An oleanane-type triterpenic acid, its preparation method and application

Abstract

This invention discloses an oleanane-type triterpene acid compound, its preparation method, and its applications. This compound is a novel oleanane-type triterpene acid with a highly oxidized C-27 methyl group, reported for the first time. The preparation method includes the extraction and separation of the triterpene acid fraction, followed by structural identification using modern spectroscopic methods. Furthermore, in vitro cytotoxicity experiments demonstrated that this compound can inhibit the proliferation of tumor cells, showing great potential in anti-tumor activity. It is expected to serve as a lead compound for the research and development of new anti-tumor drugs and provides a material basis for the study of the chemical composition and pharmacological activity of *Asparagus cochinchinensis*.

Description

Technical Field

[0001] This invention relates to a triterpenic acid, its preparation method, and its application, specifically to oleanane-type triterpenic acids, their preparation method, and their application. Background Technology

[0002] Astilbe grandis (Stapf ex Wils), also known as red astragalus or hairy cold medicine, is a plant belonging to the genus Astilbe in the family Saxifragaceae. It is a commonly used traditional Chinese medicine, with its rhizome recorded in traditional Chinese medicine texts as being used to treat injuries from falls and blows, rheumatoid arthritis, colds due to wind-heat, coughs, and snake bites, with significant effects. Furthermore, literature research has revealed that the chemical components of Astilbe plants are mostly coumarins, flavonoids, terpenes, sterols, acidic components, and volatile oils, possessing pharmacological activities such as anti-inflammatory analgesia, cytotoxicity, antitumor, and antioxidant properties. Additionally, the highly oxidized triterpenic acid at the C-27 position found in this plant differs from the commonly found triterpenic acid at the C-28 position, exhibiting superior antitumor activity. Literature such as "Study on Triterpenoids in the Root of Astilbe" and "Study on Chemical Constituents of Astilbe" discloses extraction methods for Astilbe, but does not disclose the preparation method and application of the highly oxidized oleanane-type triterpenoid acid at the C-27 position as described in this invention. Summary of the Invention

[0003] Purpose of the invention: The present invention aims to provide a method for preparing oleanane-type triterpenic acids; another purpose of the present invention is to provide an application of oleanane-type triterpenic acids in the preparation of antitumor drugs.

[0004] Technical solution: The oleanane-type triterpenic acid of this invention is extracted and isolated from Astilbe davidii, and its structural formula is as follows:

[0005]

[0006] This invention provides a method for preparing the above-mentioned oleanane-type triterpenic acid, which mainly includes the following steps:

[0007] (1) The rhizome of *Lactuca indica* was crushed, and the solution was added to soak, filtered, and the filtrate was concentrated to obtain *Lactuca indica* ethanol extract.

[0008] (2) Disperse the extract in water and extract it several times with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively.

[0009] (3) The ethyl acetate extract was separated using a silica gel column and eluted with a dichloromethane-methanol system to obtain 7 fractions Fr.AG;

[0010] (4) The components Fr.B were separated by elution using a silica gel column to obtain Fr.B1-B7;

[0011] (5) Fr.B4 was eluted with a silica gel column using a petroleum ether-acetone gradient to obtain Fr.B4.1-Fr.B4.20;

[0012] (6) The Fr.B4.20 component was analyzed by semi-preparative high performance liquid chromatography with acetonitrile-water as the mobile phase and ultraviolet detector for full wavelength detection to obtain the compound.

[0013] Further, the solvent volume concentration used in step (1) is 90-95%, soaking for 7-10 days, and repeating the soaking, filtration, and concentration of the filtrate 5-7 times, and then vacuum-concentrating to obtain the extract.

[0014] Further, in step (4), the elution separation conditions of the silica gel column are as follows: a petroleum ether-ethyl acetate system is used, with solvent volume ratios of 3:1, 2:1, 1:1 and 1:2 in sequence for gradient elution, and the silica gel used is 200-300 mesh.

[0015] Further, in step (6), the semi-preparative high performance liquid chromatography separation conditions are acetonitrile-water isocratic elution at a volume ratio of 80:20, 3 mL / min, 30 °C, and 20 min each time, and the peaks are combined and concentrated according to the elution time.

