CN115160335A

CN115160335A - Phthalide dimer and preparation method and application thereof

Info

Publication number: CN115160335A
Application number: CN202210856953.2A
Authority: CN
Inventors: 谭玉柱; 彭成; 闫洪玲
Original assignee: Chengdu University of Traditional Chinese Medicine
Current assignee: Chengdu University of Traditional Chinese Medicine
Priority date: 2022-07-20
Filing date: 2022-07-20
Publication date: 2022-10-11
Anticipated expiration: 2042-07-20
Also published as: CN115160335B

Abstract

The invention discloses a phthalide dimer and a preparation method and application thereof, and particularly relates to the technical field of medicines. The method comprises naturally drying and pulverizing rhizoma Ligustici Chuanxiong fibril, adding ethanol water solution, percolating, and concentrating the extractive solution under reduced pressure to obtain extract; dispersing the extract with water, repeatedly extracting with petroleum ether, mixing extractive solutions, and recovering solvent under reduced pressure to obtain petroleum ether extract; subjecting the extract to silica gel column, gradient eluting with petroleum ether/ethyl acetate, and collecting Fr.1-5 components under different Rf values; extracting the five parts respectively to obtain a racemic mixture (+/-) -1 and optically pure bodies thereof (+) -1b, (-) -1a, a compound 2 and a compound 3. The anti-inflammatory activity visible compound has obvious inhibition effect on the generation of NO of RAW264.7 macrophage induced by LPS, and is the phthalide compound which has the strongest inhibition effect on the model and is discovered at present. The invention further enriches the chemical component library of phthalide components by researching the fibrous root of the non-medicinal part of the ligusticum wallichii, and lays a foundation for the discovery of natural anti-inflammatory compounds.

Description

Phthalide dimer and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a phthalide dimer and a preparation method and application thereof.

Background

The extent of inflammatory response affects the incidence of cardiovascular events and is the pathological basis for the development of vascular lesions. A large number of researches show that the phthalide components have better anti-inflammatory activity. The monophthalide such as Z-ligustilide, senkyunolide A, neocnidilide and the like can inhibit the expression of various proinflammatory mediators, block the transmission of inflammatory signal channels and play a role in resisting inflammation. A part of phthalide dimers, such as ansaspirolide, gaobennolide A, (-) -6-3'a,7-6' -isowallicidide and the like, can remarkably inhibit LPS from inducing RAW264.7 macrophage to generate NO, and the EC50 is less than 5 mu M.

Ligusticum wallichii (Ligusticum chuanxiong Hort.) is a perennial herb of Umbelliferae and is one of the main sources of natural phthalide components. As one of the medicinal material varieties in the bulk Sichuan producing areas, the total Sichuan yield in 2019 reaches 1.9 ten thousand tons, and the biomass of non-medicinal parts is rich. Subject group prophase, the stem and leaf of chuanxiong rhizome are subjected to systematic chemical component research and pharmacodynamic evaluation, and a plurality of phthalide dimers with novel structures and vasodilation activity are found from the results. Recently, the UHPLC/Q-Orbitrap MS technology is used for comparing the chemical components of methanol extracts at different parts of the ligusticum wallichii, and a large amount of phthalide dimers are also found in the fibrous roots of the ligusticum wallichii, so that the method is worthy of further mining.

Disclosure of Invention

Therefore, the invention provides a phthalide dimer and a preparation method and application thereof, so as to further research the phthalide dimer in the fibrous root of ligusticum wallichii.

The invention researches the fibrous root of Ligusticum wallichii, and 3 phthalide dimers with polymerization positions of 3.3'a and 8.6' are obtained by separation. Wherein, the racemic mixture (+/-) -1 is a pair of new enantiomers (+/-) -chorixiongdiolide R11, which is called racemate (+/-) -1 for short and is named as (+) -chorixiongdiolide R11 (1B), and is called optical pure body (+) -1B and (-) -chorixiongdiolide R11 (1 a) for short, and is called optical pure body (-) -1a for short, and the other 2 are known compounds of tokinolide B (2) and ansaspirolide (3). Anti-inflammatory activity tests of the racemic mixture (+/-) -1 and the optically pure body (+) -1b, the optically pure body (-) -1a and the compounds 2 and 3 show that the compounds can obviously inhibit LPS (LPS) from inducing RAW264.7 macrophages to generate NO, and the EC50 is 0.05,0.07,1.19,2.89,0.92 mu M respectively. In racemic mixture (. + -.) -1, (+) -choanxinygdiolide R11 (1B) and (-) -choanxinygdiolide R11 (1 a), the structures of compound 2tokinolide B and compound 3ansaspirolide were as follows:

in order to achieve the above purpose, the invention provides the following technical scheme:

according to a first aspect of the present invention, there is provided a method for preparing a phthalide dimer, comprising:

step one, naturally drying fibrous roots of the ligusticum wallichii in the shade, crushing, adding an ethanol aqueous solution, percolating and extracting, and concentrating an extracting solution under reduced pressure to obtain an extract;

step two, adding water into the extract for dispersion, repeatedly extracting with petroleum ether, combining the extract liquor, and recovering the solvent under reduced pressure to obtain a petroleum ether extract;

step three, the petroleum ether extract is taken to pass through a silica gel column to carry out petroleum ether/ethyl acetate gradient elution, and five components Fr.1-5 are respectively collected under different Rf values;

step four, enabling the component Fr.4 to pass through a silica gel column chromatographic column, carrying out gradient elution by using petroleum ether/ethyl acetate, and obtaining 6 components of the component Fr.4.1-4.6 under different Rf values;

