CN111521703B

CN111521703B - Method for identifying isopentenyl dihydrostilbene by liquid chromatography-mass spectrometry and structure and application of novel compound

Info

Publication number: CN111521703B
Application number: CN202010375586.5A
Authority: CN
Inventors: 马超美; 小松加门子; 蒙昊聪; 朱姝; 范宇红; 叶日贵服部征雄
Original assignee: Inner Mongolia University
Current assignee: Inner Mongolia University
Priority date: 2020-04-25
Filing date: 2020-04-25
Publication date: 2022-06-17
Anticipated expiration: 2040-04-25
Also published as: CN111521703A

Abstract

The invention discloses a liquid chromatography-mass spectrometry method for identifying isopentenyl dihydro stilbene and a structure and application of a novel compound discovered by the method. According to the principle that the dihydrostilbene compounds are easy to break between alpha-alpha' bonds, under a negative ionization mode, fragments with more phenol groups (except o-phenols) show stronger ion peaks, and under a positive ionization mode, fragments with less phenol groups show stronger ion peaks, and under a positive ionization mode, the neutral loss of isopentenyl dihydrostilbene is 56 daltons. The invention discloses a method for finding isopentenyl dihydrostilbene from a detected object by utilizing a liquid chromatography-mass spectrometry combined technology combining neutral loss (56 daltons) and sub-ion scanning, application of the method in analyzing the isopentenyl dihydrostilbene in animal and plant extracts, tissues, cells and preparations, and application of novel isopentenyl dihydrostilbene compounds, namely structures and biological activities of glycyrrhiza stilbene D (glycyrrhiza stilbene D) and glycyrrhiza stilbene E (glycyrrhiza stilbene E), in the aspects of medicines, foods, cosmetics, livestock feeds and the like.

Description

Method for identifying isopentenyl dihydrostilbene by liquid chromatography-mass spectrometry and structure and application of novel compound

Technical Field

The invention belongs to the technical field of natural medicinal chemistry, and particularly relates to a technical method for finding a component of isopentenyl dihydrostilbenes (dihydrostilbenes) in extracts and preparations of animals and plants by liquid-mass combination. Also relates to the preparation, structure and biological activity of new isopentenyl dihydro stilbene component-glycyrrhiza stilbene D (glycyrrhiza stilbene D) and glycyrrhiza stilbene E (glycyrrhiza stilbene E), and the application thereof in the aspects of medicines, (functional) foods, cosmetics, livestock feeds and the like.

Background

Stilbene (stilbene) compounds, such as resveratrol, are natural products found in many medicinal plants, vegetables and fruits. Some plants, such as licorice, contain a component of the prenyl dihydrostilbene class, i.e., a- α' linkage saturated and linked to a prenyl substituent [ Biondi DM, Rocco C, rubetto g. dihydrostilbene derivatives from Glycyrrhiza glabra leaves.j Nat Prod, 2005, 68: 1099-; siracusa L, Saija A, Cristani M, Cimino F, D' Arrigo M, Trombeta D, Rao F, Ruberto G. Photoomplexes from quality (Glycyrrhiza glabra L.) leaves- -chemical conversion and evaluation of the anti-inflammatory, anti-genetic and anti-inflammatory activity, Fitoterapia, 2011, 82: 546-556.]. Recently, Wang et al [ Wang L, Zhang K, Han S, Zhang L, Bai H, Bao F, Zeng Y, Wang J, Du H, Liu Y, Yang Z.Constitutes isolated from the leaves of Glycyrrhiza uralensis and the anti-inflammatory activity on LPS-induced RAW264.7 cells. molecules, 2019, 24: 1923.] four novel prenyldihydrostilbenes were isolated from Glycyrrhiza uralensis leaves and found to have potent anti-inflammatory activity. Based on the biological activity of the isopentenyl dihydro stilbene, a rapid detection method thereof needs to be developed. The invention firstly shows the splitting mode of the isopentenyl dihydro stilbene, namely that the neutral loss of 56 daltons is the characteristic of the isopentenyl dihydro stilbene, and fragments generated by alpha-alpha' bond breakage provide information for the type, quantity and position of a substituent group. Then, using licorice leaf extract as an example, 10 prenyl dihydrostilbene components were identified by mining prenyl dihydrostilbene through 56 dalton neutral loss scan and ion scan. The new compounds are separated and purified by chromatography, and the nuclear magnetic resonance spectroscopy is used for confirming the structures of 2 new dihydro stilbene derivatives, namely, glycyrrhiza stilbene D (glycyrrhiza stilbene D) and glycyrrhiza stilbene E (glycyrrhiza stilbene E). These results indicate that LC-MS techniques combining neutral loss scanning with daughter ion scanning are a viable approach to find new prenyldihydrostilbenes from samples.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for identifying isopentenyl dihydrostilbene from animal and plant extracts, tissue and cell metabolites, preparations or related foods, medicines, cosmetics, feeds and the like (a method for finding the isopentenyl dihydrostilbene component based on liquid-proton combined neutral loss scanning bonder ion scanning), and structures, preparation methods and alpha-glucosidase activity inhibition of new natural products, namely glycyrrhiza stilbene D and glycyrrhiza stilbene E.