[0016] Furthermore, the oleanane-type triterpenic acids were first separated using silica gel column chromatography, and then purified by high performance liquid chromatography to obtain pure oleanane-type triterpenic acids. The screening of the two-step separation and purification conditions provides strong support for the preparation of the target compound.

[0017] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: A novel oleanane-type triterpene acid with a highly oxidized C-27 methyl group was isolated from *Astilbe davidii*. Spectroscopic techniques confirmed that it is a new compound, and this is the first time it has been isolated from *Astilbe davidii*. Furthermore, the antitumor activity of the aforementioned oleanane-type triterpene acid is disclosed for the first time. In vitro cytotoxicity experiments showed that this compound significantly inhibited the proliferation of human non-small cell lung cancer cells, human colon cancer cells, and human large cell lung cancer cells, indicating its potential for research and development as a new antitumor drug. Attached Figure Description

[0018] Figure 1 This is the high-resolution mass spectrum of compound 1 of the present invention;

[0019] Figure 2 This is the proton spectrum of compound 1 of the present invention;

[0020] Figure 3 This is the carbon spectrum of compound 1 of the present invention;

[0021] Figure 4 This is the HMBC and COSY correlation signal spectrum of compound 1 of the present invention;

[0022] Figure 5 This is the NOESY correlation signal spectrum of compound 1 of the present invention;

[0023] Figure 6 This is the ECD spectrum of compound 1 of the present invention;

[0024] Figure 7 The present invention relates to the rhizome of the medicinal plant Astilbe chinensis, and the structural formula and ECD spectrum of compound 1. Detailed Implementation

[0025] The technical solution of the present invention will be further explained below with reference to specific examples.

[0026] Example 1

[0027] The extraction and separation steps of the oleanane-type triterpenic acid are as follows:

[0028] (1) Use a pulverizer to grind the dried rhizomes of Astilbe davidii (see...) Figure 7 The mixture was crushed, soaked in a 95% ethanol solution for 7 days, filtered, and concentrated. The soaking, filtration, and concentration were repeated 5 times. The mixture was then concentrated under vacuum to obtain the ethanol extract of Astilbe davidii.

[0029] (2) Disperse the extract in water and extract it several times with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively.

[0030] (3) The ethyl acetate extract was separated using a silica gel column. Gradient elution was first performed using a dichloromethane-methanol system to obtain 7 fractions Fr.AG;

[0031] (4) Fr.B was separated by elution with a silica gel column using a petroleum ether-ethyl acetate system, with solvent volume ratios of 3:1, 2:1, 1:1 and 1:2 in sequence, and the silica gel used was 200-300 mesh, to obtain Fr.B1-B7;

[0032] (5) Fr.B4 was eluted with a silica gel column using a petroleum ether-acetone gradient to obtain Fr.B4.1-Fr.B4.20;

[0033] (6) The Fr.B4.20 fraction was analyzed by semi-preparative high performance liquid chromatography (HPLC) with acetonitrile-water as the mobile phase. Acetonitrile-water was eluted isocratically at a volume ratio of 80:20, at a rate of 3 mL / min, at 30 °C, for 20 min each time. The elution times were combined and concentrated, and the fractions were detected at all wavelengths using a UV detector to obtain compound 1, with the following structure:

[0034]

[0035] Example 2

[0036] The extraction and separation steps of the oleanane-type triterpenic acid are as follows:

[0037] (1) The dried rhizomes of Astilbe were crushed by a pulverizer, and then soaked in a 92% ethanol solution for 8 days. The solution was then filtered and concentrated. The soaking, filtration and concentration were repeated 6 times. The solution was then concentrated under vacuum to obtain an ethanol extract of Astilbe.

[0038] (2) Disperse the extract in water and extract it several times with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively.

