CN113072604A

CN113072604A - Preparation method of iridoid glycoside in herba Hedyotidis Diffusae and application of iridoid glycoside in preparing antiinflammatory medicine

Info

Publication number: CN113072604A
Application number: CN202110362903.4A
Authority: CN
Inventors: 施树云; 史方瑛; 童超英; 王岱杰
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2021-07-06
Anticipated expiration: 2041-04-02
Also published as: CN113072604B

Abstract

The invention discloses a preparation method of iridoid glycoside in oldenlandia and application thereof in preparing anti-inflammatory drugs, wherein, the extract of oldenlandia is processed by adopting a diagnostic ion filtration method, the peak time of the corresponding chromatogram of iridoid glycoside is obtained according to diagnostic ions, and the iridoid glycoside in oldenlandia is extracted by utilizing high-speed counter-current chromatography according to the peak time. The method can directionally extract iridoid glycoside from Hedyotis Diffusa, the extracted iridoid glycoside has high purity and anti-inflammatory activity, and avoids complicated sample extraction steps.

Description

Preparation method of iridoid glycoside in herba Hedyotidis Diffusae and application of iridoid glycoside in preparing antiinflammatory medicine

Technical Field

The invention relates to extraction of plant active ingredients, in particular to a preparation method of iridoid glycoside in oldenlandia diffusa and application of iridoid glycoside in preparing anti-inflammatory drugs.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Natural products have been used medically for hundreds of years and are good exogenous candidates (e.g. antioxidants, anti-inflammatory agents, antibacterial agents, antineoplastic agents). However, natural products often contain large amounts of compounds, and conventional extraction, isolation and activity measurement methods are time consuming and laborious, sometimes wasting a lot of effort in isolating non-target components. Therefore, rapid screening and purification of active ingredients in complex natural products remains a challenge.

High performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS/MS) has been widely used to identify biologically active compounds with high resolution, well-defined precursor and product ion information. However, mass spectrometry data analysis becomes a rather cumbersome and difficult task.

Oldenlandia diffusa is a medicinal plant of the rubiaceae family, and has been used for hundreds of years for the treatment of inflammation-related disorders such as urethritis, hepatitis and appendicitis. Chemical composition research shows that the oldenlandia contains iridoid glycoside, flavonoid, phenolic acid and anthraquinone. According to the research of the inventor, iridoid glycoside is probably the main component of the anti-inflammatory effect of the oldenlandia diffusa, but the iridoid glycoside in the oldenlandia diffusa is difficult to directionally extract at present.

Disclosure of Invention

In order to solve the defects of the prior art, the invention aims to provide a preparation method of iridoid glycoside in oldenlandia diffusa and application of iridoid glycoside in preparing anti-inflammatory drugs.

In order to achieve the purpose, the technical scheme of the invention is as follows:

on the one hand, the preparation method of the iridoid glycoside in the oldenlandia diffusa adopts a diagnostic ion filtration method to treat the extract of the oldenlandia diffusa, obtains the peak time of the corresponding chromatogram of the iridoid glycoside according to diagnostic ions, and extracts the iridoid glycoside in the oldenlandia diffusa according to the peak time by utilizing high-speed counter-current chromatography.

Iridoid glycoside iridoid is a kind of organic matter, belong to monoterpene, have unique parent skeleton, have similar fragmentation ways in the mass spectrometric detection process, can produce the same fragment ion, regard this fragment ion as the diagnostic product ion, can analyze the compound with iridoid glycoside parent skeleton, the invention utilizes this characteristic to adopt the diagnostic ion filtration method to position iridoid glycoside in the herba Hedyotidis Diffusae, know the time of appearance peak of the corresponding chromatogram of various iridoid glycosides, then adopt the high-speed countercurrent chromatography to extract the component under the corresponding time according to the time of appearance peak obtained, can obtain iridoid glycoside. The problems of non-adsorbability, peak tailing, inactivation and pollution of a solid support can be solved by adopting high-speed counter-current chromatography and liquid-liquid distribution, so that the purity and activity of the extracted compound are ensured.

