CN110642914B - Triterpenoid and application thereof - Google Patents

Abstract

The invention discloses a triterpenoid compound, which has a structural general formula as follows:

or

Description

Triterpenoid and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a novel triterpenoid and application thereof.

Background

Cancer is one of the global conditions of high mortality, a global focus of continued interest, and thus, the struggle with cancer presents a significant challenge to modern scientific technology. Research in the field of anticancer drugs is still a hot issue. Natural drugs, especially those derived from plants, have a wide variety of chemical structures and biological activities, and have been a major source of diseases prevention and treatment in humans. Many drugs applied clinically are directly or indirectly derived from natural products, and the natural products can be used not only as semi-synthetic precursors of drugs, but also as templates of chemical synthesis of drugs, thereby providing a new idea for the design of new drugs. Natural products have become one of the main sources for the discovery of new drugs or lead compounds.

Peganum harmala L is a whole plant of perennial herb of Peganum harmala (Peganum) of tribulus, which is named because the plant can emit a special odor similar to the body odor of a camel. The peganum harmala is distributed in arid or semiarid desert regions with slight salinization, such as Asia, Europe, North Africa and the like, and is an important desert vegetation plant. The peganum harmala herb or seed is a minority medicinal material with a long history in minority nationalities in northwest China and is listed in a medicine standard Uygur medicine booklet of Ministry of health. Peganum harmala is neutral in nature, bitter in taste, pungent and toxic, and mainly treats diseases such as weak tendons and vessels, joint pain, cough, excessive phlegm, coma, headache and the like. Folk often uses the whole herb to treat arthritis, innominate swelling and pain, epilepsy, psychosis, etc.; the seed can be used for treating cough, asthma, palpitation, dysphoria, numbness of limbs, etc.

The peganum harmala plant contains rich chemical components, the main active component of the peganum harmala plant is a alkaloid compound, and the content of alkaloid in seeds is up to 4-6%. In addition, the peganum harmala seed also contains a small amount of chemical components such as flavonoids, triterpenes, anthraquinones and sterols and glycosides. Modern pharmacological experiment research shows that the peganum harmala seed has the activities of resisting tumor, malaria, inflammation, tobacco mosaic virus, selective kinase inhibition and the like. The existing research is not thorough enough for researching the chemical components of the peganum harmala seeds, more researches are carried out on the alkaloid components rich in the peganum harmala seeds, and few researches are carried out on the triterpenes and flavonoids contained in the peganum harmala seeds. Therefore, the triterpenoids in the peganum harmala seeds are worthy of further research, development and utilization.

Disclosure of Invention

The invention aims to provide a novel triterpenoid, and application and a preparation method thereof. In order to achieve the purpose, the invention adopts the following technical scheme.

The technical scheme of the first aspect of the invention is as follows:

a triterpenoid compound has a structural general formula as follows:

wherein R1 is independently acetoxy,

r2 is independently hydrogen, oxygen or hydroxy,

r3 is independently hydroxy, hydroxymethyl or

R4 is independently acetoxy or carboxyl,

r5 is independently hydroxy or acetoxy,

r6 is independently hydroxymethyl or

R7 is independently carboxy or acetoxy.

Further, the structural formula is:

the technical scheme of the second aspect of the invention is as follows:

the triterpenoid in the technical scheme of the first aspect of the invention is applied to the preparation of antitumor drugs.

Furthermore, the anti-tumor medicine also contains pharmaceutically acceptable auxiliary materials.

Furthermore, the tumor refers to at least one of cervical cancer, liver cancer or gastric cancer of human.

The third aspect of the invention has the technical scheme that:

an anti-tumor drug contains the triterpenoid in the technical scheme of the first aspect of the invention.

Furthermore, the anti-tumor medicine also contains pharmaceutically acceptable auxiliary materials.

Furthermore, the tumor refers to at least one of cervical cancer, liver cancer or gastric cancer of human.

The technical scheme of the fourth aspect of the invention is as follows:

the preparation method of the triterpenoid in the technical scheme of the first aspect of the invention comprises the following steps:

1) taking peganum harmala seeds, heating and refluxing the peganum harmala seeds with ethanol, combining extracting solutions, and concentrating the extracting solutions under reduced pressure to obtain an extract;

2) suspending the extract with water, acidifying, and extracting with chloroform to obtain non-alkaloid layer;

3) separating the non-alkaloid layer by chromatography to obtain triterpenes.

Further, the acidification means adding hydrochloric acid to adjust the pH value to 1-2.

The invention has the beneficial effects that:

the invention extracts 7 novel triterpenoids from peganum harmala seeds, identifies the triterpenoids by physicochemical constants and modern wave spectrum means, has definite chemical structures of the 7 novel triterpenoids, and provides powerful reference data for further researching and developing and utilizing the value of the triterpenoids in the peganum harmala seeds. Animal pharmacodynamic tests show that the 7 novel triterpenoids provided by the invention have good in-vitro anti-tumor activity, have different degrees of tumor inhibition effects on the cytotoxicity of three human tumor cells, namely HeLa (human cervical cancer cells), HepG2 (human liver cancer cells) and SGC-7901 (human gastric cancer cells), and can be further used as new anti-tumor drugs for research and development.

Drawings

FIGS. 1, 2 and 3 show the inhibitory effect of triterpenoids on different tumor cells.

FIGS. 4, 5 and 6 show a hydrogen spectrum, a carbon spectrum and an infrared spectrum of Compound 1 of the present invention.