[0039] (3) The ethyl acetate extract was separated using a silica gel column. Gradient elution was first performed using a dichloromethane-methanol system to obtain 7 fractions Fr.AG;

[0040] (4) Fr.B was separated by elution with a silica gel column using a petroleum ether-ethyl acetate system, with solvent volume ratios of 3:1, 2:1, 1:1 and 1:2 in sequence, and the silica gel used was 200-300 mesh, to obtain Fr.B1-B7;

[0041] (5) Fr.B4 was eluted with a silica gel column using a petroleum ether-acetone gradient to obtain Fr.B4.1-Fr.B4.20;

[0042] Fr.B4.20 fraction was analyzed by semi-preparative high performance liquid chromatography (HPLC). Acetonitrile-water was used as the mobile phase, and acetonitrile-water was eluted isocratically at a volume ratio of 80:20 at a rate of 3 mL / min at 30 °C for 20 min each time. The fractions were combined and concentrated according to the elution time, and detected at all wavelengths using an ultraviolet detector to obtain compound 1.

[0043] Example 3

[0044] The extraction and separation steps of the oleanane-type triterpenic acid are as follows:

[0045] (1) The dried rhizomes of Astilbe were crushed by a pulverizer, and then soaked in a 90% ethanol solution for 10 days, filtered, and the filtrate was concentrated. The soaking, filtration, and concentration of the filtrate were repeated 7 times. The Astilbe ethanol extract was obtained by vacuum decompression concentration.

[0046] (2) Disperse the extract in water and extract it several times with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively.

[0047] (3) The ethyl acetate extract was separated using a silica gel column. Gradient elution was first performed using a dichloromethane-methanol system to obtain 7 fractions Fr.AG;

[0048] (4) Fr.B was separated by elution with a silica gel column using a petroleum ether-ethyl acetate system, with solvent volume ratios of 3:1, 2:1, 1:1 and 1:2 in sequence, and the silica gel used was 200-300 mesh, to obtain Fr.B1-B7;

[0049] (5) Fr.B4 was eluted with a silica gel column using a petroleum ether-acetone gradient to obtain Fr.B4.1-Fr.B4.20;

[0050] Fr.B4.20 fraction was analyzed by semi-preparative high performance liquid chromatography (HPLC). Acetonitrile-water was used as the mobile phase, and acetonitrile-water was eluted isocratically at a volume ratio of 80:20 at a rate of 3 mL / min at 30 °C for 20 min each time. The fractions were combined and concentrated according to the elution time, and detected at all wavelengths using an ultraviolet detector to obtain compound 1.

[0051] Example 4: Structural Identification of Compounds

[0052] The structure of compound 1 from Example 1 was identified. Compound 1 is a white powder, readily soluble in methanol. Its UV spectrum shows absorption at 195.5, 223.5, and 312.5 nm, suggesting the possible presence of a conjugated system. It produces a pink color in 10% sulfuric acid-ethanol, exhibits a positive Liebermann-Burchard reaction, and a negative Molish reaction, indicating that compound 1 may be a triterpenoid or steroid compound and does not contain sugar. IR(KBr)u max， (cm -1 The values ​​3360, 2930, 2860, 1680, 1600, 1450, 1170, 828, 521 suggest that the structure may contain functional groups such as hydroxyl, carboxyl, double bonds, carbonyl, and benzene rings. Figure 1 As shown, HR-ESI-MS at m / z: 617.3796 [MH] - There is a molecular ion peak (C) at this location. 39 H 53The theoretical calculated value of O6 is 617.3842. Combined with the carbon spectrum, the molecular formula of this compound can be determined to be C6. 39 H 54 O6, and the degree of unsaturation is 12.

[0053] like Figure 2 As shown, 1 The H-NMR spectrum shows numerous and complex signals in the high-field region, with obvious splitting, suggesting the presence of multiple methylene groups. It also contains seven distinct methyl proton signals, located at δ¹⁸ ppm. H 1.41 ppm (3H,s), 1.33 ppm (3H,s), 1.33 ppm (3H,s), 0.96 ppm (3H,s), 0.94 ppm (3H,s), 0.89 ppm (3H,s), 0.87 ppm (3H,s). Three olefin proton characteristic signals (δ) are present in the low-field region. H 5.63 ppm, 1H,t; 6.29 ppm, 1H,d; 7.57 ppm, 1H,d) and four characteristic signals of benzene ring protons (δ H The peak values ​​were 7.48 ppm, 2H, d; 6.81 ppm, 2H, d), and all protons on the benzene ring showed dd peaks, suggesting para-substitution of the benzene ring. Two δ peaks also appeared. H The proton characteristic signals of 4.67 ppm (1H,dd) and 4.36 ppm (1H,t) suggest that these two proton signals may be protons on the carbon atom.