In order to solve the technical problem, the invention adopts the following scheme:

carrying out sub-ion scanning on the pure isopentenyl dihydro stilbene to find out the cracking rule of the pure isopentenyl dihydro stilbene;

scanning with 56 dalton neutral loss to obtain chromatogram of isopentenyl-containing component in the extract;

identifying the structure of the isopentenyl dihydro stilbene by utilizing sub-ion scanning;

carrying out targeted separation and purification on the new isopentenyl dihydro stilbene and determining the structure by a nuclear magnetic resonance method;

the invention also provides 2 new cyclopentenyl dihydro stilbene components-glycyrrhiza stilbene D and glycyrrhiza stilbene E chemical structures:

in another aspect of the invention, compositions of glycyrrhizin D and/or glycyrrhizin E are provided;

the invention also provides a pharmaceutical preparation containing the glycyrrhizin D and/or the glycyrrhizin E;

the glycyrrhiza stilbene D and the glycyrrhiza stilbene E have stronger alpha-glucosidase inhibition activity. The alpha-glucosidase inhibitor can delay carbohydrate absorption and reduce postprandial blood glucose level. Some α -Glucosidase inhibitors also have antiviral effects (Kelly L. Warfield. targeting Endoplasmic Reticulum α -glucidase I with a Single-Dose Iminosugar Treatment Protects against viral infection Leth Influenza and Dengue Virus Infections. J. Med. Chem. 2020. https:// dx. doi. org/10.1021/acs. jmedchem.0 c00067). Therefore, the invention also relates to the application of the composition containing the glycyrrhizin D and/or the glycyrrhizin E in the treatment and/or prevention of diabetes and viral diseases.

In a preferred embodiment of the present invention, which utilizes the neutral loss scanning and conjugator ion scanning of LC-MS to find the isopentenyl dihydrostilbene in the sample, the screening and identification of the isopentenyl dihydrostilbene component comprises the following steps:

1) the sample is extracted with a solvent, preferably methanol or ethanol with a concentration of 70% or more. Filtering the extract by a micro-filter to obtain a sample solution for liquid-mass analysis;

2) performing liquid-mass spectrometry, preferably using a triple quadrupole mass spectrometer, a reversed phase silica gel C18 analytical column, performing gradient elution with water-methanol or water-acetonitrile, and optimizing a chromatographic elution program;

3) performing 56 dalton neutral loss scanning on the sample liquid by using the optimized chromatographic elution program in the step 2 and a mass spectrum detector in a positive ionization mode to mine compounds with isopentenyl groups, so as to obtain a chromatogram map;

4) subjecting the parent ions corresponding to the chromatographic peaks obtained in step 3) above to negative and positive ion scans using the same chromatographic elution procedure as in step 3 above, at a fragment voltage of 20-200V, preferably 60-170V, and a collision energy of 0-40eV, preferably 5-20 eV;

5) analyzing the mass spectrogram obtained in the 3-4) according to the rule that the dihydrostilbene compounds are easy to break among alpha-alpha' bonds, fragments with more phenol groups (except o-phenols) show stronger ion peaks in a negative ionization mode, and fragments with less phenol groups show stronger ion peaks in a positive ionization mode, inferring the number and the structure of the isopentenyl dihydrostilbene compounds in the sample, and quantifying according to peak areas.