wherein, the component Fr.4.5 is prepared by C18 medium pressure, is eluted with methanol water gradient, and is eluted with normal phase reproSil 100Si column chromatography with n-hexane/isopropanol isocratic to obtain a compound 2tokinolide B;

step five, carrying out an coarsening section on the component Fr.5 by adopting an MCI column, carrying out gradient elution according to the first methanol and water, and obtaining 7 components of the component Fr.5.1-5.7 under different Rf values;

the component Fr.5.5 is subjected to silica gel column chromatography, petroleum ether/ethyl acetate gradient elution is carried out, and 6 components of the component Fr.5.5.1-Fr.5.5.6 are obtained under different Rf values;

the component Fr.5.5.6 adopts gel HW-40C and methanol isocratic elution to obtain 5 components of the component Fr.5.5.6.1-Fr.5.5.6.5 under different Rf values;

the component Fr.5.5.6.4 is subjected to reversed phase C18 column chromatography and isocratic elution with first methanol water to obtain a compound 3ansaspirolide;

passing the component Fr.5.6 through macroporous adsorption resin NM-200, performing gradient elution by using a second methanol water, and obtaining 6 components Fr.5.6.1-Fr.5.6.6 under different Rf values;

fraction fr.5.6.3 was isocratically eluted with a second methanol water by reverse C18 column chromatography to give racemic mixture (±) -1chuanxiong diolide r11;

and sixthly, resolving the racemic mixture (+/-) -1 by adopting a chiral column Daicel Chiralpak IC through preparative HPLC to obtain a pair of enantiomers (+) -choanxinygiolide R11 and (-) -choanxinygiolide R11.

Further, in the first step, the ethanol water solution is an ethanol solution with a concentration of 95%.

Further, in the second step, the temperature of the warm water is room temperature;

and/or in the second step, the repeated extraction times of the petroleum ether are 5-6.

Furthermore, in the third step, the mesh number of the silica gel column is 200-300 meshes;

and/or, in the third step, the gradient elution condition of petroleum ether/ethyl acetate is volume ratio, and the ratio of petroleum ether to ethyl acetate is = 99: 1-1: 99;

the gradient is specifically: 99;

and/or, in the third step, the determination of different Rf values is carried out by TLC detection.

Further, in the fourth step, the gradient elution condition of petroleum ether/ethyl acetate is volume ratio, and the ratio of petroleum ether to ethyl acetate is = 99: 1-1: 99;

the gradient is specifically: 99;

the determination of different Rf values is carried out by TLC detection;

and/or the condition of methanol-water gradient elution is volume ratio, the ratio of methanol to water is =60: 40-100: 0, and the flow rate is 30mL/min;

the gradient is specifically: initially methanol to water =60:40 automatic elution program is carried out, the time is adjusted, the concentration of the methanol is gradually increased along with the time, and the concentration of the methanol is increased to 100 percent after 1 hour.

And/or the isocratic elution condition of n-hexane/isopropanol is volume ratio, n-hexane/isopropanol =80:20, and the flow rate is 5.0mL/min.

Further, in the fifth step, the condition of the first methanol-water gradient elution is volume ratio, and the ratio of methanol to water is =70: 30-100: 0;

the gradient is specifically: the automatic elution procedure was initially carried out with methanol to water =70 to 30, the time was adjusted so that the methanol concentration gradually increased over time, after 1h to 100%.

And/or, the petroleum ether/ethyl acetate gradient elution condition is volume ratio, and the petroleum ether/ethyl acetate ratio is = 99: 1-1: 99;

the gradient is specifically: 99;

and/or, the first methanol water isocratic elution condition is volume ratio, methanol: water = 73: 27;

and/or the condition of the second methanol-water gradient elution is volume ratio, wherein the ratio of methanol to water is 60: 40-100: 0;

And/or the second methanol water isocratic elution condition is volume ratio, methanol: water =80:20, flow rate is 2mL/min

The determination of the different Rf values was carried out by TLC detection.

Further, in the sixth step, under the conditions of preparative HPLC, the mobile phase is n-hexane: isopropanol =80:20, flow rate 0.6mL/min, detection wavelength set to 254nm.

According to a second aspect of the present invention, there is provided a phthalide dimer, which is racemic mixture (±) -1, compound 2 or compound 3 prepared as above.

According to a third aspect of the present invention, there is provided the use of the above phthalide dimer for the preparation of a therapeutic anti-inflammatory agent.

The invention has the following advantages:

the method for preparing phthalide dimer of the invention can separate and purify phthalide dimer with 3 polymerization positions of 3.3'a,8.6', wherein the three phthalide dimers are racemic mixture (+/-) -1chuanxingdiolide R11 as a pair of enantiomers, compound 2tokinolide B and compound 3ansaspirolide respectively. Subsequent anti-inflammatory activity shows that the racemate (+/-) -1 and the optical pure body (+) -1b thereof, the optical pure body (-) -1a thereof and the compounds 2 and 3 have obvious inhibition effects on LPS-induced RAW264.7 macrophage NO generation, and the three compounds are phthalide compounds which are discovered at present and have the strongest inhibition effect on the model.