Since oldenlandia diffusa contains not only iridoid glycoside but also other compounds with the same parent skeleton such as flavonoid and anthraquinone, other plants can also be extracted with the same parent skeleton by a similar method, and on the other hand, the application of the preparation method in directional extraction of compounds with the same parent skeleton in plants.

In a third aspect, the use of a process as described above for the preparation of an anti-inflammatory agent.

In a fourth aspect, an application of iridoid glycoside in oldenlandia diffusa in preparing anti-inflammatory drugs.

The invention has the beneficial effects that:

1. the invention adopts a diagnostic ion filtration method to obtain the peak time of the corresponding chromatogram of the iridoid glycoside in the oldenlandia diffusa, and the iridoid glycoside is extracted by high-efficiency countercurrent chromatography by utilizing the peak time.

2. The method can be used for screening and separating iridoid glycoside from herba Hedyotidis Diffusae in a targeted manner, the extracted iridoid glycoside has high purity and anti-inflammatory activity, and complicated sample extraction steps are avoided.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a total ion flux graph of HPLC-QTOF-MS of Hedyotis diffusa extract in an example of the present invention, wherein A is negative ion mode and B is total ion flux graph with diagnostic ion m/z 191.03;

FIG. 2 is a diagram showing the MS/MS spectrum (A) and the cleavage route (B) of deacetyl asperuloside in the example of the present invention;

FIG. 3 is a chromatogram of extracting iridoid glycoside from herba Hedyotidis Diffusae in the example of the present invention, wherein A is HSCCC chromatogram, and B is HPLC chromatogram of separated components;

FIG. 4 is a structural diagram of iridoid glycoside compound extracted from 6 in the example of the present invention.

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In view of the existing problem that the iridoid glycoside is difficult to directionally extract from oldenlandia diffusa and the extraction steps are complicated, the invention provides a preparation method of iridoid glycoside in oldenlandia diffusa and application of iridoid glycoside in preparing anti-inflammatory drugs.

According to a typical embodiment of the invention, the method for preparing the iridoid glycoside in the oldenlandia diffusa is provided, the extract of the oldenlandia diffusa is treated by adopting a diagnostic ion filtration method, the peak time of the corresponding chromatogram of the iridoid glycoside is obtained according to diagnostic ions, and the iridoid glycoside in the oldenlandia diffusa is extracted according to the peak time by utilizing high-speed countercurrent chromatography.

According to the invention, firstly, diagnosis ions are determined according to the cracking way of iridoid glycoside, iridoid glycoside in hedyotis diffusa is positioned by a diagnosis ion filtration method according to the diagnosis ions, the peak output time of the corresponding chromatogram of various iridoid glycosides is obtained, and then components under the corresponding time are extracted by adopting high-speed counter-current chromatography according to the obtained peak output time, so that iridoid glycoside can be obtained. Meanwhile, the purity and activity of the iridoid glycoside extracted are ensured by adopting high-speed counter-current chromatography.

Iridoid glycoside has the properties of being easily soluble in water and methanol, soluble in ethanol, acetone and n-butanol, and insoluble in lipophilic organic solvents such as chloroform, diethyl ether and benzene. For better extraction of iridoid glycosides from oldenlandia diffusa, in some embodiments, the extract of oldenlandia diffusa is extracted from oldenlandia diffusa by ethanol extraction.

In one or more embodiments, the oldenlandia diffusa powder or granules are heated and refluxed with an ethanol water solution, and the liquid after reflux extraction is evaporated to dryness to obtain the oldenlandia diffusa extract. The ratio of the spreading hedyotis herb powder or particles to the ethanol water solution is 1: 4.5-5.5, g: mL, can ensure that the target compound is completely extracted. The volume fraction of ethanol in the ethanol water solution is 70-80%, and the target compound can be better dissolved.

The diagnostic ion filtration method is generally carried out by a high performance liquid chromatography-quadrupole time of flight tandem mass spectrometry (HPLC-QTOF-MS/MS) method.