The advantages and the novel findings of the invention are as follows:

a liquid-mass identification method for discovering new isopentenyl dihydrostilbene is invented, namely a method for discovering new isopentenyl dihydrostilbene components from a sample by using a liquid-mass combination technology of neutral loss (56 daltons) and sub-ion scanning. The rule of utilization is: the dihydrostilbene compounds are easy to break at alpha-alpha' bonds, and in a negative ionization mode, the fragments with more phenol groups (except o-phenols) show stronger ion peaks, and in a positive ionization mode, the fragments with less phenol groups show stronger ion peaks. In the positive ionization mode, the neutral loss of the prenyldihydrostilbene is 56 daltons. The invention provides a basis and a method for discovering more isopentenyl dihydro stilbene-containing animal and plant resources and new isopentenyl dihydro stilbene compounds.

The obtained isopentenyl dihydro stilbene derivatives-glycyrrhiza stilbene D and glycyrrhiza stilbene E with new structures are expected to have activity on obesity, diabetes or virus infection.

Drawings

FIG. 1 is a mass spectrum and a cleavage path (A-E) of a prenyldihydrostilbene compound (1-5) in a negative ionization mode;

FIG. 2 is a graph of MS/MS and cleavage pathways for a representative prenyldihydrostilbene compound (5) in positive ionization mode at different collision energies of 40eV (A), 30eV (B), 20eV (C), 10eV (D);

FIG. 3 is a MS/MS graph (A-D) of prenyldihydrostilbene compounds (1-4) at a positive ionization mode collision energy of 10 eV;

FIG. 4 is a liquid-mass chromatogram of the Liquorice leaf extract with neutral loss (56 daltons) scan (A), and MS/MS spectra and cleavage of compounds 19-23 in negative ionization mode;

FIG. 5 is the chemical structure of the licorice leaf component;

*: compounds identified by LC-MS and verified by nuclear magnetic resonance after purification;

#: compounds preliminarily identified by LC-MS;

other compounds identified structures by comparing liquid-quality parameters with those of the pure control obtained at the earlier stage;

FIG. 6 is HMBC spectrum of the new compound glycyrrhizin D;

FIG. 7 is the HMBC spectrum of the new compound glycyrrhizin E.

Detailed Description

The analytical identification method of the prenyldihydrostilbene derivative of the present invention is described in further detail below.

Example 1: preparation of sample solution for liquid-mass analysis and general conditions for liquid-mass analysis

Extracting dried folium Glycyrrhizae with chromatographic grade methanol at a solid-to-liquid ratio of 100 mg: 4ml under ultrasound for 20 min. The supernatant was passed through a sep-pak C18 column and through a 0.22 μm filter. The middle section of the filtrate was collected as a sample solution for liquid-mass analysis.

A column of ZORBAX RRHD Eclipse Plus C18(50 mm. times.2.1 mm, 1.8 μm) was chromatographed using an Agilent 1290 definition UHPLC-DAD system and an Agilent 6340 triple quadrupole mass spectrometer. The sample volume was 1. mu.L, and the flow rate was 0.4 ml/min. The mobile phase takes 0.1% formic acid as a solvent A and acetonitrile as a solvent B, and the elution procedure is as follows: 0min 20% B, 6.5min 30% B, 7.5min 33% B, 12.5min 45% B, 15min 55% B, 17min 100% B, 18min stop, equilibration time: and 2 min. Capillary 4.5kv, gas temperature 350 ℃, gas flow 11l/min, atomizer 45 psi.

Example 2: mass spectrum cracking law of isopentenyl dihydro stilbene

And carrying out sub-ion scanning analysis on the pure isopentenyl dihydro stilbene in a negative ionization mode and a positive ionization mode to obtain an MS/MS (Mass Spectrometry) diagram. The results show that these compounds are susceptible to alpha-alpha' bond cleavage, resulting in the corresponding fragment ions (FIGS. 1-3).

In the negative ionization mode, the peak at m/z 191 (the phenyl moiety containing two phenolic hydroxyl groups) is strong and the peak at m/z 207 (the other phenyl moiety containing one phenolic hydroxyl group) is weak for compound 1 (FIG. 1A). Similar phenomena also occurred in the MS/MS plots for compounds 3 (FIG. 1C) and 4 (FIG. 1D). Both phenyl groups of compound 2 (fig. 1B) contain a phenolic hydroxyl group, showing a weak ionic peak; compounds 1 and 3-5 have structures in which at least one phenyl group has two phenolic hydroxyl groups attached, and both show strong ionic peaks at m/z 191 (FIGS. 1A, 1C and 1D). These results indicate that in the negative ionization mode, fragments containing more phenolic groups, especially when the two phenolic hydroxyl groups are in the meta or para positions, show stronger ionic peaks.