FIGS. 7, 8 and 9 show a hydrogen spectrum, a carbon spectrum and an infrared spectrum of Compound 2 of the present invention.

FIGS. 10, 11 and 12 show a hydrogen spectrum, a carbon spectrum and an infrared spectrum of Compound 3 of the present invention.

FIGS. 13, 14 and 15 show a hydrogen spectrum, a carbon spectrum and an infrared spectrum of Compound 4 of the present invention.

FIGS. 16, 17 and 18 show a hydrogen spectrum, a carbon spectrum and an infrared spectrum of Compound 5 of the present invention.

FIGS. 19, 20 and 21 show a hydrogen spectrum, a carbon spectrum and an infrared spectrum of Compound 6 of the present invention.

FIGS. 22, 23 and 24 show a hydrogen spectrum, a carbon spectrum and an infrared spectrum of Compound 7 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to examples. It will also be understood that the following examples are included merely for purposes of further illustrating the invention and are not to be construed as limiting the scope of the invention, as the invention extends to insubstantial modifications and adaptations of the invention following in the light of the principles set forth herein. The specific process parameters and the like of the following examples are also only one example of suitable ranges, and the skilled person can make a selection within the suitable ranges through the description herein, and are not limited to the specific data of the following examples.

Example 1 extraction of triterpenes

Taking 30.8kg of dry peganum harmala seed medicinal material, heating and refluxing the dry peganum harmala seed medicinal material by using 70% ethanol for 3 times, extracting for 2 hours each time, combining extracting solutions, and concentrating under reduced pressure to obtain an extract. Suspending the extract with water, adding hydrochloric acid to adjust pH to 1.0, and extracting with equal volume of chloroform for 3 times to obtain non-alkaloid layer. Wherein the non-alkaloid layer is separated by means of repeated silica gel column chromatography, reversed-phase MPLC column chromatography, Sephadex LH-20 column chromatography, semi-preparative reversed-phase HPLC column chromatography and the like to obtain the compounds 1-7.

EXAMPLE 2 identification of Compound 1

By physicochemical constants and spectral means (HRESIMS, Mass),¹H-NMR、¹³C-NMR, HSQC, HMBC and¹H-¹h COSY spectrum) of compound 1, and fig. 4, 5, and 6 are a hydrogen spectrum, a carbon spectrum, and an infrared spectrum of compound 1 of the present invention.

Compound 1 is white amorphous powder (chloroform), has light blue fluorescence absorption under ultraviolet lamp 365nm, has dark spot under 254nm, and develops pink with 10% sulfuric acid-ethanol developer by heating reaction. In IR (KBr), v_max 3408(OH)，2962、1725(C＝O)，1384、1261、1097、1025、859、802cm^-1The primary absorption is here. HR-ESI-MS spectrum shows excimer peak HR-ESI-MS M/z 705.4361[ M + H ]]⁺(calcd for C₄₃H₆₁O₈705.4360) indicates a molecular weight of 704, whose formula is C₄₃H₆₀O₈。

¹H-NMR(600Hz,CDCl₃) In the spectrum, 2 methoxy groups are present in the high field regionThe matrin signals are respectively delta_H3.94(3H, s), 3.68(3H, s), methyl Hydrogen Signal δ of 1 acetyl group_H2.02(3H, s) and 5 quaternary carbons have methyl proton signals of δ_H0.82(3H, s), 0.88(3H, s), 0.97(3H, s), 1.70(3H, s), where δ_H1.70(3H, s) is 30-CH for lupane triterpenes₃The characteristic hydrogen signal of (a), the compound is preliminarily presumed to be lupane triterpenoid; delta_H7.08(1H, dd, J ═ 8.2,1.8Hz), 7.03(1H, d, J ═ 1.8Hz), 6.92(1H, d, J ═ 8.2Hz) are olefinic hydrogen proton signals on aromatic rings, and 1, 3, 4 substitutions are presumed to be present on the benzene rings; delta_H7.58(1H, d, J ═ 15.9Hz), 6.28(1H, d, J ═ 15.9Hz) are proton signals for a group of double bonds, which are presumed to be attached to the 1-position of the phenyl ring based on the coupling constants; delta_H4.75(1H, s), 4.61(1H, s) are presumed to be proton signals of a group of terminal double bonds; delta_H4.61(1H, s), 4.46(1H, s), 4.44(1H, s) are presumed to be proton signals on the vicinal oxygen carbon; the compound is preliminarily presumed to be lupane-type triterpene from hydrogen spectrum information.

¹³C-NMR(150Hz,CDCl₃) In the spectrum, a total of 43 carbon signals are given, of which δ_C176.8, 171.1, 167.4 is the carbonyl carbon signal, which is presumed to be the carboxylic acid or ester carbonyl carbon signal by chemical shift; delta_C150.3, 110.1 and delta_C144.8, 114.8 are two sets of double bond carbon signals, where δ_C150.3, 110.1 are characteristic carbon signals of 20, 29-position of lupane triterpenoid; delta_C148.1, 146.9, 127.0, 123.1, 115.9, 109.6 are 6 SPs²A hybridized carbon signal; delta_C80.8, 63.3 are the continuous oxygen carbon signals, where delta_C80.8 is the characteristic carbon signal of 3-position beta hydroxyl substitution; delta_C51.9, 51.5 are 2 methoxy carbon signals, delta_C21.4 is the acetylmethyl carbon signal; delta_C16.5, 16.7, 16.8, 19.6, 28.0 are 5 methyl carbon signals; the combination of the hydrogen spectrum data and the above carbon spectrum chemical shift value can predict that the compound has a ferulic acid acyl substitution.