In the chemical structure of the phthalide dimer prepared by the invention, a racemic mixture (+/-) -1 and a compound 2 have the same planar structure, the configuration of only 2 chiral centers is different, and the relative configuration of four chiral carbons of the compound 3 is consistent with that of the racemic mixture (+/-) -1 except that one intra-ring double bond is added. The high similarity in structure of the three compounds results in their action at the same protein active site, thereby producing similar biological effects. The research reveals the strong anti-inflammatory potential of the phthalide dimer with the polymerization position of 3.3'a,8.6', and an anti-inflammatory activity test can be further developed for the phthalide dimer with the polymerization position in the later period, so that the action mechanism is clear.

The invention further enriches the chemical component library of phthalide components through the research on fibrous roots of the non-medicinal part of the ligusticum wallichii, lays a foundation for the discovery of natural anti-inflammatory compounds, and is beneficial to the sustainable development of traditional Chinese medicine resources.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, the proportions, the sizes, and the like shown in the specification are only used for matching with the contents disclosed in the specification, so that those skilled in the art can understand and read the present invention, and do not limit the conditions for implementing the present invention, so that the present invention has no technical essence, and any modifications of the structures, changes of the proportion relation, or adjustments of the sizes, should still fall within the scope of the technical contents disclosed in the present invention without affecting the efficacy and the achievable purpose of the present invention.

FIG. 1 is a HRESIMS spectrum of a racemic mixture (. + -.) -1 provided by the present invention;

FIG. 2 is a UV diagram of the racemic mixture (. + -.) -1 provided by the present invention;

FIG. 3 Process for preparing the racemic mixture (+/-) -1 of the present invention ¹ H NMR chart;

FIG. 4 Process for preparing the racemic mixture (+ -) -1 according to the present invention ¹³ C NMR chart;

FIG. 5 is an HSQC diagram of the racemic mixture (. + -.) -1 provided by the present invention;

FIG. 6 Process for preparing racemic mixture (+ -) -1 according to the present invention ¹ H- ¹ HCOSY diagram;

FIG. 7 HMBC diagram of the racemic mixture (. + -.) -1 provided by the present invention;

FIG. 8 NOESY plot of the racemic mixture (. + -.) -1 provided by the present invention;

FIG. 9 is a diagram of the signals associated with the major COSY, HMBC, NOESY of the racemic mixture (. + -.) -1 provided by the present invention;

FIG. 10 ECD diagram of the racemic mixture (. + -.) -1 provided by the present invention;

FIG. 11 preparation of Compound 2 according to the present invention ¹ A HNMR map;

FIG. 12 preparation of Compound 3 provided by the present invention ¹ A HNMR map;

FIG. 13 preparation of Compound 3 provided by the present invention ¹³ A CNMR map.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Instruments and materials:

bruker Avance III 600-NMR spectrometer (Bruker, germany); a Synapt G2HDMS mass spectrometer (Waters corporation, usa); MH-LC-52 type medium-high pressure liquid phase preparation (Sesbane Ruisi Beijing technologies, inc.); COSMOSIL Cholester preparative columns (5 μm,10 250mm, nacalai Tesque, japan); repuriL 100Si normal phase preparative chromatography column (5 μm, 10X 250mm,5 μm, 10X 250mm, dr. Maisch, germany); daicel Chiralpak IC chiral analytical column (5 μm, 4.6X 250mm, daicel Co., japan); HS-1200P18 rapid purification preparative liquid chromatography (Tianjin Bora Ai Jieer science and technology, inc.); perkin-Elmer 341 pulses polarimeter (Perkin Elmer, USA); ECD spectrometer (JASCO corporation, japan); RE-52A rotary evaporator (Shanghai Yangrong Biochemical Instrument plant); yopu UPT series ultrapure water (Chengdoupu electronics, inc.); 15AC cell culture chamber (SANYO company); clean bench (suzhou air technology ltd); GI54DS autoclave (Zealway corporation, usa); ALLEGRA X-12 high speed centrifuge (BECKMAN COULTER, USA); an adjustable pipettor, multiskan FC microplate reader (Thermo Fisher, USA); CKX53 inverted microscope (olympus, japan); c18 chromatography on silica gel (40-60 μm, welch, USA); CHP 20PMCI resin (75-150 μm, mitsubishi chemical, japan); toyopear1 HW-40C gel (TOSOH Co., japan); NM200 macroporous adsorbent resin packing (suzhou nano micro science ltd); silica gel GF254 thin layer plate, 200-300 mesh silica gel (Qingdao ocean factory); chromatographically pure methanol, n-hexane, isopropanol (SIGMA, usa); deuterated chloroform (Alorich, USA); other reagents were analytically pure (Dow Colon Chemicals, inc.); DMEM medium (Gibco, usa); fetal bovine serum (Zhejiang Hangzhou Biotechnology, inc.); double antibody (shanghai bi yuntian biotechnology limited); CCK-8 kit (Shanghai Tao Shu Biotech, inc.); NO kit (shanghai bi yuntian biotechnology limited); lipopolysaccharide (LPS, sigma, usa); trypsin-EDTA digest (beijing solibao technologies ltd); petri dishes, 96-well plates (corning, usa); curcumin (JHS, sigma, usa); dimethyl sulfoxide (DMSO, MP Biomedicals, USA).

RAW264.7 cells were derived from the cell bank of the national academy of sciences of shanghai.

The whole medicinal material of the Ligusticum wallichii is collected from Meishan city of Sichuan province in 5 months in 2020, is identified as the Ligusticum wallichii Ligusticum chuanxiong Hort. Plant by professor Hu Changjiang of Chengdu Chinese medicine university, and the voucher specimen is stored in a national key laboratory 1001 of southwestern characteristic Chinese medicine resources of Chengdu Chinese medicine university.