In positive ionization mode, a loss of neutrality of the small molecule is observed. All prenyldihydrostilbenes present fragment ions (fig. 2, 3) that lose 56 daltons (isobutene), which is more pronounced at lower collision energies (10 or 20) (fig. 2D and 2C) than at higher collision energies (30 and 40) (fig. 2B and 2A).

Compounds

1 and 2 contain a 2, 2-dimethyl-3-hydroxy-3, 4-dihydropyran ring in structure, exhibiting a neutral loss of 18 daltons (H)₂O) and fragment ions of 72 daltons (2-methyl-propen-1-ol) (fig. 3A and 3B). Fragments resulting from the cleavage of the α - α' bond are also present in the positive ionization mode, and fragments with fewer phenolic groups exhibit a stronger ion peak in the positive ionization mode than in the negative ionization mode. M/z 189 (a moiety containing one phenolic hydroxyl group) of compound 3 was stronger than the other fragments (fig. 3C).

Example 3: neutral loss scanning and ion scanning detection of isopentenyl compound in liquorice leaves

Since the 56 dalton neutral loss is characteristic of isopentenyl compounds, we performed 56 dalton neutral loss scans on the licorice leaf extract samples to find isopentenyl compounds unidentified therein. As a result, a chromatogram shown in FIG. 4A was obtained. Peaks corresponding to compounds 1 and 3-12 were determined by comparison with the previously obtained compound (Ye R, Fan YH, Ma CM. identification and interpretation of α -glucosidase-inhibiting dihydrostilbenes and flavanoids from Glycyrrhiza uralensis leaves. J agricultural Food Chem.2017, 65: 510. 515; Fan YH, Ye R, Xu HY, Feng XH, Ma CM. structures and in vitro anti-inflammatory drugs Sc. J. Food 2019, 84: 1225. 1230). Compounds 2, 13-18 (FIG. 5) were also isolated from Glycyrrhiza glabra leaves before, but were not present in the neutral loss chromatogram due to the absence of isopentenyl groups in the structures of these compounds.

A daughter ion scan was performed for the other major compounds that were not referenced for comparison, where the structures of the 5 compounds (19-23) were deduced from the MS/MS spectra shown in fig. 4.

The highest peak in the resulting chromatogram was the peak with retention time 14.6 minutes (20) scanned with a neutral loss of 56 daltons in liquid-mass combination (fig. 4A). The chromatographic peak shows m/z 381 in negative ionization mode. Scanning the daughter ion of 381 yielded 3 compounds (19, 5, and 20), each with a daughter ion of m/z 191.

Description

19 and 20 substituent types and numbers on 2 phenyl groups of isopentenyl dihydrostilbene are the same as in compound 5, i.e. 2 hydroxyl groups and 1 isopentenyl substitution per phenyl group, as shown in figure 5.

Compound 22 occurs at M/z 313 [ M-H]^-Peak, a strong ion peak corresponding to the structure of the dihydroxybenzyl group appears at m/z 123. Due to the high strength of the fragment ion, the two hydroxyl groups are attached in the meta position. The other phenyl group was substituted by one isopentenyl group and 2 hydroxyl groups, deduced from the molecular weight, which were assigned to the ortho position due to the weak daughter ion at m/z 191. The structure of compound 22 is shown in figure 5.

Compounds

21 and 23 both exhibit M/z 397 [ M-H]^-The peak, ion appearing at m/z 191, corresponds to phenyl group with one isopentenyl group and 2 hydroxyl substitutions, with the hydroxyl group assigned to the meta position due to the high ionic strength of m/z 191 of compound 23, while m/z 191 of compound 21 is weakly assigned to the ortho position. According to the molecular weight and [ M-H₂O]^-The same substitution pattern on the other phenyl group as in compound 1 was inferred (fig. 1A). Thus, the structures of

compounds

21 and 23 are shown in FIG. 5.