In HMBC spectra, δ_HProton signal and δ of 4.61(63.3)_C167.4, 41.6, 24.2 carbons were remotely related, determining feruloyl linkagesPosition C-27(δ 63.3); delta_HProton Signal and δ of 2.02(21.4)_C171.0, 80.9, and determining that the hydroxyl group of C-3(80.9) is acetylated to ester; delta_HProton signal and delta of 3.68(51.5)_C176.8 remote correlation, δ_HProton signal and delta of 1.41(36.8)_CThere is a remote correlation between 176.8, 56.5, 30.6, which identifies C-28 as a methyl carboxylate substitution. The compound 1 is identified as 3 beta-acetoxy-27- (4-hdroxy-3-Methoxy-E-cinnamyl-oxy) -en-28-oic acid methyl ester based on the information. Process for preparation of Compound 1¹H and¹³the C-NMR data are shown in Table 1, and the structural formula is:

EXAMPLE 3 identification of Compound 2

By physicochemical constants and spectral means (HRESIMS, Mass),¹H-NMR、¹³C-NMR, HSQC, HMBC and¹H-¹h COSY spectrum) of compound 2, and fig. 7, 8, and 9 are a hydrogen spectrum, a carbon spectrum, and an infrared spectrum of compound 2 of the present invention.

The compound 3 is white amorphous powder (chloroform), has yellow fluorescence absorption under ultraviolet lamp 365nm, has no dark spot under 254nm, and develops brown color by heating reaction with 10% sulfuric acid-ethanol developer. In IR (KBr), v_max 3527(OH)，2944、1731(C＝O)，1472、1374、1246、1184、1058、1034、1019、978cm^-1The primary absorption is here. HR-ESI-MS spectrum shows excimer ion peak M/z 515.3735[ M + H ]]⁺(Calcd for C₃₂H₅₃O₅515.3736) indicating a molecular weight of 514 and a molecular formula of C₃₂H₅₂O₅。

¹H-NMR(600Hz,CDCl₃) In the spectrum, the methyl proton signals on the 6 quaternary carbons are respectively given as delta in the high field region_H0.70(3H, s), 0.83(3H, s), 0.85(3H, s), 0.89(3H, s), 0.91(3H, s), 0.96(3H, s), presumably the compound may be a triterpenoid; delta_H2.08(3H, s) is an acetylmethyl proton signal; delta_H5.85(1H, t, J ═ 3.3Hz) is the alkene hydrogen proton signal; delta_H4.62(1H, t, J ═ 3.3Hz) is the proton signal on the vicinal oxygen carbon; delta_H 3.83(1H,d,J＝11.7Hz)、δ_H3.19(1H, d, J ═ 11.7Hz) is the proton signal on the same oxocarbon; 2.93(1H, dd, J ═ 13.7,4.3Hz) is presumed to be the proton signal on the quaternary carbon.

¹³C-NMR(150Hz,CDCl₃) In the spectrum, a 32 carbon signal is given. Delta_C183.6, 171.1 are carbonyl carbon signals, and δ can be estimated from the chemical shift values_C183.6 is the carbon signal of the carboxylic acid, delta_C171.1 is estercarbonyl; delta_C78.1 is the characteristic carbon signal for the substitution of the alpha hydroxyl group in the 3 position, and for the beta substitution, the chemical shift is approximately delta _C 80；δ_C63.1 is the hydroxymethyl carbon signal; delta_C129.7, 137.8 are¹²The characteristic carbon signals of the 12 and 13 oleanenes, the chemical shift values of the 12 and 13 carbon are about 122 and 144, the 12 carbon of the compound is shifted to the low field by about 7ppm, the 13 carbon is shifted to the high field by about 6ppm, the oleanane type triterpene theoretically has methyl proton signals on 7 quaternary carbons, the hydrogen spectrum of the compound only gives the methyl proton signals on 6 quaternary carbons, and the carbon spectrum correspondingly gives the chemical shift delta_C63.1, and thus presumably this compound is an oleanane-type triterpene substituted at the 27-position with a hydroxyl group.

The compound is determined to be oleanane type triterpene with 27-position substituted by hydroxyl, 28-position substituted by carboxylic acid and 3-position hydroxyl acetylated into ester through verification by HSQC spectrum and HMBC spectrum, and the compound 3 is identified to be 3 alpha-acetoxy-27-hydroxyolean-12-en-28-oic acid. Process for preparation of Compound 2¹H and¹³the C-NMR data are shown in Table 1, and the structural formula is:

EXAMPLE 4 identification of Compound 3

By physicochemical constants and spectral means (HRESIMS, Mass),¹H-NMR、¹³C-NMR, HSQC, HMBC and¹H-¹h COSY spectrum) was performed on compound 3, fig. 10,11. 12 is a hydrogen spectrum, a carbon spectrum and an infrared spectrum of the compound 3 of the present invention.