4-hydroxy-3-butylphthalaide: 4-hydroxy-3-butylphthalide

Example 1

This example provides a method for preparing a racemic mixture (±) -1 phthalide dimer:

1. extraction and separation

Collecting natural dry rhizoma Ligustici Chuanxiong fibrous root (50 kg), pulverizing, percolating with 95% ethanol water (400L) at room temperature, and concentrating the extractive solution under reduced pressure to obtain extract (5 kg). Dispersing the extract in room temperature water, extracting with petroleum ether for 5-6 times, combining the extractive solutions, and recovering the solvent under reduced pressure to obtain petroleum ether fraction. Taking petroleum ether part (3 kg), passing through silica gel column (200-300 meshes), gradient eluting with petroleum ether and ethyl acetate (99: 1-1: 99), and detecting by TLC to obtain solutions A-E.

Specific gradient: 99

And (3) performing a coarsening section on the E component (149.158 g) by using an MCI column, and performing gradient elution according to methanol: water (70: 30-100: 0, specific gradient: an automatic elution program is performed by using methanol: water =70 at the beginning, the time is adjusted, the concentration of the methanol is gradually increased along with the time, and the concentration of the methanol is increased to 100%. V/v after 1 h), so as to obtain 7 sub-components (E1-E7). E6 is subjected to macroporous adsorption resin NM-200, and an automatic elution procedure is carried out by using methanol/water (60-100: 0, a specific gradient is firstly carried out by using methanol/water = 60. E6.3 was isocratically eluted through reverse phase C18 column chromatography using methanol: water (80: 20, v/v,2 mL/min) to give the racemic mixture (. + -.) -1 (5.78 mg).

2. Chiral resolution

The optical rotation of the racemic mixture (+/-) -1 is close to 0, the racemic mixture is considered, a chiral column Daicel Chiralpak IC is adopted, n-hexane and isopropanol (80/20, v/v and 0.6 mL/min) are taken as a mobile phase, the detection wavelength is set to 254nm, and a pair of enantiomer optical pure bodies (+) -1 b/optical pure body (-) -1a is obtained through resolution.

3. Structural identification

The characteristics are as follows: yellow oil (c 0.30, meOH).

As shown in FIG. 1, HRESIMS m/z 403.1876[ 2] M + Na] ⁺ Indicating a molecular formula of C ₂₄ H ₂₈ O ₄ (calcd for C ₂₄ H ₂₈ O ₄ Na ⁺ M/z 403.1880) with an unsaturation of 11.

As shown in FIG. 2, UV has an absorption maximum at 281 nm.

As shown in figure 3 of the drawings, ¹ h NMR showed 2 methyl signals [ Delta ] attached to methylene _H 0.84(t,J＝7.2Hz,H ₃ -11),δ _H 0.84(t,J＝7.2Hz,H ₃ -11')](ii) a 4 alkene hydrogen proton signals [ delta ] _H 7.40(d,J＝6.6Hz,H-7'),δ _H 6.25(ddd,J＝9.6,2.6,1.3Hz,H-7),δ _H 6.04(ddd,J＝9.6,5.0,3.3Hz,H-6),δH 4.25(dd,J＝8.7,6.5Hz,H-8')]. In addition, at δ _H And the interval between 1.0 and 3.5 shows the multiplet signals of a plurality of methylene protons and methine protons which are partially overlapped.

As shown in the figure 4 of the drawings, ¹³ c NMR showed 24 carbon signals, in combination HSQC showed 2 methyl groups (. Delta.) as shown in FIG. 5 _C 13.7,14.2); 8 methylene groups (. Delta.) _C 21.6,22.9,23.0,23.2,24.8,24.9,27.2,32.3); 6 methine radicals [ containing 2 sp ³ Hybrid carbon (. Delta.) _C 37.4,48.5) and 4 olefinic carbons (. Delta.) (S.) _C 107.5,117.6,129.4,140.7); 8 quaternary carbons [ containing 2 sp ³ Hybrid carbon (. Delta.) _C 52.2,δ _C 92.6 4 olefinic carbons (. Delta.) _C 128.1,134.4,148.1,158.4) and 2 ester carbonyls (. Delta.) (S) _C 163.9,169.4)]。.

As shown in figure 6 of the drawings, ¹ H- ¹ HCOSY shows C-4-C-5-C-6-C-7 (C-4 '-C-5' -C-6 '-C-7') and C-8-C-9-The carbon chain connection sequence of C-10-C-11 (C-8 ' -C-9' -C-10' -C-11 ') and C-8-C-6' shows that the compound is the basic framework of phthalide dimer. .

As shown in FIG. 7, the HMBC spectra showed that H-4', H-6' were associated with C-3, and H-5', H-7' were associated with C-8 suggesting that the dimer polymerized as C-3-C-3'a, C-8-C-6'. H-8' is related to NOESY of H-4, indicating Δ ^3',8' The double bond is in the Z configuration. The above data with isotokinolide B ^[1] Similarly, it was determined that their planar structures were consistent.

As shown in FIG. 8, NOESY showed that H-4'/H-5', H-5'/H-8, H-4'/H-4, H-4/H-8 are related, indicating that H-4', H-5' and H-8 are on the same side of the six-membered ring and close to C-4, revealing that the relative configuration of the dimer is 3S,8R,6'S,3' aR, with a different stereoconfiguration than isotokinolide B; the specific signal display is shown in fig. 9. Further carrying out chiral resolution, and determining the absolute configuration of the compound to be (+) 1b3S,8R,6'S,3' aR by ECD; (-) 1a3R,8S,6'R,3' aS (shown in FIG. 10).