In order to confirm the structure of the main compounds of the liquid-mass preliminary identification, the compounds are taken as targets, and preparative separation, purification and identification under the guidance of the liquid-mass are carried out.

Example 4: preparative extraction and separation of isopentenyl dihydrostilbene compounds

Glycyrrhiza glabra leaf (200g) was extracted with methanol with ultrasound.The filtrate was concentrated in vacuo onto a silica gel column and eluted with petroleum ether-ethyl acetate. The component B is obtained from petroleum ether-ethyl acetate 8: 2 elution part, and the component C is obtained from petroleum ether-ethyl acetate 7: 3 elution part. Performing ODS column chromatography on the component B, eluting with water-methanol, purifying 75% methanol eluate with Sephadex-LH 20, eluting with methanol, and further purifying with preparative high performance liquid chromatography to obtain compound 20(20 mg). The preparation type high performance liquid phase conditions comprise a flow rate of 10ml/min and a detection wavelength of 280 nm, and the mobile phase takes water as a solvent A and CH₃CN is solvent B, and the elution procedure is 0.01min 30% B, 60 min 50% B, 90-100min 100% B. The C component was separated by ODS column chromatography, and eluted from 70% methanol to give C1F 2. C1F2 was further separated by ODS column, 40-50% CH₃The CN eluate fraction was purified by preparative high performance liquid chromatography to give compounds 19(4mg) and 21(2 mg).

Example 5: nuclear magnetic resonance spectrum for confirming structure of isopentenyl dihydro stilbene compound

The NMR data of the pure compounds 19 to 21 obtained by isolation are shown in Table 1.

TABLE 1 NMR data for Compounds 19-21

*: these attributes may be exchanged.

Pure at 19 deg.C¹In HNMR, the proton signals for δ 6.13(d, J ═ 2.4Hz, H-6) and 6.15(d, J ═ 2.4Hz, H-4) show two hydrogens in the meta position on one phenyl group; a pair of bimodal aromatic proton signals J at δ 6.46(H-6 ') and 6.56 (H-5') 7.8Hz, indicating that the other phenyl group has a vicinal dihydro structure. Delta.2.65 (2H, CH)₂- α) and 2.66(2H, CH)₂The two methylene proton signals at- α ') correlate with the two aromatic carbon signals at δ 142.9(C-1) and 131.9 (C-1'), indicating a dihydrostilbene skeleton. There were significant methyl signals at δ 1.63(3H, s, H-11), 1.65(3H, s, H-11 '), 1.69(3H, s, H-10) and 1.73(3H, s, H-10'). In the HMBC spectrum, methyl signals at δ 1.63 and 1.69 and methylene at δ 3.20The signal (d, J ═ 6.6Hz, H-7) correlates with the aromatic carbon signals at δ 124.3(C-8) and 129.4(C-9), indicating the presence of isopentenyl groups in the structure of 19. Another pair of methyl signals at δ 1.65, 1.73 and methylene proton signals at δ 3.32(d, J ═ 6.6Hz, H-7 ') correlate with carbon signals at δ 123.7(C-8 ') and 130.1(C-9 '), indicating the presence of another isopropenyl group in 19. The substitution positions of the two isopentenyl groups were determined by correlation of H-7 with C-1, C-2 and C-3 and H-7 'with C-1', C-2 'and C-3' in the HMBC spectra. From the above evidence, compound 19 was identified as having the structure shown in fig. 5. This is a new compound, named glycyrrhiza stilbene D, and its HMBC spectrum is shown in figure 7.

Process for preparation of Compound 20¹HNMR shows a pair of meta hydrogen signals on the phenyl rings at δ 6.51(d, J ═ 1.8Hz, H-6) and 6.40 (d, J ═ 1.8Hz, H-2), and two symmetric hydrogen signals on the phenyl rings δ 6.18 (2H, s, H-2 ', 6'). In addition, two isopentenyl groups were found in the nmr spectra and their positions were determined using HMBC. Nmr data indicate the structure of compound 20 as shown in figure 5. This compound has been reported to be present in Glycyrrhiza species (Biondi DM, Rocco C, Ruberto G. New hydrostilene derivatives from the leaves of Glycyrrhiza glabra and evaluation of the anti-inflammatory activity. J Nat Prod 2003, 66: 477-480).