Compound 4 is white amorphous powder (chloroform) with light blue fluorescence absorption under UV lamp 365nm, and reacts with 10% sulfuric acid-ethanol developer to develop purple color. In IR (KBr), v_max 3556(OH)，2949、2875、1723(C＝O)，1372、1247、1154、1015、982、885cm^-1The primary absorption is here. HR-ESI-MS spectrum shows excimer ion peak M/z 545.3468[ M + H ]]⁺(calcd for C₃₂H₄₉O₇545.3478) indicating a molecular weight of 544 and a molecular formula of C₃₂H₄₈O₇。

¹H-NMR(600Hz,CDCl₃) In the spectrum, the methyl proton signals on the quaternary carbons are respectively delta in the high field region_H0.72(3H, s), 0.84(3H, s), 1.13(3H, s), 1.66(3H, s), where δ_H1.66(3H, s) is the characteristic hydrogen signal of the methyl group at position 30 of lupane triterpenoid, which was initially assumed to be lupane triterpenoid; delta_H2.06(3H, s) is the acetylmethyl proton signal, Δ_H3.75(3H, s) is the methoxy proton signal; delta_H4.61(1H, s), 4.75(1H, s) are proton signals of a group of terminal double bonds; delta_H4.61(1H, s) presumably proton signal on the oxygen-linked carbon; delta_H2.56(1H, ddd, J ═ 13.1,6.3,2.2Hz), 2.94(1H, t, J ═ 11.3Hz), 3.11(1H, td, J ═ 10.6,4.9Hz) are presumed to be proton signals on the quaternary carbons.

¹³C-NMR(150Hz,CDCl₃) In the spectrum, a total of 32 carbon signals are given. Low field region delta_C213.8 is the keto carbonyl carbon signal; delta_C175.8, 170.8 for ester carbonyl or carboxylic carbon signals; delta_C148.9 and 111.1 are characteristic carbon signals of 20 and 29 positions of lupane triterpenoids; delta_C78.3, 73.9, 62.0 are the continuous oxygen carbon signals, where delta_C78.3 is the characteristic carbon signal of 3-position alpha hydroxyl substitution, and delta can be known by combining DEPT135 DEG spectrum_C73.9 is the continuous oxygen quaternary carbon signal, delta_C62.0 is the oxygen-linked tertiary carbon signal; delta_C52.0 is the methoxy carbon signal, delta_C21.4 is the acetylmethyl carbon signal; delta_C 15.4、18.7、216, 24.6, 27.9 are 5 methyl carbon signals, lupane triterpenes theoretically have methyl proton signals on 7 quaternary carbons, the compound hydrogen spectrum gives only methyl proton signals on 6 quaternary carbons, combined with delta_C73.9 is the quaternary carbon-linked oxygen signal, presumably the compound may have a missing horn methyl group or be substituted by a hydroxyl group.

In HMBC spectra, delta_HProton signal and delta of 2.31(39.7)_C55.7, 213.8 have a remote dependence on carbon, δ_HProton signal and delta of 1.72(29.7)_CThe carbons of 55.7, 175.8, 213.8 have a remote dependence, δ_C55.7, 39.7, 29.7 are the 17, 15, 22 carbon signals, respectively, determining delta_C213.8 ketocarbonyl is the 16 carbon signal; delta_HProton Signal and δ of 1.13(24.6)_CThe carbons of 42.2, 62.0, 73.9 have a remote dependence, δ_HProton Signal and δ of 1.16(42.2)_CThe carbons of 24.6, 49.8, 73.9 have a remote dependence, δ_HProton Signal and δ of 1.25(24.4)_CThe carbons of 38.3, 62.0, 73.9 have a remote dependence, δ_C24.4, 24.6, 42.2 for the 12, 26, 7 carbon signals, respectively, determining δ_C73.9 with the oxygen quaternary carbon is a 14-carbon signal, thus the compound has the methyl group at position 27 deleted and the hydrogen on the quaternary carbon at position 14 replaced by a hydroxyl group; delta_HProton signal and delta of 1.41(49.8)_C15.4, 42.2, 62.0 carbon has a remote dependence and delta_HProton Signal and δ of 1.25(24.4)_CThe carbons at 38.3, 62.0, 73.9 have long range correlation, and the chemical shift of the 11 position substituted by hydroxyl can be determined as delta_C 62.0；δ_H2.06(21.4) proton Signal and δ_C171.0, 78.3, determining the acetylation of the hydroxyl group at position 3 to form an ester; delta_HProton signal and delta of 3.75(52.0)_C175.8 has a long range correlation, delta_HProton signal and delta of 1.72(29.7)_CThe carbons of 55.7, 175.8, 213.8 were remotely correlated, confirming methyl esterification of the 28-position carboxylic acid. The relative configuration of the 2-position and the 3-position of the compound is determined by an NOESY spectrogram, NOE effect is observed in the NOESY spectrogram at 1.17(H-11) and 1.41(H-9 alpha), H-3 and 0.84(H-24 beta), and the relative configuration of the compound is determined to be 3 alpha-acetoxy-11 beta and 14 alpha-dihydroxy-olean.

To sum up the findingsThe compound 3 is 3 alpha-acetoxy-11 alpha, 14 alpha-dihydroxy-olean-20 (29) -en-16-on-28-oic acid methyl ester. Process for preparation of Compound 3¹H and¹³the C-NMR data are shown in Table 1, and the structural formula is:

EXAMPLE 5 identification of Compound 4

By physicochemical constants and spectral means (HRESIMS, Mass),¹H-NMR、¹³C-NMR, HSQC, HMBC and¹H-¹h COSY spectrum) of compound 4, and fig. 13, 14, and 15 are a hydrogen spectrum, a carbon spectrum, and an infrared spectrum of compound 4 of the present invention.

The compound 4 is white block crystal (chloroform) with yellowish fluorescence absorption under ultraviolet lamp 365nm, and reacts with 10% sulfuric acid-ethanol developer under heating to give purple color. In IR (KBr), v_max 3514(OH)，2949、2863、1707(C＝O)，1639(C＝C)，1377、1273、1186、1147、880cm^-1The primary absorption is here. HR-ESI-MS spectrum shows excimer ion peak M/z 529.3538[ M + H ]]⁺(Calcd for C₃₂H₄₉O₆529.3529) indicating a molecular weight of 529 and a molecular formula of C₃₂H₄₈O₆。

¹H-NMR(600Hz,CDCl₃) In the spectrum, the methyl proton signals on 5 quaternary carbons in a high field region are respectively delta_H0.84(3H, s), 0.88(3H, s), 0.91(3H, s), 1.27(3H, s), 1.66(3H, s), where δ_H1.66(3H, s) is the characteristic hydrogen signal for the methyl group at position 30 of lupane triterpenes; delta_H4.60(1H, s), 4.73(1H, s) are proton signals of a group of terminal double bonds; delta_H4.62(1H, s) presumably proton signal on the oxygen-linked carbon; delta_H2.07(3H, s) is the acetylmethyl proton signal, Δ_H3.75(3H, s) is the methoxy proton signal.

¹³C-NMR(150Hz,CDCl₃) In the spectrum, a total of 32 carbon signals, δ, are given_C148.9 and 110.9 are characteristic carbon signals of 20 and 29 positions of lupane triterpenes, and are confirmed by combining hydrogen spectraDetermining the structure of the parent nucleus of the compound as lupane triterpenoid; delta_CThe carbonyl carbon signals of 212.4, 175.8 and 170.7 can be found to be delta according to chemical shifts_C212.4 is the keto carbonyl carbon signal, delta_C175.8, 170.7 ester carbonyl or carboxylate carbon signals; delta_C78.4, 73.2 are the continuous oxygen carbon signals, where delta_C78.4 is the characteristic carbon signal of 3-position alpha hydroxyl substitution, and delta can be known by combining DEPT135 DEG spectrum_C73.2 is the oxygen-connected quaternary carbon signal; delta_C52.0 is the methoxy carbon signal, delta_C21.3 is the acetylmethyl carbon signal; delta_C14.9, 18.9, 21.9, 28.0, 30.7 are 5 quaternary carbon methyl carbon signals; lupane triterpenes theoretically have methyl proton signals on 7 quaternary carbons, and the hydrogen spectrum of the compound gives only methyl proton signals on 6 quaternary carbons, combined with delta_C73.9 is the quaternary carbon-linked oxygen signal, presumably this compound may have a deletion of the angle methyl group.

In HMBC spectra, delta_HProton Signal and δ of 2.30(39.7)_CThe carbons 34.0, 55.7, 73.2, 175.8 are remote dependent, δ_HProton Signal and δ of 1.76(42.0)_CThe carbons of 50.2, 59.4, 73.2 have a remote dependence, δ_HProton Signal and δ of 1.27(30.7)_CThe carbons of 14.9, 38.7, 42.0, 59.4, 73.2 have a remote dependence, δ_C73.2 and delta_HRemote correlations were found for 1.76(C-7,42.0), 2.30(C-15,39.7), 1.27(C-26,30.7) to determine δ_C73.2 is the 14 carbon signal, consistent with the above reasoning that this compound has a methyl deletion, i.e. the methyl is deleted at position 27 and a hydroxyl substitution is present at position 14. Delta_HProton Signal and δ of 1.54(24.9)_CThe carbons of 212.4, 54.9, 38.6 have a remote dependence, δ_HProton signal and delta of 2.94(53.2)_CThe carbons of 212.4, 29.7, 24.9 have a remote dependence, δ_C212.4 and delta_H 1.54(C-12,24.9)、δ_H2.94(C-13,53.2) all have remote correlation, and the 11 position is determined to be ketone carbonyl; delta_HProton Signal and δ of 2.07(21.4)_C170.7, 78.4 carbon has remote correlation, determining 3-position hydroxyl group is acetylated into ester; delta_HProton signal and delta of 3.75(52.0)_C175.8 has a remote correlation and delta_H2.08(29.7)Proton signal and delta_CThe carbons at 31.7, 48.5, 55.7, 175.8 were remotely correlated to determine the methyl esterification of the 28-position carboxylic acid.

The compound is identified as 3 alpha-acetoxy-14 alpha-hydroxy-olean-20 (29) -en-11-on-28-oic acid methyl ester. Process for preparation of Compound 4¹H and¹³the C-NMR data are shown in Table 2, and the structural formula is:

EXAMPLE 6 identification of Compound 5

By physicochemical constants and spectral means (HRESIMS, Mass),¹H-NMR、¹³C-NMR, HSQC, HMBC and¹H-¹h COSY spectrum) of compound 5, and fig. 16, 17, and 18 are a hydrogen spectrum, a carbon spectrum, and an infrared spectrum of compound 5 of the present invention.

Compound 5 is white amorphous powder (chloroform), has blue fluorescence absorption under ultraviolet lamp 365nm, has dark spot under 254nm, and is heated to react with 10% sulfuric acid-ethanol developer to develop purple color. In IR (KBr), v_max 3514(OH)，2942、1707(C＝O)，1631、1601、1515(C＝C)，1465、1261、1159、1031、803cm^-1The primary absorption is here. HR-ESI-MS spectrum shows excimer ion peak M/z 663.4238[ M + H ]]⁺(Calcd for C₄₁H₅₉O₇663.4261) indicating a molecular weight of 662 and a molecular formula of C₄₁H₅₈O₇。

¹H-NMR(600Hz,CDCl₃) In the spectrum, the methyl proton signals of 6 quaternary carbons in the high field region are respectively delta_H 0.72(3H,s)、0.82(3H,s)、0.83(3H,s)、0.90(3H,s)、0.92(3H,s)、0.93(3H,s)；δ_H3.94(3H, s) and 3.68(3H, s) are 2 methoxy proton signals; delta_H7.54(1H, d, J ═ 15.9Hz), 6.92(1H, dd, J ═ 8.1Hz) are proton signals for a group of exocyclic double bonds, δ_H6.21(1H, d, J ═ 15.9Hz), 7.05(1H, dd, J ═ 8.1,1.8Hz), 7.01(1H, d, J ═ 1.8Hz) are olefinic hydrogen proton signals on aromatic rings, and it is presumed from the coupling constants that there are 1, 3, 4-position substitutions in the benzene ring, and 1-position and ringAn external double bond. Delta_H4.33(1H, d, J ═ 12.6 Hz); 4.33(1H, d, J ═ 12.6Hz), 3.37(1H, t, J ═ 2.5Hz) are presumed to be proton signals on the vicinal oxygen carbons.

¹³C-NMR(150Hz,CDCl₃) In the spectrum, a total of 41 carbon signals, δ, are given_C178.3, 167.1 are carbonyl carbon signals; delta_C137.7 and 127.0 are characteristic carbon signals of 13 and 12 positions of the oleanolic triterpenes; delta_C144.7, 114.8 are carbon signals of a group of exocyclic double bonds; delta_C148.0, 146.9, 127.1, 123.1, 116.0, 109.5 are 6 SPs²The hybridized carbon signal can be known to have 1, 3 and 4-position substitution of the aromatic ring according to the combination of chemical shift and hydrogen spectrum, and the compound is presumed to have a ferulic acid acyl substitution; delta_C76.1, 65.9 are the continuous oxygen carbon signals, where delta_C76.1 is the characteristic carbon signal of alpha hydroxyl substitution at the 3-position; delta_C51.8 is the methoxy carbon signal; delta_C15.4, 18.1, 22.3, 23.7, 28.3, 33.0 are 6 methyl carbon signals. In HMBC spectra, delta_H 4.34(65.9)、δ_HProton signal and delta of 4.21(65.9)_C167.1, 137.7, 45.4, 40.2 carbons were remotely related, and the ferulic acid acyl group attachment position was determined to be 27 (delta)_C 65.9)，δ_HProton signal and δ of 3.64(51.8)_C178.3 remote correlation, δ_HProton Signal and δ of 1.96(23.5)_C178.3, 46.7, 23.1 are remotely related, and C-28 is determined to be substituted by methyl carboxylate. The compound is identified as 3 alpha-hydroxy-27- (4-hydroxy-3-Methoxy-E-cinnamyl) -12-en-28-oi-c acid methyl ester. Process for preparation of Compound 5¹H and¹³the C-NMR data are shown in Table 2, and the structural formula is:

EXAMPLE 7 identification of Compound 6

By physicochemical constants and spectral means (HRESIMS, Mass),¹H-NMR、¹³C-NMR, HSQC, HMBC and¹H-¹h COSY spectrum) of compound 6, and fig. 19, 20, 21 are hydrogens of compound 6 of the present inventionSpectra, carbon spectra, infrared spectra.

The compound 6 is white amorphous powder (chloroform), has no fluorescence absorption under 365nm ultraviolet lamp, has dark spot under 254nm, and is heated to react with ethanol sulfate reagent to give purple color. In IR (KBr), v_max 3455(OH)，2948、1714(C＝O)，1027、1183、977、885cm^-1The primary absorption is here. HR-ESI-MS spectrum shows excimer ion peak M/z515.3749[ M + H ]]⁺(Calcd for C₃₂H₅₁O_7,515.3736) indicating a molecular weight of 514 and a molecular formula of C₃₂H₅₀O₅。

¹H-NMR(400Hz,CDCl₃) In the spectrum, five quaternary carbon methyl proton signals are respectively delta in a high field region_H0.82(3H, s), 0.83(3H, s), 0.87(3H, s), 0.95(3H, s), 1.68(3H, s), where δ_H1.68(3H, s) is 30-CH for lupane triterpenes₃The characteristic hydrogen signal of (a), the compound is preliminarily presumed to be lupane triterpenoid; methyl proton signal delta of 1 acetyl group_H 2.04(3H,s)，δ_H4.75(1H, s), 4.61(1H, s) are proton signals of a group of terminal double bonds; 4.46(1H, dd, J ═ 10.9,5.3Hz) is the vicinal oxymethylene proton signal; 4.23(1H, d, J ═ 12.4Hz) and 3.81(1H, d, J ═ 12.4Hz) are the signals for the oxymethylene protons.

¹³C-NMR(101Hz,CDCl₃) In the spectrum, a total of 32 carbon signals are given, of which delta_C181.4, 171.1 is the carbonyl carbon signal, which is presumed to be the carboxylic acid or ester carbonyl carbon signal from chemical shift; delta_C150.3, 110.1 are double bond carbon signals, where delta_C150.3, 110.1 are characteristic carbon signals of 20, 29-position of lupane triterpenoid; delta_C81.0, 61.2 are the continuous oxygen carbon signals, where delta_C81.0 is the characteristic carbon signal, δ, for the substitution of the beta hydroxy group in position 3_C61.2 is the hydroxymethyl carbon signal; delta_C21.4 is the acetylmethyl carbon signal. Binding compared with the compound 3-acetoxy-27-hydroxyup-20 (29) -en-28-oic acid methyl ester¹H-NMR and¹³C-NMR the compound lacks one methoxy group and the carbonyl carbon signal in the compound is delta_C181.4 and 3-acetoxy-27-hydroxyup-20(29) one of the carbonyl carbon signals δ of en-28-oic acid methyl ester_C176.8 shifted low by about 4.5ppm, suggesting that the C-28 position of the compound is a carboxylic acid substitution. The compound is determined to be lupane-type triterpene with 28-position substituted by carboxylic acid, 27-position substituted by hydroxyl and 3-position acetylated into ester by verification through HSQC spectrum and HMBC spectrum, and is identified to be 3-acetoxy-27-hydroxyup-20 (29) -en-28-oic acid. Process for preparation of Compound 6¹H and¹³the C-NMR data are shown in Table 3, and the structural formula is:

EXAMPLE 8 identification of Compound 7

By physicochemical constants and spectral means (HRESIMS, Mass),¹H-NMR、¹³C-NMR, HSQC, HMBC and¹H-¹h COSY spectrum) of compound 7, and fig. 22, 23, and 24 are a hydrogen spectrum, a carbon spectrum, and an infrared spectrum of compound 7 of the present invention.

The compound 7 is amorphous white powder (chloroform), has blue fluorescence absorption under ultraviolet lamp 365nm and dark spot under 254nm, and can be heated to react with 10% sulfuric acid-ethanol developer to develop purple color. In IR (KBr), v_max 3442(OH)，2943、2876、1724(C＝O)，1642、1251、1015、976cm^-1The primary absorption is here. HR-ESI-MS spectrum shows excimer peak HR-ESI-MS M/z 543.3680[ M + H ]]⁺(calcd for C₃₃H₅₁O₆542.3607) indicating a molecular weight of 514, the formula C₃₃H₅₀O₆。

¹H-NMR(400Hz,CDCl₃) In the spectrum, the methyl proton signals of 6 quaternary carbons in the high field region are respectively delta_H 0.83(3H,s)、0.88(3H,s)、0.88(3H,s)、0.94(3H,s)、0.97(3H,s)、1.05(3H,s)；δ_H2.08(3H, s) is the acetyl proton signal; 3.65(3H, s) is the methoxy proton signal; delta_H5.97(1H, s) is an olefinic hydrogen proton signal; delta_H4.61(1H, s) is the proton signal on the oxygen-linked carbon; delta_H 4.20(1H,d,J＝14.4Hz)；3.5(1H, d, J ═ 14.4Hz) presumably signals two protons on the same vicinal oxygen carbon.

¹³C-NMR(101Hz,CDCl₃) In the spectrum, a total of 33 carbon signals, δ_C201.3 presumably the ketocarbonyl carbon signal, delta_C177.3, 171.0 is the carboxylic acid or ester carbonyl carbon signal; delta_C78.0 is the characteristic carbon signal for alpha hydroxy substitution at the 3-position; delta_C63.1 is the hydroxymethyl carbon signal; delta_C132.4, 160.8 are characteristic carbon signals of oleanolic triterpenes at 12 and 13 positions, the carbon chemical shifts of 12 and 13 positions are 122 and 144 in general, but 13 position of the compound PH-90 is shifted to a low field by about 16ppm, 12 position is shifted to a low field by about 10ppm, the oleanolic triterpenes theoretically should have 7 angular methyl groups, and PH-90 only has 6 angular methyl groups, and a hydroxymethyl signal delta is added_C63.1, and a ketocarbonyl signal δ_C201.3, it is therefore assumed that the compound PH-90 is substituted by hydroxy in position 27 and by ketocarbonyl in position 11; combined with HMBC spectrum, delta_HProton signal and delta of 5.97(132.4)_C 41.2、δ_CThere is a remote correlation of 62.0 carbons, and δ_HProton signal and δ of 3.06(62.0)_CThe existence of remote correlation among carbons of 16.6, 20.8 and 201.3 determines that the ketone carbonyl is connected at the 11 position; delta_H 4.20(63.1)、δ_HProton Signal and δ of 3.50(63.1)_CThe carbons of 24.7, 45.9, 46.0, 160.8 were remotely related, and the hydroxymethyl linkage position was determined to be 27 (. delta.) (Δ)_C 63.1)；δ_HProton signal and delta of 3.65(52.1)_C177.1 carbons have a long range correlation, δ_H 1.93(22.6)、δ_HProton Signal and δ of 1.74(31.6)_C177.1 all have remote correlations, determining the 28-position as a carboxylic acid methyl ester substitution; delta_HProton signal and delta of 2.08(21.6)_C171.0 there is a remote correlation and it is determined by combining HSQC spectra that the alpha hydroxyl group at position 3 is acetylated to an ester. The compound is identified as 3 alpha-acetoxy-27-hydroxyolean-12-en-16-on-28-oic acid methyl ester. Process for preparation of Compound 7¹H and¹³the C-NMR data are shown in Table 3, and the structural formula is:

table 1: of Compounds 1 to 3 of the present invention¹H-NMR and¹³C-NMR spectral data