The separation of iridoid glycosides may be effected in the presence of different mobile phases (e.g., methanol-acetic acid water, acetonitrile-acetic acid water, methanol-formic acid water and acetonitrile-formic acid water), in some embodiments, in the chromatographic mobile phase of the diagnostic ion filtration method, formic acid water as mobile phase a and acetonitrile as mobile phase B. Can ensure the separation of iridoid glycoside, and can further ensure the content of iridoid glycoside when the volume fraction of formic acid in the mobile phase A is 0.05-0.15 percentAnd (5) separating effect. Different elution procedures, including isocratic and gradient elution, also affect the separation. The gradient elution is formed as follows: 0-5min, 15% -20% B; 5-20min, 0% -44% B; the flow rate is 0.7-0.9 mL/min. Can ensure the complete separation of iridoid glycoside. Column selection for diagnostic ion filtration₁₈And the separation effect is better.

In ESI-MS (electrospray ionization mass spectrometry), the detection mode is divided into a negative ion mode and a positive ion mode, and in some embodiments, the mass spectrometry employs the negative ion mode. The positive ion mode has fewer fragment ions, and the negative ion mode has higher MS response and lower background noise, so that the iridoid glycoside can be screened more favorably.

According to the research of the invention, the skeleton of different types of iridoid glycosides in oldenlandia diffusa comprises the monothioglucoside derivative, paederoside, geniposide and asperuloside, so that the determination of diagnostic ions is complex, and in some embodiments, the diagnostic ions m/z are 190.0000-193.0000. Experiments show that although the parent nucleus ion of the iridoid glycoside is about 165.0000, the characteristic fragment ion m/z of iridoid and iridoid 4, 6-lactone is about 191.0000, so the iridoid glycoside in the oldenlandia diffusa can be better positioned by adopting the diagnostic ion.

In some embodiments, the capillary voltage is 3.0 to 4.0kV, the ion energy is 4.5 to 5.5eV, and the atomizer pressure is 30 to 40psi in the mass spectrum. Under the condition, fragment ions are more abundant, so that diagnostic ions can be better judged, and the positioning effect of iridoid glycoside is further improved.

In the high-speed counter-current chromatography, all solvents in a solvent system are uniformly mixed, then the mixture is subjected to static liquid separation, the upper layer solution is used as a stationary phase, and the lower layer solution is used as a mobile phase. In some embodiments, the solvent system for high-speed countercurrent chromatography consists of ethyl acetate, n-butanol, and water. The solvent system can better separate and purify iridoid glycoside in the oldenlandia diffusa, wherein the partition coefficient of the solvent system influences the separation degree of each compound, and in some embodiments, the volume ratio of ethyl acetate, n-butanol and water is 4.45-4.55: 0.45-0.55: 4.5-5.5. The condition can better separate the iridoid glycoside.

In some embodiments, the high-speed countercurrent chromatography flow rate is 1.75-1.85 mL/min. Experiments show that the iridoid glycoside can be better separated at the flow rate.

In some embodiments, the detection wavelength is 252 to 256nm in high-speed countercurrent chromatography.

In some embodiments, after the high-speed countercurrent chromatography, the fractions are further separated using a Sephadex LH-20 column using a methanol solvent. Can obtain high-purity iridoid glycoside.

In another embodiment of the invention, the application of the preparation method in directional extraction of the compounds with the same parent frameworks in the plants is provided.

In a third embodiment of the invention, there is provided a use of the above-mentioned preparation method for preparing an anti-inflammatory agent.

In a fourth embodiment of the invention, the application of iridoid glycoside in oldenlandia diffusa for preparing anti-inflammatory drugs is provided.

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

The reagents used in the following examples are all commercially available.

Examples

First, crude extraction of oldenlandia diffusa

Pulverizing dried herba Hedyotidis Diffusae (200.00g), extracting with 80 deg.C 75% ethanol under reflux for 2 hr, and repeating for 3 times. Filtration, combining the filtrates, evaporating to dryness at 50 deg.C under reduced pressure using a rotary evaporator to obtain 32.31g of crude extract, and storing at 4 deg.C for use.

Second, HPLC-QTOF-MS/MS analysis

HPLC analytical column using C₁₈Column (250mm × 4.6mm i.d., 5 μm), temperature set at 25 ℃, elution with water (a) and acetonitrile (B) with addition of a 0.1% formic acid gradient, injection volume 20 μ L, gradient conditions set: 15% -20% B (0-5min), 20% -44% B (5-20 min), the flow rate is set to 0.8 mL/min.