Process for preparation of Compound 21¹The H-NMR spectrum shows two pairs of meta-coupled phenyl hydrogen signals at δ 6.16(1H, J ═ 1.8Hz, H-2), 6.12(1H, J ═ 1.8Hz, H-6) and 6.45(1H, d, J ═ 2.4Hz, H-2 '), 6.34(1H, d, J ═ 2.4Hz, H-6'). In HMBC spectra, methylene proton signals (CH) at δ 2.64 and 2.65₂- α and CH₂α ') correlates with the two aromatic carbon signals at δ 141.3 and 132.8 (C-1 and C-1'), indicating that it has a dihydrostilbene skeleton. There are 4 methyl signals at δ 1.21/1.30 (3H, s, H-10, 11 each), 1.70(3H, s, H-10 ') and 1.72(3H, s, H-11'). The methyl signals at δ 1.70 and 1.72 show a remote correlation in the HMBC spectra with the pair of olefin carbon signals at δ 122.9(C-8 ') and 131.1 (C-9'). The methylene proton signal at δ 3.24(2H, d, J ═ 7.5Hz, H-7') is also related to HMBC for both olefin carbons. As described aboveThe information shows that one isopentenyl group is present in 21 structures of the compound. Two more methyl signals at δ 1.21/1.30 and a methylene signal at δ 2.53/2.49 correlate with HMBC at δ 69.3(C-8) and the oxygen-containing carbon at 76.1(C-9), indicating the presence of a 2, 2-dimethyl-3-hydroxy-3, 4-dihydropyran ring. Due to the HMBC correlation between H-7 ' and C-4 ', C-5 ' and C-6 ', the location of the isopentenyl group was determined at C-5 '. Since H-7 is associated with HMBC at C-3, C-4 and C-5, 2-dimethyl-3-hydroxy-3, 4-dihydropyran rings are formed at the C-4 and C-5 positions. The structure of 21 is shown in FIG. 5, as confirmed by the above NMR analysis. This is a new compound, named glycyrrhizin E, whose HMBC spectrum is shown in FIG. 7.

Example 6: quantitative analysis of chemical components of licorice leaves

Control compounds (1-18) were dissolved in DMSO at a concentration of 20. mu.g/ml for each compound, and then three-fold serial dilutions were made in DMSO. Quantification was performed in liquid-mass Multiple Reaction Monitoring (MRM) mode using the optimized fragment voltage and collision energy for each compound (table 2).

TABLE 2 Mass Spectrometry quantitative determination conditions

The quantitative results shown in table 3 show that licorice leaves collected in joldos, inner mongolia in 2018 and 2019 contain compounds 19-21 and most of the previously reported compounds. The content of the isopentenyl flavonoid compound (10) and the two isopentenyl dihydro stilbenes compounds (5 and 20) is the highest, and the content of the isopentenyl flavonoid compound and the two isopentenyl dihydro stilbenes compounds in 1g of dried liquorice leaves exceeds 100 micrograms.

TABLE 3 content of Compounds in Licorice leaves

Note: nd: the content is below the limit of quantitation.

Example 7: inhibitory Activity on alpha-glucosidase

Inhibition of alpha-glucosidase experiments were performed in 96-well plates with test compounds dissolved in DMSO. Each well contained 10. mu.l of the test solution (control well added with the same amount of DMSO), 80. mu.l of 2 mmol of 4-nitrophenyl α -D-glucopyranoside (dissolved in 100 mmol of potassium phosphate buffer at pH 7.09), and 10. mu.l of 0.40U/ml Bacillus Stearthermophilus α -glucosidase. After incubation at 37 ℃ for 20 minutes, absorbance at 405nm was measured using a microplate reader. The inhibition rate calculation formula is as follows: inhibition% [ (% absorbance of control well-absorbance of sample well)/absorbance of control well. Three tests were performed with each compound at final concentrations of 100, 10, 1 and 0.1 μ g/ml. 50% inhibitory concentration IC₅₀Values were obtained from the inhibition-concentration curves.