Physicochemical properties and spectral data of racemic mixture (+ -) -1: yellow oil;

UV(MeOH)λ _max (logε)＝281(1.15)nm；

HRESIMS m/z 403.1876[M+Na] ⁺ (calcd for C ₂₄ H ₂₈ O ₄ Na ⁺ ,m/z 403.1880)；

(+)(3S,8R,6'S,3'aR)chuanxiongdiolide R11 (1b)；

ECD(c0.02,MeOH)＝Δ _ε213 –3.41,Δ _ε221 +3.62,Δ _ε241 –62.58,Δ _ε286 +28.42.(–)(3R,8S,6'R,3'aS)chuanxiongdiolideR11(1a)；ECD(c0.01,MeOH)＝Δ _ε210 +40.28,Δ _ε222 –2.61,Δ _ε239 +34.83,Δ _ε285 –14.94. ¹ HNMR and ¹³ the CNMR data are shown in Table 1.

TABLE 1

Example 2

This example provides a method for the preparation of the compound 2tokinolide b:

1. extraction and separation

Collecting natural and shade dried rhizoma Ligustici Chuanxiong fibril (50 kg), pulverizing, percolating with 95% ethanol water (400L) at room temperature, and concentrating the extractive solution under reduced pressure to obtain extract (5 kg). Dispersing the extract with room temperature water, extracting with petroleum ether for 5-6 times, combining the extracts, and recovering the solvent under reduced pressure to obtain petroleum ether fraction. Taking petroleum ether part (3 kg), passing through silica gel column (200-300 meshes), gradient eluting with petroleum ether and ethyl acetate (99: 1-1: 99), and detecting by TLC to obtain solutions A-E.

Specific gradient: 99

Subjecting the solution D (156 g) to silica gel column chromatography, gradient eluting with petroleum ether and ethyl acetate (99: 1-1: 99, v/v), and detecting by TLC to obtain 6 components (D1-D6).

Specific gradient: 99;

the component D5 was prepared under medium pressure in C18, and was subjected to an automatic elution procedure using methanol/water (60-100: 0, with a specific gradient: methanol/water =60:40 at the beginning, with time adjusted, the methanol concentration gradually increased over time, and after 1h the methanol concentration increased to 100%. V/v,30 mL/min), and then subjected to normal phase ResproSil 100Si column chromatography with n-hexane/isopropanol (80: 20, v/v,5.0 mL/min) at an equal rate to give Compound 2 (4.96 mg).

2. Structural identification

Compound 2: a pale yellow oily liquid.

The molecular formula is as follows: c ₂₄ H ₂₈ O ₄ ，HR-ESI-MS m/z：403.1871[M+Na] ⁺ 。

As shown in figure 11 of the drawings, ¹ H NMR(CDCl ₃ ,600MHz)δ:7.51(1H,d,J＝6.7Hz,H-7'),6.16(1H,dt,J＝9.6,2.1Hz,H-7),5.92(1H,dt,J＝9.6,4.2Hz,H-6),4.62(1H,dd,J＝8.8,6.8Hz,H-8'),3.10(1H,m,H-6'),2.49(1H,m,H-4'b) 2.28 (2H, m, H-5), 2.17 (2H, m, H-4), 2.11 (1H, m, H-5'b), 2.05 (1H, m, H-9'b), 1.96 (1H, m, H-9'a), 1.66 (1H, m, H-8), 1.46 (1H, m, H-9 b), 1.34 (1H, m, H-4'a), 1.30 (1H, m, H-9 a), 1.29 (1H, m, H-10 b), 1.28 (1H, m, H-5'a), 1.26 (2H, m, H-10'), 1.13 (1H, m, H-10 a), 0.88 (3H, t, 7J, 3H-7, 11J, 11H-7 Hz). The above spectral data are consistent with the literature reports ^[2] Thus, compound 2 was identified as tokinolide B.

Example 3

This example provides a method for preparing 3 ansaspirolide:

1. extraction and separation

Specific gradient: 99

And (3) performing a coarsening section on the E component (149.158 g) by using an MCI column, and performing gradient elution according to methanol: water (70: 30-100: 0, specific gradient: an automatic elution program is performed by using methanol: water =70 at the beginning, the time is adjusted, the concentration of the methanol is gradually increased along with the time, and the concentration of the methanol is increased to 100%. V/v after 1 h), so as to obtain 7 sub-components (E1-E7). E5 was subjected to silica gel column chromatography and eluted with a petroleum ether/ethyl acetate (99: 11: 99, specific gradient 99. E5.6 gel HW-40C, methanol isocratic elution, get 5 subfractions (E5.6.1-E5.6.5). E5.6.4 was isocratically eluted via reverse phase C18 column chromatography with methanol: water (73: 27, v/v,2 mL/min) to give compound 3 (8.41 mg).

2. Structural identification

Compound 3: a pale yellow oily liquid.

The molecular formula is as follows: c ₂₄ H ₂₆ O ₄ ，

HR-ESI-MS m/z：379.2001[M+H] ⁺ 。

As shown in the schematic view of figure 12, ¹ H NMR(CDCl ₃ ,600MHz)δ:7.88(1H,dd,J＝7.5,1.0Hz,H-7),7.76(1H,td,J＝7.5,1.0Hz,H-5),7.61(1H,td,J＝7.5,1.0Hz,H-6),7.53(1H,d,J＝7.5Hz,H-4),7.47(1H,d,J＝6.6Hz,H-7'),3.25(1H,m,H-6'),3.21(1H,dd,J＝8.7,6.5Hz,H-8'),2.26(1H,t,J＝7.0Hz,H-8),2.21(1H,m,H-4'b),2.04(1H,m,H-9'b),1.91(1H,m,H-5'b),1.74(1H,m,H-9'a),1.57(1H,m,H-5'a),1.49(1H,m,H-4'a),1.20(1H,m,H-10b),1.16(2H,m,H-9),1.07(1H,m,H-10'b),1.00(1H,m,H-10'a),0.98(1H,m,H-10a),0.73(3H,t,J＝7.0Hz,H-11),0.68(3H,t,J＝7.4Hz,H-11')；

as shown in figure 13 of the drawings, in which, ¹³ CNMR(151MHz,CDCl ₃ ) Delta.: 169.0 (C-1), 164.1 (C-1 '), 149.3 (C-3 a), 148.0 (C-3'), 140.9 (C-7 '), 134.1 (C-7'a), 134.1 (C-5), 129.9 (C-7 a), 128.2 (C-6), 125.9 (C-7), 122.2 (C-4), 107.6 (C-8 '), 91.5 (C-3), 52.1 (C-3'a), 51.3 (C-8), 38.4 (C-6 '), 32.7 (C-9), 27.0 (C-9'), 25.5 (C-4 '), 25.2 (C-5'), 22.5 (C-10), 21.5 (C-10 '), 14.1 (C-11), 13.6 (C-11'). The above spectral data and literature ^[3] The reports are consistent, so compound 3 was identified as ansaspirolide.

Comparative example

Collecting natural and shade dried rhizoma Ligustici Chuanxiong fibril (50 kg), pulverizing, percolating with 95% ethanol water (400L) at room temperature, and concentrating the extractive solution under reduced pressure to obtain extract (5 kg). Heating the extract for dispersing water, sequentially extracting the extract for 5 to 6 times by using petroleum ether, ethyl acetate and n-butyl alcohol, combining extraction liquids of all parts, and recovering a solvent under reduced pressure to obtain extraction parts. Petroleum ether part (3 kg) is taken to pass through silica gel column (200-300 meshes), and gradient elution is carried out by using petroleum ether and ethyl acetate (99: 1 → 1: 99), thus obtaining components A-E respectively. Performing UHPLC/Q-OrbitrapMS analysis on the components A to E, triggering data dependent acquisition (PIL-DDA) through a parent ion list to establish a characteristic molecular network (FBMN), and selecting a target polar section E component for further separation. The E component (149.158 g) was subjected to a coarsening section using an MCI column, and eluted in a gradient of methanol to water (70: 30-100: 0,v/v) to give 7 subcomponents (E1-E7). Subjecting the fractions E1-E7 to UPLC-MS analysis, selecting the target sub-fractions for further separation

E5 is subjected to silica gel column chromatography and gradient elution by petroleum ether and ethyl acetate (99: 1-1: 99, v/v) to obtain 6 components (E5.1-E5.6). E5.4 was eluted isocratically via reverse phase C18 column chromatography in methanol: water (55: 45, v/v,2 mL/min) to give compounds 25 (3.00 g) and 26 (3.61 mg); e5.6 gel HW-40C, methanol isocratic elution, get 5 components (E5.6.1-E5.6.5). E5.6.3 was isocratically eluted via reverse phase C18 column chromatography with methanol: water (77: 23, v/v,2 mL/min) to give compounds 4 (3.16 mg), 12 (13.15 mg) and 13 (2.62 mg); e5.6.4 was isocratically eluted via reverse phase C18 column chromatography with methanol: water (73: 27, v/v,2 mL/min) to give compounds 5 (7.84 mg), 6 (4.23 mg), 8 (4.56 mg) and 3 (8.41 mg); e5.6.5 was isocratically eluted via reverse phase C18 column chromatography in methanol: water (70: 30, v/v,2 mL/min) to give compound 27 (4.52 mg).

E6 passes through macroporous adsorption resin NM-200, and is subjected to gradient elution by methanol and water (60: 40-100: 0,v/v) to obtain 6 components (E6.1-E6.6). E6.4 was eluted isocratically through reverse phase C18 column chromatography in methanol: water (80: 20, v/v,8 mL/min) to give 15 (8.66 mg) and 16 (8.93 mg); e6.5 was eluted isocratically via reverse phase C18 column chromatography using methanol: water (75: 25, v/v,2 mL/min) to give compounds 7 (5.42 mg) and 23 (5.15 mg); e6.6 using gel HW-40C, methanol isocratic elution, get 7 fractions (E6.6.1-E6.6.7). E6.6.3 was isolated via a C18 medium pressure column with methanol: eluting with water (60: 40-100: 0,v/v,30 mL/min) in gradient, and separating by PTLC to obtain 14 (4.24 mg) and 17 (20.17 mg); e6.6.4 was isocratically eluted via reverse phase C18 column chromatography with methanol: water (80: 20, v/v,8 mL/min) to give compounds 18 (2.55 mg), 19 (4.14 mg) and 20 (9.23 mg).

The component B (140 g) is subjected to silica gel column chromatography and gradient elution with petroleum ether and ethyl acetate (99: 11: 99, v/v) to obtain 8 components (B1-B8). The component B3 is passed through NM-200 macroporous adsorbent resin, and gradient eluted with methanol and water (60: 40100: 0,v/v) to obtain 7 components (B3.1-B3.7). B3.1 was isocratically eluted through reverse phase C18 column chromatography with methanol: water (55: 45, v/v,2 mL/min) to give compounds 28 (4.95 mg) and 29 (6.43 mg); b3.2 was eluted isocratically via reverse phase C18 column chromatography in methanol: water (50: 50, v/v,2 mL/min) to give compounds 11 (3.28 mg), 30 (2.07 mg) and 31 (8.11 mg); b3.5 was prepared by medium pressure half-prep, methanol: water (70: 30, v/v,20 mL/min) isocratic elution to give compound 32 (47.24 mg); b3.4 is subjected to medium-pressure half-preparation to obtain 5 components (B3.4.1-B3.4.5). B3.4.1 was subjected to reverse phase C18 column chromatography eluting isocratically with methanol: water (60: 40, v/v,2 mL/min) to give compound 33 (18.45 mg); b3.4.2 and B3.4.3 were prepared by PTLC to give compounds 9 (4.59 mg) and 21 (8.07 mg), respectively.

Subjecting the component D (156 g) to silica gel column chromatography, and gradient eluting with petroleum ether and ethyl acetate (99: 1-1: 99, v/v) to obtain 6 components (D1-D6). D2 was prepared at C18 medium pressure, gradient eluted with methanol: water (60: 0100: 0,v/v,30 mL/min) followed by reversed phase C18 column chromatography isocratic eluted with methanol: water (70: 30, v/v,2 mL/min) to give compound 34 (2.89 mg); d5 was prepared under C18 medium pressure, eluted with a methanol: water gradient (60: 40-100: 0,v/v,30 mL/min) followed by normal phase Repsil 100Si column chromatography eluting isocratically with n-hexane: isopropanol (80: 20, v/v,5.0 mL/min) to give compounds 10 (2.79 mg), 22 (2.48 mg), 2 (4.96 mg) and 24 (1.49 mg).

Gradient conditions were the same as in examples 1-3.

Test example 1

This test example performed an anti-inflammatory cell experiment using the phthalide dimers prepared in examples 1 to 3 and comparative example 1; curcumin was used as a control.

1. Cell culture

RAW264.7 cells in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin at 37 ℃ with 5% CO ₂ Culturing in the incubator, and subculturing when the cell growth reaches about 80%.

2. Preparation of sample solution

The test compound was dissolved in DMSO to prepare a 120mM stock solution, which was diluted to an appropriate concentration in DMEM medium immediately before use.

3. Cell viability assay

Taking cells in logarithmic growth phase at 1.0 × 10 ⁵ PermL the cell suspension was inoculated into a 96-well plate at 100. Mu.L/well, set at 37 ℃ and contains 5% CO ₂ Culturing in an incubator for 24h to stabilize the cells. The various concentrations of the subject racemic mixture (. + -.) -1, optically pure (+) -1b, optically pure (-) -1a, compound 2, compound 3, and Curcuma longa of the vegetation of example 1 were added separatelyThe culture was continued for 24h in 96-well plates with negative and zero control (medium only, no cells). Add 10. Mu.L of CCK-8 reagent per well and continue to stand at 37 ℃ with 5% CO ₂ Incubating for 1h in the incubator in the dark, and measuring the absorbance OD value by using a multifunctional microplate reader under the wavelength of 450 nm. The cell viability was calculated by substituting the OD into the equation. The experiment was repeated 3 times. The survival rate is calculated by the formula:

cell survival rate = [ (administration group OD value-zero group OD value)/(negative group OD value-zero group OD value) ] × 100%

Measurement of NO production inhibition Rate

Taking cells in logarithmic growth phase at 5.0 × 10 ⁵ PermL the cell suspension was inoculated into a 96-well plate at 100. Mu.L/well, set at 37 ℃ and contains 5% CO ₂ Culturing for 16h in an incubator to ensure that the cells are paved on the bottom of the plate by 80 percent. Except for the normal control group without any drug treatment, only LPS (0.1. Mu.g/mL) was added to the cells of the LPS model group, and LPS (0.1. Mu.g/mL) and different concentrations of the test samples were added to each of the administration groups. After further culturing for 24h, 50. Mu.L of supernatant was taken from each well and put in a 96-well plate, 50. Mu.L of Griess Reagent I and 50. Mu.L of LGriess Reagent II were added, and reacted at room temperature for 15min, and the OD value of absorbance at 540nm was measured with a microplate reader.

The OD value is substituted into a formula to calculate the inhibition rate. The experiment was repeated 3 times. The formula for calculating the inhibition rate is as follows:

inhibition rate = [ (model group OD value-administration group OD value)/(model group OD value-normal group OD value) ] × 100%

In order to eliminate the cytotoxicity of the compound on RAW264.7 macrophage, the CCK-8 method is used for detecting the cell survival rate after RAW264.7 macrophage is treated for 24 hours by using different concentrations of a compound to be detected. As shown in Table 2, most compounds had no significant effect on cell proliferation at a concentration of 100. Mu.M, and some compounds showed greater cytotoxic effects, and IC for determining cytotoxicity was further diluted ₅₀ The value is obtained. All isolated compounds were subjected to an in vitro LPS-stimulated RAW264.7 macrophage NO production inhibition experiment at tested concentrations with cell viability higher than 80%.

The cytotoxicity of the test compounds on RAW264.7 cell line and the inhibitory effect on LPS-stimulated NO production (n = 3) are shown in table 2.

TABLE 2

As can be seen from Table 2, the racemate (+/-) -1 and the optically pure (+) -1b thereof, the optically pure (-) -1a thereof and the compounds 2 and 3 all have obvious inhibition effects on LPS-induced RAW264.7 macrophage NO generation, and EC ₅₀ 0.05,0.07,1.19,2.89,0.92. Mu.M, respectively. The anti-inflammatory activity of the tested compound is obviously superior to that of positive medicament curcumin. Therefore, the phthalide dimer compound prepared by the invention has good anti-inflammatory effect.

The invention selects Ligusticum wallichii fibrous root to carry out chemical composition research, separates and identifies 3 phthalide dimers with polymerization positions of 3.3'a, 8.6'. Wherein the racemic mixture (. + -.) -1 is a pair of enantiomers. The anti-inflammatory activity shows that the raceme (+/-) -1 and the optical pure body (+) -1b thereof, the optical pure body (-) -1a thereof and the compounds 2 and 3 have obvious inhibition effect on LPS induced RAW264.7 macrophage NO generation, and the three compounds are phthalide compounds which have the strongest inhibition effect on the model. In terms of chemical structure, a racemic mixture (+/-) -1 and a compound 2 have the same planar structure, the configuration of only 2 chiral centers is different, and the relative configuration of four chiral carbons of the compound 3 is consistent with that of the racemic mixture (+/-) -1 except that one intra-ring double bond is added. The high similarity in structure of the three compounds may result in their action at the same protein active site, thereby producing similar biological effects. The invention discloses strong anti-inflammatory potential of a phthalide dimer with a polymerization position of 3.3'a,8.6', and an anti-inflammatory activity test can be further developed aiming at the phthalide dimer with the polymerization position at a later stage to define an action mechanism. The invention further enriches the chemical component library of phthalide components through the research on fibrous roots of the non-medicinal parts of the ligusticum wallichii, lays a foundation for the discovery of natural anti-inflammatory compounds, and is beneficial to the sustainable development of traditional Chinese medicine resources.

Reference documents:

[1] luxinhua (Luxinhua); zhang Jinjuan; zhang Xuexia; liang Hong; zhao Yuying, separation and structural identification of ligustilide dimer in radix Angelicae sinensis. Chinese medicine journal 2008,19,2196-2201.

[2] Tang Fei; hongling; yang Li; sun Guyi; hu Changjiang; peng Cheng; ao Hui; tan Yuzhu, and the chemical components of phthalides in stems and leaves of rhizoma Ligustici Chuanxiong, and the research and development of natural products 2022,1-15.

[3] Wei Wei; xu Wei; 3238 Zxft 3238, research on chemical components of normalized planted rhizoma Ligustici Chuanxiong, and 3262 Zxft 3262 (15), 3017-3025.

Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A preparation method of phthalide dimer is characterized by comprising the following steps:

wherein, the component Fr.4.5 is prepared by C18 medium pressure, is eluted by methanol-water gradient, and is eluted by normal phase ReProSil 100Si column chromatography with normal hexane/isopropanol isocratic to obtain a compound 2tokinolide B;

step five, carrying out a coarsening section on the component Fr.5 by adopting an MCI column, carrying out gradient elution according to the first methanol and water, and obtaining 7 components of the component Fr.5.1-5.7 under different Rf values;

the component Fr.5.5.6 adopts gel HW-40C, methanol isocratic elution, under different Rf values, 5 components of the component Fr.5.5.6.1-Fr.5.5.6.5 are obtained;

the component Fr.5.5.6.4 is subjected to reversed phase C18 column chromatography and eluted isocratically with first methanol water to obtain a compound 3ansaspirolide;

component Fr.5.6.3 is isocratically eluted with a second methanol water by reverse C18 column chromatography to give racemic mixture (+ -) -1-chuanxingdiolide R11;

2. The method for preparing the phthalide dimer according to claim 1, wherein in the first step, the ethanol aqueous solution is 95% ethanol solution.

3. The method for preparing phthalide dimer according to claim 1, wherein in the second step, the petroleum ether is extracted repeatedly for 5-6 times.

4. The method for preparing the phthalide dimer according to claim 1, wherein in the third step, the mesh number of the silica gel column is 200-300 meshes;

and/or, in the third step, the determination of different Rf values is performed by TLC detection.

5. The method for preparing phthalide dimer according to claim 1, wherein in step four, the gradient elution condition of petroleum ether/ethyl acetate is volume ratio, and the ratio of petroleum ether to ethyl acetate is = 99: 1-1: 99;

and/or, the determination of different Rf values is by TLC detection;

and/or, the methanol-water gradient elution conditions are volume ratio, methanol: water =60: 40-100: 0, and the flow rate is 30mL/min;

6. The method for preparing phthalide dimer according to claim 1, wherein in the fifth step, the first methanol-water gradient elution condition is volume ratio, methanol-water =70: 30-100: 0;

and/or, the first methanol-water isocratic elution condition is volume ratio, and methanol-water = 73: 27;

and/or the second methanol water isocratic elution condition is volume ratio, methanol: water =80:20, and flow rate is 2mL/min.

7. The method for preparing phthalide dimer according to claim 1, wherein in step six, the conditions of preparative HPLC are as follows, and the mobile phase is n-hexane: isopropanol =80:20, flow rate 0.6mL/min, detection wavelength set to 254nm.

8. A phthalide dimer, wherein said phthalide dimer is racemic mixture (±) -1, compound 2, or compound 3 prepared by the method of any of claims 1-7.

9. An application of phthalide dimer in preparing anti-inflammatory medicine is disclosed.