CN113105522B

CN113105522B - Oriental water plantain triterpenes compound, preparation method, structure characterization method and application thereof

Info

Publication number: CN113105522B
Application number: CN202110367052.2A
Authority: CN
Inventors: 范思洋; 杨倩; 刘曦子; 韩诗怡; 李寅庆
Original assignee: Shenwei Pharmaceutical Group Co Ltd
Current assignee: Shenwei Pharmaceutical Group Co Ltd
Priority date: 2021-04-06
Filing date: 2021-04-06
Publication date: 2022-04-01
Anticipated expiration: 2041-04-06
Also published as: CN113105522A

Abstract

The invention provides a triterpenoid of rhizoma alismatis, a preparation method, a structure characterization method and application thereof. The method comprises the steps of taking dried tubers of the alisma orientale as raw materials, extracting the tubers with ethanol with specific concentration, extracting with petroleum ether and dichloromethane, roughly separating dichloromethane phases obtained by extraction with petroleum ether-ethyl acetate with different proportions, eluting and separating with methanol-water with different proportions, and finally separating and purifying by semi-preparative liquid chromatography to obtain alisma orientale monomeric compounds with two novel structures shown in formulas I and II. The method for separating and purifying the triterpenoids of the oriental waterplantain rhizome can obtain a plurality of high-purity triterpenoids with novel structures at one time, provides high-purity raw materials for the industrial development of triterpenoid preparations of the oriental waterplantain rhizome, and has the advantages of simple separation method, high separation efficiency and extremely high popularization value.

Description

Oriental water plantain triterpenes compound, preparation method, structure characterization method and application thereof

Technical Field

The invention relates to the technical field of separation and purification of active ingredients of traditional Chinese medicines, in particular to a triterpenoid compound of rhizoma alismatis, and a preparation method, a structure characterization method and application thereof.

Background

Alismatis rhizoma is dried tuber of Alisma orientalis (Sam.) Juz and Alismatis rhizoma (A. plantago-aquatica Linn.) belonging to Alismaceae. Rhizoma alismatis has a long medicinal history, is listed as a top grade in Shen nong Ben Cao Jing, has the effects of promoting diuresis and excreting dampness, clearing heat, dissolving turbidity and reducing blood fat, and is applied to various traditional Chinese medicine clinical compound and Chinese patent medicines. Pharmacological research shows that the alcohol extract, the water extract and the monomer compound of the alisma have various pharmacological activities of diuresis, calculus resistance, kidney protection, blood sugar reduction, blood fat reduction, anti-inflammation and the like. The triterpenoids are compounds with the largest proportion of the alisma rhizome and the most reported pharmacological activity, and the skeleton of the triterpenoids is mainly the protopanaxarene type tetracyclic triterpene, which is also a characteristic component of the medicinal material. The structure of the tetracyclic triterpene in the rhizoma alismatis is variable, and the tetracyclic triterpene mainly comprises a fatty chain connected with a 17-bit protopanaxane, a fatty chain with an oxygen bridge connected with a 17-bit protopanaxane, a ring formed by the fatty chain connected with the 17-bit protopanaxane and a 16-bit nortriterpene and the like. So far, the obtained alisma triterpenes have 97 kinds, and account for 43% of all known compounds (226) in alisma.

Different alisma monomers have different pharmacological activities. In order to reveal the material basis of the traditional Chinese medicine taking the triterpenes of the alisma as the main active ingredient for treating diseases and fully utilize the pharmacological activity of the triterpenes of the alisma, the triterpenes of the alisma need to be separated and purified, but the triterpenes of the alisma with similar structures exist in a large number of the triterpenes, so that the separation of the triterpenes monomer compounds of the alisma is very difficult. The triterpenoid of the alisma orientale has various pharmacological activities, and the separation of the new triterpenoid of the alisma orientale can provide a material basis for the development of new drugs of the alisma orientale. Therefore, how to realize the separation and purification of the triterpenoids in the alisma orientale is of great significance.

Disclosure of Invention

Aiming at the technical problems, the invention provides a triterpenoid of the alisma rhizome, a preparation method, a structure characterization method and application thereof.

The rhizoma alismatis is rhizoma alismatis, and the medicinal material part is a dry tuber.

In order to solve the technical problems, the technical scheme provided by the invention is as follows:

a triterpenoid of Alismatis rhizoma has the following structural formula:

compared with the prior art, the invention provides two alisma triterpenoids with brand-new structures, enriches the database of the alisma triterpenoids, and provides a material basis for the activity research of the alisma triterpenoids and the development of new drugs with single components.

The invention also provides a preparation method of the alisma triterpenoid, which comprises the following steps:

step one, adding petroleum ether into an alcohol extraction concentrated solution of the rhizoma alismatis for extraction, and collecting a water phase; adding dichloromethane into the obtained water phase for extraction, collecting dichloromethane phase, and concentrating under reduced pressure to dry to obtain Alismatis rhizoma dry extract;

step two, adding the rhizoma alismatis dry extract into a silica gel chromatographic column, eluting with a petroleum ether-ethyl acetate solution with a volume ratio of 6:1-5:1, discarding the eluent, then sequentially eluting with petroleum ether-ethyl acetate solutions with a volume ratio of 16:5-14:5 and 11:5-9:5 respectively, monitoring by thin-layer chromatography, collecting the eluent, and evaporating to dryness to obtain a sample a and a sample b;

step three, adding the sample a and the sample b into a reverse phase silica gel chromatographic column respectively, eluting with methanol-water solution with the volume ratio of 9:11-11:9, discarding the eluent, eluting with methanol-water with the volume ratio of 9:4-5:2, monitoring by thin layer chromatography, collecting the eluent, and obtaining a component a 'and a component b' respectively;

step four, respectively separating and purifying the component a 'and the component b' by semi-preparative liquid chromatography to respectively obtain compounds shown in formula I and formula II; wherein, the chromatographic column is a C18 column, the mobile phase is acetonitrile-water solution, the detection wavelength is 205-215nm, and isocratic elution is carried out.

Compared with the prior art, the invention has the following beneficial effects:

the alisma orientale contains almost hundreds of alisma orientale triterpenes with similar structures and physicochemical properties, so that the separation of the alisma orientale triterpenes monomer substances is difficult to realize, and the content of the alisma orientale triterpenes monomer substances in the lily is very low, so that a plurality of alisma orientale triterpenes monomers with higher purity are difficult to obtain by a simple separation method.

According to the method for separating and purifying the triterpenoid monomeric compounds of the alisma orientale, a large amount of impurities in the alisma orientale can be effectively removed by selecting a specific elution solvent and an elution sequence and controlling appropriate parameters, and various triterpenoids of the alisma orientale can be effectively separated to obtain two triterpenoids of the alisma orientale with high purity and novel structures. The method for separating and purifying the triterpenoids of the oriental waterplantain rhizome can obtain a plurality of triterpenoids with novel structures at one time, provides high-purity raw materials for the industrial development of triterpenoid preparations of the oriental waterplantain rhizome, and has the advantages of simple separation method, high separation efficiency and extremely high popularization value.

Preferably, in the first step, the alcohol extraction concentrated solution of alisma orientale is prepared by the following method: slicing rhizoma Alismatis dried tuber, adding 55-65% ethanol water solution with volume concentration 12-18 times of the mass of rhizoma Alismatis, heating and reflux-extracting for 2-4 times, and concentrating under reduced pressure to obtain ethanol extract of rhizoma Alismatis.

Preferably, in the step one, the time for each heating reflux extraction is 1.5-2.5 h.

Preferably, in the first step, the temperature of the concentration under reduced pressure is 55-65 ℃, and the concentration under reduced pressure is carried out until the ratio of the volume of the concentrated solution to the dried rhizoma alismatis tubers is 1:1 (v/w).

By the extraction method, a large amount of impurities in the rhizoma alismatis can be removed, and the subsequent separation and purification efficiency can be improved.

Preferably, in step one, the boiling range of the petroleum ether is 60-90 ℃.

Preferably, in the first step, petroleum ether is added for extraction for 2-4 times.

Preferably, in the step one, the volume of the petroleum ether added in each extraction is 1 to 1.5 times of the volume of the alcohol extraction concentrated solution of the rhizoma alismatis.

Preferably, in step one, dichloromethane is added for extraction 2-4 times.

Further preferably, in step one, the volume of dichloromethane added per extraction is 1-1.5 times the volume of the aqueous phase.

The preferable extraction method can further remove the dry impurities in the alisma alcohol extract, thereby being beneficial to improving the subsequent elution effect.

Preferably, in the second step, the filler particle size of the silica gel chromatography column is 200-300 meshes.

Preferably, in the third step, the reversed phase silica gel chromatographic column is an octadecylsilane chemically bonded silica chromatographic column.

More preferably, the specification of the filler in the reverse silica gel chromatographic column is that the particle size is 50 μm and the pore diameter is 12 nm.

Optionally, in the third step, the sample a and the sample b are dissolved by methanol water and then added into the reverse silica gel chromatographic column respectively.

Preferably, the methanol water contains 50% by volume of methanol.

Preferably, in step four, the flow rate of the semi-preparative liquid chromatography is 2.8-3.2 mL/min.

Preferably, in the fourth step, the mobile phase is 38-42% acetonitrile-water solution, and the eluent with ultraviolet absorption value greater than 50mA after 32min is collected to obtain the alisma triterpenoid shown in the formula I.

Preferably, in the fourth step, the mobile phase is 76-80% acetonitrile-water solution, and the eluent with the ultraviolet absorption value larger than 50mA after being collected for 26min is obtained to obtain the alisma triterpenoid shown in the formula II.

The proportion of the mobile phase is volume ratio.

Preferably, in the fourth step, the C18 column is A YMC-Pack ODS-A column having A size of 10mm X250 mm and A packing particle size of 5 μm.