Of the isopentenyl dihydrostilbenes from Liquoria leaves previously reported (Fan YH, Ye R, Xu HY, Feng XH, Ma CM. structures and in vitro anti-inflammatory fibrosus activities of substituted dihydrostilbenes and flavanoids from Glycyrrhiza uralensis leaves. J Food Sci 2019, 84: 1225. epsilon. 1230.)₅₀3 μ g/ml) has the strongest inhibitory activity against α -glucosidase. Thus, the activity of the newly isolated compounds 19-21 was tested and compared to compound 5. All the newly isolated isopentenyl dihydrostilbenes showed inhibitory activity on α -glucosidase: 19 (IC)₅₀＝4μg/ml)、20(IC₅₀＝8μg/ml)、21(IC ₅₀4 μ g/ml).

Compounds

19 and 21 showed comparable inhibitory activity to compound 5.

In conclusion, the invention discloses a method for rapidly detecting isopentenyl dihydrostilbene by liquid-mass combination, and proves that liquorice leaves contain rich phenolic components. 23 phenolic components are identified from the licorice leaves, wherein 10 isopentenyl dihydro stilbene components and 13 phenolic components are flavonoids. The new compounds predicted by the liquid-mass method are purified by chromatography and then are verified by the analysis of nuclear magnetic resonance spectrum. The newly separated isopentenyl dihydro stilbene has stronger inhibition effect on alpha-glucosidase.

The invention discloses a method for rapidly detecting isopentenyl dihydrostilbene compounds in animal and plant samples (neutral loss scanning of 56 daltons, and conjugator ion scanning), which can comprehensively identify isopentenyl dihydrostilbene in animal and plant extracts, animal and plant tissues and cell metabolites. The research result provides reference for the development and utilization of the tested substance as a medicine or a health-care food, and also provides a valuable source for preparing a lead compound with biological activity.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention should not be limited thereto, and it is understood that the technical solutions obtained by simple changes or substitutions by those skilled in the art are within the technical scope of the present invention.

Claims

1. The method for identifying the isopentenyl dihydro stilbene derivative by liquid chromatography-mass spectrometry is characterized by comprising the following steps: the isopentenyl dihydro stilbene derivative is as follows:

the method comprises the following steps:

1) extracting a plant sample by using methanol, and filtering an extracting solution by using a micro-filter to obtain a sample solution for liquid chromatography-mass spectrometry combined analysis;

2) carrying out liquid chromatography-mass spectrometry analysis on the sample solution, wherein the analysis conditions are as follows: chromatographic conditions are as follows: c18 column, 50mm × 2.1mm, 1.8 μm, mobile phase a with 0.1% formic acid and mobile phase B with acetonitrile, gradient elution: 0min, 20% B, 6.5min, 30% B, 7.5min, 33% B, 12.5min, 45% B, 15min, 55% B, 17min, 100% B, flow rate 0.4 ml/min; mass spectrum conditions: a triple quadrupole mass spectrometer, an electrospray ion source, a capillary 4.5kv, a gas temperature of 350 ℃, a gas flow of 111/min, and an atomizer 45 psi;

the liquid chromatography-mass spectrometry combined analysis comprises the following steps: firstly, carrying out 56 dalton neutral loss scanning in a positive ionization mode, wherein the fragment voltage is 90V, and the collision energy is 10eV, so as to obtain a chromatogram map of the isopentenyl compound in a sample solution; then, respectively scanning parent ions corresponding to chromatographic peaks of the various isopropenyl dihydrostilbene derivatives in the chromatogram under a negative ionization mode and a positive ionization mode, wherein the fragment voltage is 60-170V, and the collision energy is 5-20eV, so as to obtain secondary mass spectrograms of the various isopropenyl dihydrostilbene derivatives; finally, the secondary mass spectrogram is analyzed according to the parent ion [ M-H ] in a negative ionization mode]^-Deducing the substitution number and substitution position of isopentenyl and hydroxyl in the derivative by the mass-to-charge ratio of the isopentenyl dihydrostilbene derivative, the mass-to-charge ratio of the daughter ion generated by the alpha-alpha' bond fracture and the strength, thereby preliminarily identifying the structure of each isopentenyl dihydrostilbene derivative;

3) and (3) separating and purifying the prenyldihydrostilbene derivative with the preliminarily identified structure, and confirming the structure by using a nuclear magnetic resonance method.

2. An isopentenyl dihydrostilbene derivative characterized by: the derivative is glycyrrhizin D, and the structural formula is as follows:

3. an isopentenyl dihydrostilbene derivative characterized by: the derivative is glycyrrhizin E, and the structural formula is as follows: