triterpenoid glycoside compound according to claim 5, characterized in that, In step S2, each solvent is extracted 3 times.
7. The method for preparing the novel triterpenoid glycoside compound according to claim 2, characterized in that, The gradient elution procedure in step S3 is as follows: using methanol-water with volume ratios of 3:7, 5:5, 7:3, and 0:1 as the mobile phase for gradient elution, with each gradient elution consisting of 4 column volumes.
8. The method for preparing the new triterpenoid glycoside compound according to claim 2, characterized in that, The gradient elution procedure in step S4 is as follows: using dichloromethane-methanol with volume ratios of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, and 0:1 as the mobile phase for gradient elution, with each gradient elution consisting of 4 column volumes.
9. The method for preparing the new triterpenoid glycoside compound according to claim 2, characterized in that, The gradient elution procedure in step S5 is as follows: using methanol-water with volume ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 10:0 as the mobile phase for gradient elution, with each gradient elution consisting of 3 column volumes.
10. The use of the novel triterpenoid glycoside compound as described in claim 1 in the preparation of a drug for treating diabetes.