In the liquid chromatography separation and purification process, the selection and combination of various chromatographic conditions are important because it directly affects the time of appearance of peaks, peak shapes, etc. of substances; the chromatographic conditions mainly comprise a chromatographic column (comprising filler, column length, column temperature and the like), a mobile phase (comprising components, flow rate and the like), a detector, detection wavelength and the like. The chromatographic conditions provided by the invention can optimize the peak emergence time, peak shape, separation effect and the like of the substances, and realize the preparation of the novel high-purity alisma triterpenoid.

The invention also provides a characterization method of the absolute configuration of the triterpenoid of the alisma rhizome, which is characterized by adopting nuclear magnetic resonance¹H-NMR、¹³And C-NMR, DEPT, NOESY, HSQC and HMBC spectrograms and an electronic circular dichrogram are used for carrying out absolute configuration identification on the obtained triterpenoid of the alisma rhizome.

The existing determination method of the absolute configuration of the natural product mainly comprises an organic synthesis method, an X-ray single crystal diffraction method, a Mosher method, an optical rotation method, a circular dichroism method and the like, the absolute configuration of the natural product can be determined only by characterization of various methods, and the method is complex to operate, long in test period and high in cost. The absolute configuration of two newly prepared alisma triterpenoids is determined by nuclear magnetism representation, Electronic Circular Dichroism (ECD) representation and a quantum chemistry theory calculation method, so that a large amount of test time and cost are saved, and the method has high practical value.

The invention also provides application of the alisma triterpenoid in preparing medicaments for promoting urination, resisting calculus, protecting kidney, reducing blood sugar and blood fat or resisting inflammation.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a photograph of Alismaketene B prepared in example 1 of the present invention¹H-NMR spectrum (400 MHz);

FIG. 2 is a photograph of Alismaketene B prepared in example 1 of the present invention¹³C-NMR spectrum (100 MHz);

FIG. 3 is a DEPT spectrum (100MHz) of Alismaketene B prepared in example 1 of the present invention;

FIG. 4 is an HSQC spectrum of Alismaketene B prepared in example 1 of the present invention;

FIG. 5 is an HMBC spectrum of Alismaketene B prepared in example 1 of the present invention;

FIG. 6 is a NOESY spectrum of Alismaketene B prepared in example 1 of the present invention;

FIG. 7 is an ECD spectrum of Alismaketene B prepared in example 1 of the present invention; wherein, 1a and 1B are two different configurations of the Alismaketene B, and the rightmost side is ECD spectrogram of the 1a, 1B and the Alismaketene B;

FIG. 8 is a Noralisulc acid A preparation of example 1 of the invention¹H-NMR spectrum (400 MHz);

FIG. 9 is a Noralisulc acid A preparation of example 1 of the invention¹³C-NMR spectrum (100 MHz);

FIG. 10 is a DEPT spectrum (100MHz) of Noralisulac acid A prepared in example 1 of the present invention;

FIG. 11 is an HSQC spectrum of Noralisulac acid A prepared in example 1 of the present invention;

FIG. 12 is a HMBC spectrum of Noralisulc acid A prepared in example 1 of the present invention;

FIG. 13 is a NOESY spectrum of Noralisulc acid A prepared in example 1 of the present invention;

FIG. 14 is an EDC spectrum of Noralisulc acid A prepared in example 1 of the present invention; wherein 2a and 2b are two different configurations of Noralisulc acid A, and the rightmost side is the ECD spectrum of 2a, 2b and Noralisulc acid A.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Example 1

A method for separating and purifying triterpenes from Alismatis rhizoma comprises:

firstly, slicing dried tubers of the rhizoma alismatis, weighing 5kg, adding 75L of 60% ethanol water solution in volume concentration, heating and refluxing for extraction for 3 times, wherein the extraction time is 2.0h each time, and concentrating under reduced pressure at 60 ℃ to 5L to obtain an ethanol extraction concentrated solution of the rhizoma alismatis;

step two, adding petroleum ether with the boiling range of 60-90 ℃ into the alcohol extraction concentrated solution of the rhizoma alismatis for extraction for 3 times, wherein the adding volume of the petroleum ether is 1 time of the volume of the concentrated solution each time, discarding a petroleum ether phase, and collecting a water phase; adding dichloromethane into the obtained water phase, extracting for 3 times, wherein the volume of dichloromethane added each time is 1 time of the volume of the water phase, mixing dichloromethane phases after extraction, and concentrating under reduced pressure to dryness to obtain rhizoma Alismatis dry extract 120.4 g;

step three, adding the rhizoma alismatis dry extract into a silica gel (200-400-mesh) chromatographic column, eluting with a petroleum ether-ethyl acetate solution with a volume ratio of 5:1, monitoring by a TLC (thin layer chromatography) and a silicotungstic acid color development method, stopping elution when the rhizoma alismatis dry extract is colorless, discarding the eluent, then sequentially eluting with petroleum ether-ethyl acetate solutions with volume ratios of 15:5 and 10:5 respectively, monitoring by a TLC (thin layer chromatography) and a silicotungstic acid color development method, collecting the eluent, and evaporating the solvent to obtain a sample a and a sample b;