A QTOF-MS system with electrospray ionization (ESI) ion source was used for MS data acquisition. The optimal parameters for iridoid glycoside identification in the negative ion mode are set as follows: the scanning range is 100-1000m/z, the spraying voltage is 3.5kV, the flow rate of the drying gas is 8.0L/min, the temperature is 200 ℃, the maximum energy of the MS/MS analysis of the atomizer pressure is 35psi, and the collision energy is controlled to be 35 eV.

Three, on-line extraction-HSCCC separation

0.8g of ground Hedyotis diffusa was inserted into a guard column (30X 10mm, inner diameter), both ends were sealed with a small amount of silica gel (100 and 200 mesh), and the guard column was connected to a six-way switching valve.

The countercurrent chromatographic separation column was completely filled with an upper phase solution (stationary phase) of ethyl acetate/n-butanol/water (4.5:0.5:5, v/v) with the six-way switching valve set to the "Load" position. Subsequently, the apparatus was set to run at 800rpm, pumping into the column at a flow rate of 1.8 mL/min. After hydrodynamic equilibrium is reached, the six-way switching valve is switched to the "Inject" position and the sample in the guard column is then eluted into the HSCCC. The detector continuously monitors the effluent at 254nm and collects the fractions manually. Then further separating the fraction by a Sephadex LH-20 chromatographic column by using a methanol solvent to finally obtain 6 high-purity compounds.

And fourthly, carrying out structural characterization on the compound by adopting a mass spectrum and a hydrogen spectrum.

Fifth, evaluation of cytotoxic and anti-inflammatory Activity

The MTT method was used to assess cytotoxicity. The macrophage Raw264.7 cell line was from the cell bank of the Chinese academy of sciences (Shanghai, China). Will be 2X 10 in total⁴The cells/mL were plated in 96-well plates and incubated overnight in 10% FBS-containing DMEM medium at 37 ℃. After removal of the cell culture medium, the raw264.7 cells were washed 3 times with PBS buffer. Then, cells were treated with six compounds at different concentrations and incubated at 37 ℃ for another 24 h. At the end of each incubation period, each well was washed 3 times with PBS buffer and 150.0 μ L of fresh MTT solution (0.5mg mL) was added^–1). Subsequently, the supernatant was removed, 150.0. mu.L DMSO was added, shaken for 15min, and incubated at 37 ℃ for 4 h. Finally, 3 replicates at 490nm were measured perOptical density of the wells.

The anti-inflammatory effects of the 6 compounds were evaluated by inhibiting Nitric Oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatants induced by lipopolysaccharide in Raw264.7 cells. NO production was measured using Griess reagent and TNF- α was measured using a quantitative enzyme-linked immunosorbent assay kit.

Sixth, statistical analysis

All analyses were performed 3 times and all data are expressed as mean ± Standard Deviation (SD). Data analysis was performed by one-way analysis of variance (ANOVA) and Tukey test. A p-value of less than 0.05 was considered to have statistically significant difference results.

Seventh, result and discussion

1. HPLC-QTOF-MS/MS (high Performance liquid chromatography-quantitative analysis) analysis of iridoid glycoside components in oldenlandia diffusa

This example optimizes the different mobile phases (methanol-acetic acid water, acetonitrile-acetic acid water, methanol-formic acid water and acetonitrile-formic acid water), elution procedures (isocratic and gradient elution), flow rates and MS conditions (scan mode, capillary voltage, optimized spray voltage, collision energy and dry gas temperature). The results show that 0.1% formic acid (a) and acetonitrile (B) exhibit more satisfactory degrees of separation in the gradient mode (0-5min, 15% -20% B, 5-20min, 20% -44% B) and a mobile phase flow rate of 0.8mL/min, as shown in fig. 1A.

In ESI-MS experiment, iridoid glycoside component in herba Hedyotidis Diffusae is analyzed in negative ion and positive ion mode to produce [ M + Na ] mainly in positive ion mode]⁺Ions and less MS/MS fragment ion products; and in the negative ion mode, the MS response is higher and the background noise is lower. Therefore, the negative ion mode is finally selected. Other operating parameters were optimized as follows: capillary voltage was 3.5kV, ion energy was 5eV, atomizer pressure was 35psi, nitrogen was dry gas, temperature and flow rate were 200 ℃ and 8.0L/min. For MS/MS spectroscopy, a range of collision energies (10-40eV) were optimized to obtain abundant fragment ions. FIG. 1B shows a total ion flow graph of Hedyotis diffusa extract with diagnostic ion m/z191.03 in negative ion mode.

2. Screening iridoid glycoside component by diagnostic ion filtration method

Natural products often exhibit a unique parent backbone with different substituents, while compounds with the same structural class contain similar fragmentation pathways with certain diagnostic ions. Therefore, the structural diagnostic ion-targeting network is used to rapidly screen for iridoid glycosides in oldenlandia diffusa. The literature has summarized different types of iridoid glycoside skeletons in oldenlandia diffusa, including monothioside derivatives, paederoside, geniposide and asperuloside. In general, [ M-H-162 ] can be detected in its MS/MS spectrum]^-、

[M-H-162-18]^-And [ M-H-162-18-44 ]]^-Indicating a continuous loss of Glc, H₂O and CO₂. Taking the example of deacetyl asperuloside as an example, the MS/MS spectrum is shown in FIG. 2A, and the cleavage pathway is shown in FIG. 2B. Apparently, the excimer ion [ MH ] at m/z 389.1065(C16H21O11)]At M/z 227.0569[ M-H-Glc]^-、209.0460[M-H-Glc-H₂O]^-、191.0326[M-H-Glc-2×H₂O]^-、165.0549[M-H-Glc-H₂O-CO₂]^-And 147.0435[ M-H-Glc-2 XH₂O-CO₂]^-. Although the fragment ion of the parent nuclear ion is at m/z 165.0549 (C)₉H₉O₃) As shown in fig. 2A. But at m/z 191.0326 (C)₁₀H₇O₄) Fragment ions of (A) are characteristic of iridoids and iridoid 4, 6-lactones. The final m/z191.03 is used as diagnostic ion of iridoid glycoside ligand derivative, and the extracted ion flow diagram of compounds 1-6 is shown in FIG. 1B.

3. Separation of target iridoid glycoside compounds

3.1 screening of two-phase solvent systems by countercurrent chromatography

Ethyl acetate/n-butanol/water solvent systems with different volume ratios were tested to obtain suitable partition coefficients (K values). As shown in Table 1, as the proportion of n-butanol increases, the K value of the objective compound increases. In view of the peak separation degree and the separation time, ethyl acetate/n-butanol/water (4.5:0.5:5, v/v) was selected.

TABLE 1 partition coefficient of the main compounds 1-6 in Oldenlandia diffusa

3.2, on-line extraction-HSCCC separation and Sephadex LH-20 chromatographic separation purification of iridoid glycoside

Crushed oldenlandia diffusa (0.8g) was loaded onto a guard column and connected to the HSCCC apparatus and tested at different flow rates (1.5, 1.8 and 2.0mL/min), with an optimal flow rate of 1.8mL/min and the isolation results shown in figure 3A. As shown in FIG. 3B, fraction a (85-95 min in FIG. 3A) gives compounds 1-3. The component b contains compounds 5 and 6, and the collection time is 192-234 min. The component c contains the compound 4, and the collection time is 244-294 min.

In order to obtain more iridoid glycoside, four continuous on-line extraction-HSCCC separation processes are carried out (the peaks of HSCCC and Sephadex LH-20 are collected according to a tube, HPLC analysis is adopted, and the peak-appearance time comparison is carried out with the peak of HPLC-QTOF-MS/MS). Separating the components a and b by a Sephadex LH-20 chromatographic column, and taking methanol as a mobile phase. By recrystallization, compounds 1(129.2mg), 2(59.6mg), 3(10.0mg), 4(18.0mg), 5(11.8mg) and 6(16.8mg) were obtained with a purity of more than 98%.

4. Structural analysis of iridoid glycoside

By MS and¹H-NMR identified the structures of 6 purified compounds, as shown in FIG. 4.

Compound 1: ESI-MS [ M-H ]]^-m/z:389.1066(–3.1ppm)；¹H NMR(400MHz,CD₃OD) δ (ppm) 5.55(1H, d, J ═ 8.0Hz, H-1), 7.35(1H, s, H-3), 3.56(1H, m, H-5), 6.20(1H, dd, J ═ 2.4,5.6Hz, H-6), 5.61(1H, dd, J ═ 2.0,5.6Hz, H-7), 2.67(1H, m, H-9), 3.57(2H, m, H-10), 4.68(1H, d, J ═ 7.8Hz, H-1 '),3.21(1H, m, H-2'), 3.26(1H, m, H-3 '),3.25(1H, m, H-4'), 3.37(1H, m, H-5 '),3.84(1H, m, 6H-6 a), 1H-6, H', 1 '6, H', 1H-6, H ', 1, H-10'). Compound 1 was identified as monoterpene.

Compound 2 ESI-MS [ M-H ]]^-m/z:389.1065(–3.3ppm)；¹H NMR(400MHz,CD₃OD) delta (ppm) 5.04(1H, d, J ═ 8.4Hz, H-1), 7.60(1H, s, H-3), 3.01(1H, d, J ═ 6.0Hz, H-5), 4.82(1H, m, H-6), 6.01(1H, brs, H-7), 2.55(1H, m, H-9), 4.20(1H, d, J ═ 15.6Hz, H-10 a),4.45(1H, d, J ═ 15.6Hz, H-10 b),4.72(1H, d, J ═ 7.8Hz, H-1 '),3.21(1H, m, H-2'), 3.27(1H, m, H-3 '),3.25(1H, m, H-4'), 3.37(1H, tym, tyh-3.85H, tye, data. Compound 2 was identified as deacetylasperuloside.

Compound 3 ESI-MS [ M-H ]]^-m/z:413.1104(-9.0ppm)；¹H NMR(400MHz,CD₃OD)δ(ppm):5.95(1H,d,J＝8.0Hz,H–1),7.30(1H,s,H–3),3.65(1H,m,H–5),5.56(1H,m,H–6),5.73(1H,m,H–7),3.30(1H,m,H–9),4.65(1H,d,J＝15.6Hz,H–10a),4.78(1H,d,J＝15.6Hz,H–10b),2.07(3H,s,–CH₃) 4.68(1H, d, J ═ 7.6Hz, H-1 '),3.19(1H, m, H-2 '),3.26(1H, m, H-3 '),3.23(1H, m, H-4 '),3.36(1H, m, H-5 '),3.98(1H, m, H-6 ' a),3.90(1H, m, H-6 ' b). Compound 3 was identified as asperuloside.

Compound 4: ESI-MS [ M-H ]]^-m/z:549.1646(7.8ppm)；¹H NMR(400MHz,CD₃OD) δ (ppm) 4.66(1H, d, J ═ 8.0Hz, H-1), 7.48(1H, s, H-3), 3.32(1H, d, J ═ 6.4Hz, H-5), 5.83(1H, brs, H-6), 5.62(1H, brs, H-7), 3.06(1H, dd, J ═ 6.0,6.6Hz, H-9), 4.19(1H, d, J ═ 16.0Hz, H-10 a),4.36(1H, d, J ═ 16.0Hz, H-10 b),3.63(3H, s, H-12), 5.30(1H, d, J ═ 7.2Hz, H-1 '),3.20(1H, m, H-2 '),3.28(1H, m, 3H-3H, 3H-12), 5.30(1H, d, J ═ 7.2Hz, H-1 ', 3.20(1H, m ', 3.28, 3H, 3.8H, 3H-1, 3H ', 8H ', 3H-6H ', 3.7H ', 8H-6H ', 1, H-6H ', 6H ',87 ',7 ', h-6 "), 6.75(2H, d, J ═ 8.0Hz, H-3", H-5 "), 6.86(1H, d, J ═ 13.2Hz, H-7"), 5.76(1H, d, J ═ 13.2Hz, H-8 "). The compound 4 is identified as 6-O- (Z) -p-coumaroyl scandoside methyl ester.

Compound 5 ESI-MS [ M-H ]]^-m/z:579.1705(-0.5ppm)；¹H NMR(400MHz,CD₃OD)δ(ppm):4.74(1H,d,J＝7.8Hz,H–1),7.56(1H,s,H–3),3.39(1H,d,J＝6.4Hz,H–5),5.90(1H,m,H–6),5.73(1H,m,H–7),3.09(1H,dd,J＝6.4,6.6Hz,H–9),4.26(1H,d,J＝15.6Hz,H–10a),4.35(1H,d,J＝15.6Hz,H–10b),3.68(3H,s,H–12),5.37(1H,d,J＝7.0Hz,H–1'),3.26(1H,m,H–2'),3.34(1H,m,H–3'),3.29(1H,m,H–4'),3.44(1H,m,H–5'),3.69(1H,m,H–6'a),3.94(1H,m,H–6'b),7.89(1H,s,H–2”),6.82(1H,d,J＝7.2Hz,H–5”),7.06(1H,d,J＝7.2Hz,H–6”),6.94(1H,d,J＝13.2Hz,H–7”),5.85(1H,d,J＝13.2Hz,H–8”),3.94(1H,s,OCH₃). The compound 5 is identified as 6-O- (Z) -feruloyl scandensoside methyl ester.

Compound 6: ESI-MS [ M-H ]]^-m/z:549.1587(-2.9ppm)；¹H NMR(400MHz,CD₃OD) δ (ppm) 4.68(1H, d, J ═ 8.0Hz, H-1), 7.51(1H, s, H-3), 3.31(1H, d, J ═ 6.8Hz, H-5), 5.84(1H, brs, H-6), 5.67(1H, brs, H-7), 3.07(1H, dd, J ═ 6.2,6.8Hz, H-9), 4.21(1H, d, J ═ 16.0Hz, H-10 a),4.39(1H, d, J ═ 16.0Hz, H-10 b),3.63(3H, s, H-12), 5.29(1H, d, J ═ 6.4Hz, H-1 '),3.20(1H, m, H-2'), 3.28(1H, m, 3H-1H, 3H-12), 5.29(1H, d, J ═ 6.4Hz, H-1 ', 3.20(1H, m', 3.28(1H, 3H, 8H, 3H-1, 7H ', 6H-6H', 6H-6H ', 7H', 6H-6H ', 6H',87 ',7', h-6 "), 6.80(2H, d, J ═ 6.8Hz, H-3", H-5 "), 7.61(1H, d, J ═ 16.0Hz, H-7"), 6.34(1H, d, J ═ 16.0Hz, H-8 "). The compound 6 is identified as 6-O- (E) -p-coumaroyl scandoside methyl ester.

5. Anti-inflammatory activity of iridoid glycosides

Oldenlandia diffusa is the main ingredient in Chinese patent medicine for treating inflammation-related diseases. This study evaluated the ability of compounds 1-6 to modulate the production of inflammatory mediators (NO and TNF-. alpha.) in Raw264.7 cells. Each compound with the concentration of 200 mug/mL has no obvious cytotoxicity on RAW264.7 cells, and the cell survival rate is over 90 percent. Thus, anti-inflammatory activity was measured at concentrations less than 200. mu.g/mL. As shown in Table 2,

compounds

1 and 3 showed stronger NO Inhibition (IC)₅₀: 94.7 + -10.5 and 78.1 + -9.2 mug/mL), while their inhibition of TNF-alpha is relatively significant (59.2 + -7.4 and 70.8 + -10.2 mug/mL). Although compounds 2 and 4-6 had NO significant inhibitory activity against NO, they had some TNF-. alpha.inhibitory activity. The introduction of a hydroxyl group at C-6 decreases the activity, while the esterification of the carboxyl group increases the activity. Thus, compound 2 was much less effective and compounds 1, 3-6 showed stronger TNF-. alpha.inhibitory activity.

TABLE 2 inhibitory Activity of iridoid glycoside monomers on lipopolysaccharide-induced NO and TNF- α production in RAW264.7 cells

Results are expressed as mean ± standard deviation from three independent experiments, with different labeled a-d lists showing significant differences (p <0.05) and indomethacin used as a positive control.

Conclusion

This example establishes an efficient method for the targeted screening and isolation of antiinflammatory cycloolefinosides in Hedyotis diffusa. Firstly, the cracking way of iridoid glycoside is systematically researched to carry out positioning diagnosis ion. Then, a diagnostic ion filtration-based HPLC-QTOF-MS/MS analysis method was established to screen for iridoid glycosides in Hedyotis diffusa. Afterwards, the online extraction-HSCCC strategy is adopted, so that the complicated offline sample extraction step is avoided, and the purification step of the target iridoid glycoside is accelerated. Results 6 major iridoid glycosides: the mixture is prepared by successfully separating and preparing the crystal orcein (1), the deacetyl asperulosidic acid (2), the asperuloside (3), the 6-O- (Z) -p-coumaroyl paederoside methyl ester (4), the 6-O- (Z) -feruloyl paederoside methyl ester (5) and the 6-O- (E) -p-coumaroyl paederoside methyl ester (6). The purified iridoid glycoside component is subjected to anti-inflammatory activity research, and the iridoid glycoside extracted by the method has anti-inflammatory activity.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of iridoid glycoside in oldenlandia diffusa is characterized in that the extract of oldenlandia diffusa is treated by adopting a diagnostic ion filtration method, the peak time of an iridoid glycoside corresponding chromatogram is obtained according to diagnostic ions, and the iridoid glycoside in oldenlandia diffusa is extracted by utilizing high-speed counter-current chromatography according to the peak time.

2. The method for preparing iridoid glycoside in oldenlandia according to claim 1, wherein the extract of oldenlandia is extracted from oldenlandia by ethanol extraction.

3. The method for preparing iridoid glycoside in oldenlandia according to claim 1, wherein the powder or granule of oldenlandia is extracted by heating and refluxing with aqueous solution of ethanol, and the liquid obtained after the reflux extraction is evaporated to dryness to obtain the extract of oldenlandia; preferably, the ratio of the spreading hedyotis herb powder or particles to the ethanol water solution is 1: 4.5-5.5, g: mL; preferably, the volume fraction of ethanol in the ethanol water solution is 70-80%.

4. The method for preparing iridoid glycoside of oldenlandia according to claim 1, wherein in the chromatographic mobile phase of diagnostic ion filtration method, formic acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B; preferably, the volume fraction of formic acid in the mobile phase A is 0.05-0.15%; preferably, the gradient elution is shaped as: 0-5min, 15% -20% B; 5-20min, 0% -44% B; the flow rate is 0.7-0.9 mL/min.

5. The method for preparing iridoid glycoside in oldenlandia according to claim 1, wherein the mass spectrum adopts negative ion mode;

or the diagnostic ion m/z is 190.0000-193.0000;

or in the mass spectrum, the capillary voltage is 3.0-4.0 kV, the ion energy is 4.5-5.5 eV, and the pressure of the atomizer is 30-40 psi.

6. The process for preparing iridoid glycosides from oldenlandia diffusa according to claim 1, wherein the solvent system of high-speed countercurrent chromatography consists of ethyl acetate, n-butanol and water; preferably, the volume ratio of the ethyl acetate to the n-butanol to the water is 4.45-4.55: 0.45-0.55: 4.5-5.5.

7. The method for preparing iridoid glycoside in oldenlandia according to claim 1, wherein the flow rate of high-speed countercurrent chromatography is 1.75-1.85 mL/min;

or in the high-speed countercurrent chromatography, the detection wavelength is 252-256 nm;

or, after the high-speed counter-current chromatography, further separating the fraction by using a Sephadex LH-20 chromatographic column and a methanol solvent.

8. Use of the preparation method of claim 1 for the targeted extraction of compounds having the same parent skeleton in plants.

9. Use of the process according to claim 1 for the preparation of an anti-inflammatory agent.

10. Application of iridoid glycoside in herba Hedyotidis Diffusae in preparing antiinflammatory medicine is provided.