[0054] like Figure 3 As shown, 13 The C-NMR spectrum showed 39 carbon signals. Combined with the DEPT spectrum, these 39 carbons were identified as 7 methyl carbons, 9 methylene carbons, 12 methine carbons, and 11 quaternary carbons. Some characteristic signal peaks of functional groups were observed in the low-field region, δ... C 180.3 ppm is the characteristic signal of the carboxyl group, δ C 168.9 ppm is the characteristic signal of the ester group, δ C 138.1 ppm, 126.5 ppm, 115.7 ppm, and 146.3 ppm are characteristic signals for the four olefin carbons, δ C 81.0 ppm and 68.3 ppm are characteristic signals for the two oxygen-bound carbons. Additionally, the characteristic signal for the benzene ring carbons is δ. C 161.2 ppm, 131.2 ppm, 127.1 ppm, 116.8 ppm, of which δ C The peak heights at 131.2 ppm and 116.8 ppm are twice that of the other peaks, and the carbon spectrum signals highly overlap. Combined with high-resolution mass spectrometry, this also confirms that the benzene ring is para-substituted, consistent with the previous proton spectrum prediction. Meanwhile, the δ¹²... CThe 161.2 ppm increase in chemical shift suggests the presence of a -OH group attached to the benzene ring. Based on the above analysis, the functional groups occupy a total of 7 unsaturations (2 double bonds, 1 ester group, 1 benzene ring, and 1 carboxyl group), and the remaining 5 unsaturations form a pentacyclic triterpenoid skeleton. Furthermore, the NMR spectral data match those of oleanane-type triterpenoids, thus suggesting that this compound is an oleanane-type triterpenic acid.

[0055] like Figure 4 As shown, 1 H- 1 The H COSY spectrum can reveal δ H 5.63(H-12) and δ H There is a related signal at 2.04 (H-11), and δ H 5.63 ppm (H-12) and δ C HMBC-related signals were observed at 49.6 ppm (C-9) / 57.7 ppm (C-14) / 50.7 ppm (C-18), thus determining the double bond position to be between C-12 and C-13; δ0.05 can be observed on the HMBC spectrum. H 4.67 ppm (H-3) and δ C 168.9 ppm (C-1'), δ H 7.57 ppm (H-3') and δ C 168.9 ppm (C-1'), δ H 6.29 ppm (H-2') and δ C The correlation signal between 127.1 ppm (C-4') indicates that the benzene ring substituent is attached at C-3, as shown in the structure diagram. δ H 4.36 ppm (H-6) and δ C The presence of HMBC-related signals at 40.4 ppm (C-8) / 38.56 ppm (C-10) / 37.9 ppm (C-4) indicates that -OH is linked to C-6. 1 H- 1 H-3(1H,δ) in H COSY H 4.67ppm) and H-2 (2H,δ) H 1.61ppm, 2.10ppm), H-6 (1H,δ) H 4.36ppm) and H-5 (1H,δ) H 1.40ppm) / H-7(2H,δ H The related signals (1.47 ppm, 1.81 ppm) further confirmed this conclusion. The carboxyl carbon (C-27, δ) C 180.3ppm) and δH The presence of HMBC-related signals at 2.04 / 1.61 ppm (2H, H-15) indicates that the carboxyl group is attached to C-14 and is designated as the carboxyl group at position 27. Simultaneously, the quaternary carbon (C-14, δ) linked to the carboxyl group... c 57.6 ppm) and CH3-26 (δ H 1.31 ppm, s) and H-12 (δ H The presence of HMBC-related signals at 5.63 ppm (brs) further confirms the above conclusion. Its relative configuration was determined by the NOSEY spectrum (e.g., Figure 5 As shown in the NOSEY spectrum, H3-25 / H3-24, H3-25 / H3-26, H3-25 / H-18, H-18 / H-12, H3-26 / H3-28, and H3-28 / H3-30 exhibit correlation signals, indicating that H3-24, H3-25, H3-26, H-12, H-18, H3-30, and H3-28 are in the β configuration; while H-3 / H3-23, H3-23 / H-6, H-6 / H-5, and H-5 / H-9 indicate that H-3, H-5, H-6, H-9, H3-23, and H3-29 are in the α configuration. Furthermore, the proton signals of the two double bonds on the substituents are δ... H 8.00 ppm (1H,d,J=15.9Hz) and δ H The concentration of 6.58 ppm (1H, d, J = 15.9 Hz) indicates that the double bond is in the trans configuration. Finally, the absolute configuration of the compound was determined by comparing the experimental ECD spectrum with the calculated ECD spectrum. Figure 6 As shown, the calculated ECD curves of the compound configurations of 3S, 6R, and 14R in the ECD spectrum are consistent with the measured CD curves. Finally, the structure of the compound was determined to be (2'E)-(3S,6R,14R)-3β-trans-p-coumaroyloxy-6b-hydroxyolean-12-en-27-oic.

[0056] Compound 1 1 H and 13 The C NMR data are shown in Table 1 below.

[0057] Table 1 Compound 1 1 H(600MHz, MeOD) and 13 C NMR (151MHz, MeOD) data (δin ppm, J in Hz)

[0058]

[0059] Example 5: Study on the in vitro antitumor effects of the compound

[0060] In vitro antitumor experiments were conducted on the two compounds isolated in Example 1. The cells used in the experiment were human non-small cell lung cancer cells (A546), human colon cancer cells (Caco-2), human large cell lung cancer cells (H460), and human ovarian cancer cells (Skov-3). The positive control drug was hydroxycamptothecin (HCPT), and the conventional CCK-8 assay was used for testing.

[0061] The specific method is as follows:

[0062] (1) Cell passage: When the cell density reaches approximately 80%-90%, the cells are passaged. The culture dish is transferred to a clean bench, the original culture medium is discarded, and the cells are washed once with 3 mL of PBS. The PBS is discarded, and 2 mL of 0.25% trypsin containing EDTA is added for digestion. Since the digestion time varies for different cell types, cell morphology should be observed under a microscope. When the cells become round and bright, they are quickly transferred to the clean bench, the trypsin is discarded, and 3 mL of the corresponding culture medium is added to stop the digestion. The bottom of the dish is repeatedly pipetted to form a single-cell suspension. 1 mL of the cell suspension is transferred to a new 10 cm culture dish, 7-8 mL of the corresponding culture medium is added, and the dish is gently shaken back and forth and left and right to mix. The culture dish is then placed in a 37°C, 5% CO2 incubator.

[0063] (2) Half-inhibition concentration IC 50 Experiment: ① Cells in the logarithmic growth phase were digested with trypsin, and culture medium was added to form a single-cell suspension. Cells were counted using a counting chamber. Both the control and experimental groups were seeded at 5000 cells / well in 96-well plates. 100 μL of culture medium was added to each well, and the 96-well plates were incubated at 37℃ in a 5% CO2 incubator for 24 h. ② Culture media containing different compound concentrations were prepared: The compounds were weighed and dissolved completely in DMSO to obtain a stock solution with an initial concentration of 2.0 mM. A certain volume of the stock solution was taken and diluted with the corresponding culture medium at concentration gradients of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM. ③ After the cells have fully adhered to the plate, remove the 96-well plate and transfer it to a clean bench. Discard the original culture medium in the experimental wells, and add 100 μL of culture medium containing different concentration gradients of compounds (6 concentration gradients, 3 replicates). Incubate the 96-well plate at 37°C in a 5% CO2 incubator for 24 hours. ④ After 24 hours, discard the original culture medium in both the experimental and control groups. Add 100 μL of culture medium containing 10% CCK-8 solution to each well. The blank group contains 100 μL of culture medium containing 10% CCK-8 solution without cells, and the control group contains 100 μL of culture medium containing 10% CCK-8 solution with normal cancer cells. Incubate at 37°C in a 5% CO2 cell culture incubator for 2 hours. ⑤ Detect the OD value of each well at 450 nm using a microplate reader. The cell growth inhibition rate is calculated using the following formula:

[0064] Inhibition rate (%) = (Average OD value of blank wells - Average OD value of control wells) / (Average OD value of blank wells - Average OD value of experimental wells) × 100%

[0065] The calculation results are shown in the table below:

[0066]

[0067] According to the experimental results of CCK-8, compound 1 has a certain inhibitory effect on all four types of tumor cells, IC50. 50 All concentrations were less than 20 μM, with more significant inhibitory effects on the growth of A549 and Caco-2 tumor cells, with half-maximal inhibitory concentrations (WMCs) of 2.50 ± 1.03 μM and 4.94 ± 0.79 μM, respectively. The inhibitory effect on A549 tumor cells was almost comparable to that of the positive control drug HCPT. Therefore, this new compound exhibits considerable inhibitory activity against tumor cells.

[0068] Therefore, the novel compounds prepared by this invention provide a new option for the search of active ingredients in antitumor drugs. At the same time, these novel compounds can be modified to improve their activity and formulated into drugs with pharmaceutically acceptable carriers for the treatment of cancer.

Claims

1. A method for preparing oleanane-type triterpenic acids, characterized in that, The oleanolic triterpenic acid has the following structural formula: ； The method for preparing the oleanane-type triterpenic acid includes the following steps: (1) The rhizome of *Lactuca indica* was crushed, and the solution was added to soak, filtered, and the filtrate was concentrated to obtain *Lactuca indica* ethanol extract. (2) Disperse the extract in water and extract it in sequence with petroleum ether, ethyl acetate and n-butanol to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract respectively; (3) The ethyl acetate extract was separated using a silica gel column and eluted with a dichloromethane-methanol system to obtain 7 fractions Fr. AG; (4) The components Fr. B were eluted and separated using a silica gel column to obtain Fr. B1-B7; (5) Fr. B4 was eluted with a silica gel column using a petroleum ether-acetone gradient to obtain Fr. B4.1-Fr. B4.20; (6) The Fr. B4.20 component was analyzed by semi-preparative high performance liquid chromatography with acetonitrile-water as the mobile phase to elute and obtain the target compound.

2. The method for preparing oleanane-type triterpenic acids according to claim 1, characterized in that: The volume concentration of the solvent in step (1) is 90-95%.

3. The method for preparing oleanane-type triterpenic acids according to claim 1, characterized in that: The soaking time in step (1) is 7-10 days.

4. The method for preparing oleanane-type triterpenic acids according to claim 1, characterized in that: In step (1), the soaking, filtration, and concentration of the filtrate are repeated 5-7 times.

5. The method for preparing oleanane-type triterpenic acids according to claim 1, characterized in that: The elution conditions for the silica gel column in step (4) are as follows: use a petroleum ether-ethyl acetate system with solvent volume ratios of 3:1, 2:1, 1:1 and 1:2 for gradient elution.

6. The method for preparing oleanane-type triterpenic acids according to claim 1, characterized in that: The silica gel used for elution separation in step (4) is 200-300 mesh.

7. The method for preparing oleanane-type triterpenic acids according to claim 1, characterized in that: The separation conditions for the semi-preparative high performance liquid chromatography described in step (6) are: acetonitrile-water isocratic elution at a volume ratio of 80:20, 3 mL / min, 30 °C, for 20 min each time, and the peaks are combined and concentrated according to the elution time.

8. The application of an oleanane-type triterpenic acid in the preparation of an antitumor drug, wherein the oleanane-type triterpenic acid has the following structural formula, and the tumor is selected from non-small cell lung cancer, large cell lung cancer, colon cancer, and ovarian cancer. 。

Images