step four, after a sample a is dissolved by 50% (v/v) methanol aqueous solution, adding the dissolved sample a into a reversed phase silica gel (ODS, S-50 μm,12nm) chromatographic column, eluting by using methanol-water solution with the volume ratio of 5:5, discarding eluent, then eluting by using methanol-water with the volume ratio of 7:3, monitoring by TLC and a silicotungstic acid color development method, collecting eluent, concentrating and drying to obtain a component a';

dissolving the sample b with 50% (v/v) methanol aqueous solution, adding into a reversed phase silica gel (ODS, S-50 μm,12nm) chromatographic column, eluting with methanol-water solution with volume ratio of 5:5, discarding eluent, eluting with methanol-water with volume ratio of 7:3, monitoring by TLC and silicotungstic acid color development method, collecting eluent, concentrating and drying to obtain a component b';

dissolving the component a' in absolute methanol (with the concentration of 50mg/mL), separating and purifying by semi-preparative liquid chromatography, collecting the eluent with the ultraviolet absorption value of more than 50mA after 32min, stopping collecting when the ultraviolet absorption value is less than 50mA, concentrating and drying the collected eluent to obtain the alisma triterpenoid with the purity of 95.2 percent, wherein the alisma triterpenoid is shown in the formula I; wherein the chromatographic column is C18 column (YMC-Pack ODS-A, 10mm × 250mm, 5 μm), the mobile phase is 40% acetonitrile-water solution, the detection wavelength is 210nm, the flow rate is 3.0mL/min, and isocratic elution is performed;

dissolving the component b' in anhydrous methanol (with the concentration of 50mg/mL), separating and purifying by semi-preparative liquid chromatography, collecting the eluate with the ultraviolet absorption value of more than 50mA after 26min, stopping collecting when the ultraviolet absorption value is less than 50mA, concentrating and drying the collected eluate to obtain the alisma triterpenoid with the purity of 97.5 percent shown in the formula II; wherein the chromatographic column is C18 column (YMC-Pack ODS-A, 10mm × 250mm, 5 μm), the mobile phase is 78% acetonitrile-water solution, the detection wavelength is 210nm, the flow rate is 2.8-3.2mL/min, and isocratic elution is performed.

Example 2

firstly, slicing dried tubers of the rhizoma alismatis, weighing 5kg, adding 60L of 65% ethanol water solution with volume concentration, heating and refluxing for extraction for 4 times, wherein the extraction time is 1.5h each time, and concentrating under reduced pressure at 55 ℃ to 5L to obtain an ethanol extraction concentrated solution of the rhizoma alismatis;

step two, adding petroleum ether with the boiling range of 60-90 ℃ into the alcohol extraction concentrated solution of the rhizoma alismatis for extraction for 2 times, wherein the adding volume of the petroleum ether is 1.5 times of the volume of the concentrated solution each time, discarding a petroleum ether phase, and collecting a water phase; adding dichloromethane into the obtained water phase, extracting for 4 times, wherein the volume of the added dichloromethane is 1 time of the volume of the water phase, mixing the dichloromethane phases after extraction, and concentrating under reduced pressure to dryness to obtain 119.8g of rhizoma alismatis dry extract;

step three, adding the rhizoma alismatis dry extract into a silica gel (200-400-mesh) chromatographic column, eluting with petroleum ether-ethyl acetate solution with the volume ratio of 6:1, monitoring by a TLC (thin layer chromatography) and a silicotungstic acid color development method, stopping elution when the rhizoma alismatis dry extract is colorless, discarding eluent, then sequentially eluting with petroleum ether-ethyl acetate solution with the volume ratio of 16:5 and 9:5 respectively, monitoring by a TLC (thin layer chromatography) and a silicotungstic acid color development method, collecting eluent, and evaporating solvent to obtain a sample a and a sample b;

step four, after a sample a is dissolved by 50% methanol water (v/v), adding the dissolved sample a into a reversed phase silica gel (ODS, S-50 μm,12nm) chromatographic column, eluting by using methanol-water solution with the volume ratio of 11:9, discarding eluent, then eluting by using methanol-water with the volume ratio of 9:4, monitoring by TLC and a silicotungstic acid color development method, collecting eluent, concentrating and drying to obtain a component a';

dissolving the sample b with 50% (v/v) methanol aqueous solution, adding into a reversed phase silica gel (ODS, S-50 μm,12nm) chromatographic column, eluting with 11:9 methanol-aqueous solution by volume ratio, discarding the eluent, eluting with 9:4 methanol-water by volume ratio, monitoring by TLC and silicotungstic acid color development method, collecting the eluent, concentrating and drying to obtain a component b';

dissolving the component a' in absolute methanol (with the concentration of 50mg/mL), separating and purifying by semi-preparative liquid chromatography, collecting the eluent with the ultraviolet absorption value of more than 50mA after 32min, stopping collecting when the ultraviolet absorption value is less than 50mA, concentrating and drying the collected eluent to obtain the alisma triterpenoid with the purity of 95.1% shown in the formula I; wherein the chromatographic column is C18 column (YMC-Pack ODS-A, 10mm × 250mm, 5 μm), the mobile phase is 42% acetonitrile-water solution, the detection wavelength is 205nm, the flow rate is 2.8mL/min, and isocratic elution is performed;

dissolving the component b' in anhydrous methanol (with the concentration of 50mg/mL), separating and purifying by semi-preparative liquid chromatography, collecting the eluate with the ultraviolet absorption value of more than 50mA after 26min, stopping collecting when the ultraviolet absorption value is less than 50mA, concentrating and drying the collected eluate to obtain the alisma triterpenoid with the purity of 97.3% as shown in the formula II; wherein the chromatographic column is C18 column (YMC-Pack ODS-A, 10mm × 250mm, 5 μm), the mobile phase is 80% acetonitrile-water solution, the detection wavelength is 205nm, the flow rate is 2.8mL/min, and isocratic elution is performed.

Example 3

firstly, slicing dried tubers of the rhizoma alismatis, weighing 5kg, adding 90L of 55% ethanol water solution with volume concentration, heating and refluxing for 2 times, wherein the extraction time is 2.5h each time, and concentrating under reduced pressure at 65 ℃ to 5L to obtain an ethanol extraction concentrated solution of the rhizoma alismatis;

step two, adding petroleum ether with the boiling range of 60-90 ℃ into the alcohol extraction concentrated solution of the rhizoma alismatis for extraction for 4 times, wherein the adding volume of the petroleum ether is 1 time of the volume of the concentrated solution each time, discarding a petroleum ether phase, and collecting a water phase; then adding dichloromethane into the obtained water phase for extraction for 2 times, wherein the volume of the added dichloromethane is 1.5 times of the volume of the water phase, combining the dichloromethane phases after extraction, and concentrating under reduced pressure to dryness to obtain 120.6g of rhizoma alismatis dry extract;

step three, adding the rhizoma alismatis dry extract into a silica gel (200-400-mesh) chromatographic column, eluting with a petroleum ether-ethyl acetate solution with a volume ratio of 5.5:1, monitoring by a TLC (thin layer chromatography) and a silicotungstic acid color development method, stopping elution when the rhizoma alismatis dry extract is colorless, discarding the eluent, then sequentially eluting with petroleum ether-ethyl acetate solutions with volume ratios of 14:5 and 11:5 respectively, monitoring by a TLC (thin layer chromatography) and a silicotungstic acid color development method, collecting the eluent, and evaporating the solvent to obtain a sample a and a sample b;

dissolving a sample a by using 50% (v/v) methanol aqueous solution, adding the dissolved sample into a reversed phase silica gel (ODS, S-50 mu m,12nm) chromatographic column, eluting by using methanol-water solution with the volume ratio of 9:11, discarding eluent, then eluting by using methanol-water with the volume ratio of 5:2, monitoring by TLC and a silicotungstic acid color development method, collecting eluent, concentrating and drying to obtain a component a';

dissolving the sample b with 50% (v/v) methanol aqueous solution, adding into reversed phase silica gel (ODS, S-50 μm,12nm) chromatographic column, eluting with methanol-water solution with volume ratio of 9:11, discarding eluate, eluting with methanol-water with volume ratio of 5:2, monitoring by TLC and silicotungstic acid color development method, collecting eluate, concentrating, and drying to obtain component b';

dissolving the component a' in absolute methanol (with the concentration of 50mg/mL), separating and purifying by semi-preparative liquid chromatography, collecting the eluent with the ultraviolet absorption value of more than 50mA after 32min, stopping collecting when the ultraviolet absorption value is less than 50mA, concentrating and drying the collected eluent to obtain the alisma triterpenoid with the purity of 94.9 percent and shown in the formula I; wherein the chromatographic column is C18 column (YMC-Pack ODS-A, 10mm × 250mm, 5 μm), the mobile phase is 38% acetonitrile-water solution, the detection wavelength is 215nm, the flow rate is 3.2mL/min, and isocratic elution is performed;

dissolving the component b' in anhydrous methanol (with the concentration of 50mg/mL), separating and purifying by semi-preparative liquid chromatography, collecting the eluate with the ultraviolet absorption value of more than 50mA after 26min, stopping collecting when the ultraviolet absorption value is less than 50mA, concentrating and drying the collected eluate to obtain the alisma triterpenoid with the purity of 97.1% as shown in the formula II; wherein the chromatographic column is C18 column (YMC-Pack ODS-A, 10mm × 250mm, 5 μm), the mobile phase is 76% acetonitrile-water solution, the detection wavelength is 215nm, the flow rate is 3.2mL/min, and isocratic elution is performed.

Example 4

Identification of absolute configuration:

1.1 the absolute configuration of the alisma triterpenoid of formula I prepared in example 1 was identified as follows:

the Alismatis rhizoma triterpenes compound is yellow amorphous powder, [ alpha ]]²⁴ _D+53.4(c 0.02, MeOH); the molecular formula is determined to be C when HRESIMS shows that m/z is 533.3499 (calculated value 533.3478)₃₀H₄₈O₅. Warp beam¹The H NMR spectrum (shown in FIG. 1) can judge that the total number of 8 methyl groups in the molecule exist in delta_H1.32(s), 1.27(s), 1.19(s), 1.17(d J ═ 7.1Hz), 1.07(s), 1.06(s), 1.05(s) and 0.89(s) at δ_HFour oxymethylene groups are present at 5.13(t, J ═ 6.5Hz), 4.10(m),3.83(m) and 3.28 (m). According to¹³The C NMR spectrum (shown in FIG. 2) indicates that there is a 30 carbon signal in the molecule, including 1 carbonyl carbon (. delta.)_C220.0), 2 olefin carbons (. delta.)_C142.6, 135.5), 4 oxygen-containing methine carbons (. delta.)_C69.9, 71.7, 76.6 and 83.6), and 1 oxygen-containing quaternary carbon (. delta.))_C73.7). According to the DEPT spectrum (as shown in FIG. 3), 4 quaternary carbons, 3 tertiary carbons, 7 secondary carbons and 8 primary carbons are additionally present in the molecule. And (3) synthesizing one-dimensional map data, wherein the compound is a tetracyclic triterpene skeleton.

According to³J_H、HThe coupling values and NOESY data determine the relative configuration of the compounds.¹Larger in H NMR spectrum³J_9,11The value (10.7Hz) indicates the axial relationship of H-9 and H-11. NOESY spectrum (shown in FIG. 6), H-18 (. delta.) (_H0.89) and H-5 (. delta.))_H2.11),H-11(δ_H3.83) correlation, H-16 (. delta.))_H5.13) and H-15 alpha (. delta.) (delta.)_H2.32) correlation, H-30 (. delta.))_H1.19) and H-9 (. delta.))_H1.75)，H-24(δ_H3.28) correlation, H-24 (. delta.))_H3.28) and H-15 beta (. delta.))_H1.33) correlation, indicating that H-5, H-11, H-16 and H-18 are in the alpha position and H-9, H-24 and H-30 are in the beta position; the correlation of Me-19/H-9, H-20/H-23, H-11/Me-21, H-16/Me-21 indicates that Me-19, H-20 and H-23 are in the beta position. The data for this compound is very similar to that for the known compound, alimasketene B23-acetate, except that the acetyl group at C-23 is substituted with a hydroxyl group to give the new compound, hence the name alimasketene B. Specific nuclear magnetic data are shown in table 1.

TABLE 1 Nuclear magnetic data (400/100MHz, CDCl) of Alismaketene B₃)

After the relative configuration of compound 1 was determined, its stereochemistry was identified. Calculating ECD maps of different conformations by using quantum chemistry theory and conformation at B3LYP/6-311g (d, p) group level in methanol, and comparing the ECD maps with experimental value [ ECD (MeOH) lambda ] of the compound_max(Δε)290(+2.98),205(–9.96)]A comparison is made. As shown in fig. 7, the ECD in the 1a conformation has a high degree of agreement with the experimental value. It is concluded therefrom that the stereostructure of compound 1 is 5R,8R,9S,10S,11S,14S,16S,20R,23S,24R and is named aismaketone B, the absolute configuration is shown below:

1.2 the absolute configuration of the alisma triterpenoid of formula II prepared in example 1 was identified as follows:

the Alismatis rhizoma triterpenes compound represented by formula II is yellow amorphous powder, [ alpha ]]²⁴ _D+57.0(c 0.04, MeOH); the molecular formula is determined to be C when HRESIMS shows that m/z is 561.2883 (calculated value 461.2844)₂₆H₄₀O₄。¹H NMR spectra (e.g. ofFIG. 8) shows that a total of 6 methyl groups are present in the molecule, respectively_H0.97(s), 1.05(s), 1.05(s), 1.06(d J ═ 7.1Hz), 1.07(s), 1.10(s), and δ_H3.85(m) an oxygen-containing methine group is present. According to¹³C NMR spectrum (shown in FIG. 9) was determined to have 26 carbon signals in the molecule, including 2 carbonyl carbons (. delta.)_C220.4, 177.0), 2 olefin carbons (. delta.)_C134.5, 137.3), 1 oxygen-containing methine carbon (. delta.)_C70.0). By integrating the one-dimensional map data, the compound is a tetracyclic triterpene skeleton and has a highly similar structure with the Alisol A, except that the C-22 position is connected with a carboxylic acid to replace a triol unit corresponding to the Alisol A. This result is supported by the correlation between H-22 and C-23, and H-20 and C-23, C-17, which are visible in HMBC maps (as shown in FIG. 12).

According to³J_H、HThe coupling values and NOESY data determine the relative configuration of the compounds. Is bigger³J_9,11The value (10.6Hz) indicates the axial relationship of H-9 and H-11. NOESY spectrum (shown in FIG. 13), H-18 (. delta.) (_H0.97) and H-11 (. delta.))_H3.85), H-11 and H-5 (. delta.))_H2.08) related, H-18 and H-15 α (δ)_H1.87) related, H-30 (. delta.))_H1.10) and H-9 (. delta.))_H1.72), H-30 and H-15 beta (. delta.))_H1.34),H-19(δ_H1.05) of the two molecules, indicating that H-5, H-11 and H-18 are in the alpha position and H-9 and H-30 are in the beta position. Specific nuclear magnetic data are shown in table 2.