Table 2: of Compounds 4 to 5 of the present invention¹H-NMR and¹³C-NMR spectral data

Table 3: of the compounds 6 and 7 according to the invention¹H-NMR and¹³C-NMR spectral data

Example 9 antitumor Activity test

The compound of the invention is used for in vitro tumor inhibition activity experiments of 3 tumor strains of a human body, wherein the 3 tumor strains comprise cytotoxicity of three human tumor cells of HeLa (human cervical cancer cells), HepG2 (human liver cancer cells) and SGC-7901 (human gastric cancer cells). Cis-dichlodidiamineplatinum (II) (cisplatin) as a positive control. Based on the results of the study by the MTT method, the compounds 3 and 7 of the present invention were examined for their inhibitory effects on HeLa cells (human cervical cancer cells) by a Clone formation experiment (Clone formation method) and a Hochest staining method.

Inhibition of tumor cell proliferation (MTT method): inoculating the tumor cells into a 96-well plate, adding a sample to be tested after culturing for 24h, and determining the inhibition rate of the sample on the proliferation of the tumor cells by an MTT method after culturing for 48 h. The cell proliferation inhibition rate was calculated according to the following formula, and the half inhibitory concentration of the test sample (IC50) was calculated using CalcuSyn software.

Clone formation experiment (Clone formation method): inoculating tumor cells into a 24-well plate, inoculating 80 cells, culturing for 12h, adding a sample to be tested after the cells adhere to the wall, culturing for 48h, adding a complete culture medium, changing the culture medium for four times, discarding the supernatant, dyeing with crystal violet for 20min, washing for 3 times with PBS (phosphate buffer solution), counting the number of formed clones, and comparing the difference of the formed clones of each group.

Fluorescence microscopy (Hochest staining): inoculating tumor cells into a 48-well plate, culturing for 12h, adding a sample to be tested after the cells adhere to the wall, and observing the cell morphology by a microscope after the cells act for 48 h; discard the supernatant, wash 3 times with PBS, fix the cells in 4% paraformaldehyde for 20 minutes, wash 3 times with PBS, add 10 ug/ml Hochest to stain for 15 minutes, wash 3 times with PBS, and observe the nuclear morphology with a fluorescence microscope.

Western Blot experiment: cells in exponential growth phase were seeded in 6-well plates, compound 2(10.0, 20.0, 40.0 μmol · L-1) and compound 6(5.0, 10.0, 20.0 μmol · L-1) were added at different concentrations, after stimulation, total proteins of each group were extracted at the corresponding time points, electrophoresed in 10% polyacrylamide gels, transferred to NC membranes, blocked, incubated, developed using ECL kit and imaged. Each set of experiments was repeated 3 times.

The inhibition rate of cell proliferation ═ (average value of OD values in negative control group-average value of OD values in sample group) ÷ (average value of OD values in negative control group-average value of OD values in blank control group) × 100%, and the experimental results are shown in table 4.

TABLE 4 inhibitory Effect of the Compounds of the present invention on the proliferation of three tumor cells

From the experimental data in table 4, it can be seen that compounds 2, 3,5, 6 and 7 have inhibitory effects on three different tumor cells, namely HeLa, HepG2 and SGC-7901, to different extents, indicating that compounds 2, 3,5, 6 and 7 can be further researched and developed as new antitumor drugs.

Compounds 2 and 6 inhibited the clonogenic capacity of HeLa cells (human cervical cancer cells), and the results are shown in FIG. 1.

FIG. 1 shows that the clone-forming ability of HeLa cells is significantly reduced after the cells are treated with compounds 2 and 6 at different concentrations. And HeLa cells of a higher concentration treatment group (20-40 mu M) can hardly form macroscopic cell clones.

The cell and nucleus morphologic apoptosis characteristics of HeLa cells induced by compounds 2 and 6 are shown in FIG. 2.

Fig. 2 shows that the results of the Hochest staining experiment show that the apoptosis characteristics of HeLa cells are more remarkable with the increase of the concentration after the HeLa cells are treated by the compounds 2 and 6 with different concentrations.

FIG. 3 shows the expression of apoptosis-related proteins by Western blot analysis. The experimental results show that compounds 2 and 6 induce HeLa cell apoptosis. Wherein "+" in the figure indicates the significance of triterpenoids relative to HeLa cell-associated proteins. Wherein^*p＜0.05，^**＜0.01，^***p＜0.001。

The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.

Claims (2)

1. The preparation method of the triterpenoid is characterized by comprising the following steps: the method comprises the following steps:

1) taking peganum harmala seeds, heating and refluxing the peganum harmala seeds with ethanol, combining extracting solutions, and concentrating the extracting solutions under reduced pressure to obtain an extract;

2) suspending the extract with water, acidifying, and extracting with chloroform to obtain non-alkaloid layer;

3) separating the non-alkaloid layer by chromatography to obtain triterpenes;

the structural formula of the triterpenoid is as follows:

2. the method of claim 1, wherein: and the acidification refers to adding hydrochloric acid to adjust the pH value to 1-2.

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