TABLE 2 Nuclear magnetic data (400/100MHz, CDCl) of Noralisolic acid A₃)

After the relative configuration of the alisma triterpenoid shown in the formula II is determined, the stereochemistry of the alisma triterpenoid is discussed. Calculating ECD maps of different conformations by using quantum chemistry theory and conformation at B3LYP/6-311g (d, p) group level in methanol, and comparing the ECD maps with experimental value [ ECD (MeOH) lambda ] of the compound_max(Δε)293(+2.44),203(–3.43)]A comparison is made. As shown in fig. 14, the ECD in the 2a conformation has a high degree of agreement with the experimental value. Thereby the device is provided withThe three-dimensional structure of the alisma triterpenoid shown in the formula II is deduced to be 5R,8R,9S,10S,11S,14S and 20R, and is named as Noralisolic acid A, and the absolute configuration of the Noralistic acid A is shown as follows:

example 5

Drugs and reagents:

HEK293/NF-kB cell line, Wuhan Ministry of research; DMEM medium, BI, germany; fetal Bovine Serum (FBS), Gibco, usa; penicilin & Streptomycin (P/S), excell, china; 0.25% pancreatin-EDTA, cultivated in Chinese; bright Glo, Promega, usa; TNF α, Peprotech, usa; dimethylsulfoxide, Sigma, usa.

The instrument comprises the following steps: Envision-PerkinElmer, usa.

The compound, Alismaketene B, was dissolved in Dimethylsulfoxide (DMSO), stored at 4 ℃, thawed out at the time of the experiment, and diluted with DMEM medium so that the concentration of Alismaketene B in the final reaction system was 50. mu.M.

The compound Noralisulac acid A was dissolved in dimethyl sulfoxide (DMSO), stored at 4 ℃, thawed at the time of experiment, and diluted with DMEM medium to give a concentration of 50. mu.M in Noralisulac acid A in the final reaction system.

The positive control drug IMD0354 was dissolved in dimethyl sulfoxide (DMSO), stored at 4 deg.C, thawed at the time of the experiment, and diluted with DMEM medium to a final concentration of 30. mu.M in the reaction system.

TNF α was stored at-20 ℃ and thawed at the time of the experiment and diluted with DMEM medium to a final reaction concentration of 20 ng/mL.

Taking out HEK293 cells to be recovered from a liquid nitrogen tank rapidly, melting in 37 ℃ water bath, adding into preheated DMEM culture medium rapidly, centrifuging at 1000 r/min for 5min, taking out the centrifuge tube, discarding supernatant, adding fresh preheated DMEM culture medium into the centrifuge tube, resuspending cells, adding cell suspension into the culture medium, adding 5% CO at 37 ℃ to obtain a suspension₂Culturing in an incubator. When the cells grow 80-90% of the culture dishThe cells were digested with 0.25% trypsin, then resuspended in fresh DMEM medium, and passaged for about 1 and 2 passages over 2-4 days. 1 day before detection, the NF-kB cells were seeded into 96-well cell plates according to the cell growth rate of 40000 cells per well, 80. mu.L of cell suspension was seeded into each well, and 5% CO was added at 37 ℃₂The incubator was used for incubation overnight. mu.L of test sample was added to each well, and 10. mu.L of 0.5% DMSO solution (DMSO in DMEM medium at a DMSO concentration of 0.5 wt%) was added to the negative control, and after 2 hours of incubation, 10. mu.L of LTNF alpha (20ng/mL) was added at 37 ℃ and 5% CO₂Incubate in the dark for 24 hours. After the incubation was completed, Bright Glo 50. mu.L/well was added, and chemiluminescence was measured on Envision to calculate the cell inhibition rate. Each concentration was repeated 2 times and averaged. The results are shown in Table 3.

The cell inhibition ratio ═ 1-B/a) × 100, where B is the luminescence value of the sample well and a is the luminescence value of the negative well.

TABLE 3 NF-kB Signal Path inhibition ratio of test samples

Compound (concentration)	Inhibition ratio (%)
		IMD-0354(30μM)	57.6±2.7
Alismaketone B(50μM)	23.1±8.1
		Noralisolic acid A(50μM)	7.5±2.8

Example 6

Drugs and reagents:

dihydroorotic acid (L-DHO), GLPBIO, usa; human dihydroorotate dehydrogenase (hDHODH), usa, GLPBIO; brequinar, usa, GLPBIO; sodium 2, 6-dichlorophenolate indophenol (DCIP), Sigma, usa; coenzyme Q10(CoQ 10), Sigma, usa; tris (hydroxymethyl) aminomethane (Tris), shanghai michelin; octyl phenyl ether of polyethylene glycol (Triton X-100), Mecanol, Shanghai; potassium chloride (KCl), chinese medicine group; dimethylsulfoxide, Sigma, usa.

The instrument comprises the following steps: microplate reader, BioTek, usa.

Buffer solution: 5mM Tris, 150mM KCl, 0.1 wt% Triton X-100, and adjusting the pH of the system to 8.10.

The compound, Alismaketene B, was dissolved in Dimethylsulfoxide (DMSO), stored at 4 ℃ and thawed at the time of the experiment and diluted with buffer to give a final concentration of Alismaketene B of 50. mu.M in the reaction system.

The compound Noralisulac acid A is dissolved in dimethyl sulfoxide (DMSO), stored at 4 ℃, thawed out during the experiment, and diluted with buffer solution, so that the concentration of the Noralisulac acid A in the final reaction system is 50 μ M.

The positive control drug Brequinar is dissolved in dimethyl sulfoxide (DMSO), stored at 4 ℃, unfrozen during the experiment, and diluted by buffer solution to make the concentration in the final reaction system be 12 nM.

hDHODH, DCIP and CoQ10 were diluted in buffer to give final concentrations of 10nM, 120. mu.M and 100. mu.M, respectively.

mu.L of buffer solution, 20. mu.L of each of hDHODH, DCIP and CoQ10, was added to each well of a 96-well plate, 20. mu.L of test sample was added to the experimental group, 20. mu.L of 0.5% DMSO solution (DMSO was diluted with buffer solution, DMSO content was 0.5 wt%) was added to the negative control group, 20. mu.L of L-DHO was added to initiate the reaction after incubation at 37 ℃ for 5 minutes, absorbance was measured at 600nm using a microplate reader, reading was performed every 31s for 6 minutes, and the inhibition rate was calculated. Each sample was repeated 2 times and averaged. The results are shown in Table 4.

Enzyme inhibition ═ 1-B/a) × 100, where B is the reaction rate of the sample wells and a is the reaction rate of the negative control. The reaction rate is that the absorbance tested at each time is used as the ordinate, the time is used as the abscissa, a straight line is drawn, and the slope of the straight line is the reaction rate.

TABLE 4 inhibition of hDHODH enzyme by test sample

Compound (concentration)	Inhibition ratio (%)
		Brequinar(12nM)	61.9±2.1
Alismaketone B(50μM)	19.6±6.7
		Noralisolic acid A(50μM)	0

In conclusion, the method for separating and purifying the triterpenoids from the alisma orientale, provided by the invention, combines the selected specific separation method with the optimal combination of process parameters, obtains two monomeric compounds with higher purity through one-time separation process, provides a material basis for research and development of a single component of the alisma orientale, does not need special equipment in the separation and purification process, is simple and convenient in process operation, has high extraction efficiency, and has wide application prospects.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The preparation method of the alisma triterpenoid is characterized in that the alisma triterpenoid has the structural formula as follows:

the preparation method comprises the following steps:

step two, adding the rhizoma alismatis dry extract into a silica gel chromatographic column, eluting with a petroleum ether-ethyl acetate solution with the volume ratio of 6:1-5:1, discarding the eluent, and sequentially using a solvent with the volume ratio of 16:5-14: 5. eluting with petroleum ether-ethyl acetate solution at a ratio of 11:5-9:5, monitoring by thin layer chromatography, collecting eluate, and evaporating to obtain sample a and sample b;

step three, adding the sample a and the sample b into a reverse phase silica gel chromatographic column respectively, eluting with methanol-water solution with the volume ratio of 9:11-11:9, discarding the eluent, eluting with methanol-water with the volume ratio of 9:4-5:2, monitoring by thin layer chromatography, collecting the eluent, and obtaining a component a 'and a component b' respectively; the reverse phase silica gel chromatographic column is an octadecylsilane chemically bonded silica chromatographic column;

step four, respectively separating and purifying the component a 'and the component b' by semi-preparative liquid chromatography to respectively obtain compounds shown in formula I and formula II; wherein the chromatographic column is a C18 column, the mobile phase is acetonitrile-water solution, the detection wavelength is 205-215nm, and isocratic elution is carried out;

wherein in the first step, the alcohol extraction concentrated solution of the alisma orientale is prepared by the following method: slicing dried rhizoma Alismatis, adding 55-65% ethanol water solution with volume concentration 12-18 times of the mass of rhizoma Alismatis, heating and reflux-extracting for 2-4 times, and concentrating under reduced pressure to obtain ethanol extract of rhizoma Alismatis;

in the fourth step, the flow rate of the semi-preparative liquid chromatography is 2.8-3.2 mL/min; the C18 column is A YMC-Pack ODS-A chromatographic column with the specification of 10mm multiplied by 250mm and the filler particle size of 5 mu m;

in the fourth step, the mobile phase is 38-42% acetonitrile-water solution, and the eluent with the ultraviolet absorption value larger than 50mA after 32min is collected to obtain the alisma triterpenoid shown in the formula I; the mobile phase is 76-80% acetonitrile-water solution, and the eluent with ultraviolet absorption value greater than 50mA after 26min is collected to obtain the alisma triterpenoid shown in formula II.