Triterpene compound as well as extraction and separation method and application thereof

Abstract

The invention provides a triterpenoid compound as well as an extraction and separation method and application thereof, and belongs to the technical field of traditional Chinese medicine separation. The preparation method comprises the following steps: by taking mairei as a raw material, extracting with ethanol to obtain a primary extract, dispersing with pure water, and extracting with petroleum ether to obtain a petroleum ether part extract; and then sequentially carrying out silica gel column chromatography, reversed-phase column separation, gel column chromatography and high performance liquid chromatography separation on the petroleum ether part extract, and extracting and separating the multiflorane type triterpenoid compounds as shown in a formula (I) and a formula (II) for the first time. Pharmacological studies show that the triterpenoid provided by the invention has strong inhibitory activity on a plurality of tumor cells, and the minimum IC50 is 0.47 M + / -0.09 M.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine separation technology, specifically to a triterpenoid compound and its extraction and separation method and application. Background Technology

[0002] Triterpenes are terpenoids with a 30-carbon skeleton, consisting of six isoprene units linked end-to-end. In plants, they mostly exist in pentacyclic or tetracyclic structures. Their oxygenated derivatives (hydroxyl, carboxyl, ketone, etc.) can inhibit tumor proliferation, induce apoptosis, block invasion and metastasis, and reverse multidrug resistance through pathways such as arresting the cell cycle, activating mitochondrial-caspase cascades, and inhibiting NF-κB and MMP-9. They have become a hot topic in the research of anti-tumor natural drugs.

[0003] Earthen altar tree ( Alangium salviifolium Also known as the tongue-cutting tree, it belongs to the genus *Illicium* of the family Illicaceae (family Illicaceae). Alangium A deciduous tree or shrub. The fruit of the *Symplocos edulis* is edible and sweet. This plant has the effects of dispelling wind and promoting blood circulation, detoxifying and inducing vomiting, and reducing inflammation and pain. The roots are commonly used as an antidote by the Li ethnic group, and the leaves can alleviate rheumatic bone pain and treat injuries. However, despite traditional applications suggesting its potential medicinal value, research on the effective active ingredients contained in *Symplocos edulis* is currently insufficient. This has resulted in the potential anti-tumor medicinal value of *Symplocos edulis* remaining at the level of traditional experience, with its pharmacodynamic material basis unclear, hindering in-depth pharmacological mechanism research and new drug development, creating a significant gap and waste in the application of this medicinal plant resource in the field of anti-tumor therapy. Therefore, the extraction, separation, and identification of triterpenoid chemical components in *Symplocos edulis*, and the discovery of its unique triterpenoid monomers, are of great significance for elucidating its traditional pharmacodynamic material basis, discovering novel anti-tumor lead compounds, and promoting the development of tumor therapeutic drugs. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a triterpenoid compound, its extraction and separation method, and its application. The triterpenoid compounds shown in formulas (I) and (II) exhibit strong inhibitory activity against the proliferation of various tumor cells and can be used to prepare drugs for the prevention or treatment of tumors.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides a triterpenoid compound, the general structural formula of which is: ; R is selected from either hydroxymethyl or carboxyl groups.

[0006] A second aspect of the present invention provides a method for separating the above-described triterpenoid compounds, comprising the following steps: The leaves of the leaves of the *Tutan* tree were extracted using an alcohol solvent, and the resulting extract was concentrated to obtain a paste. The extract was dispersed in water, extracted with petroleum ether, and concentrated to obtain the petroleum ether extract fraction. The petroleum ether extract was subjected to silica gel column chromatography with a gradient elution using a petroleum ether-ethyl acetate solvent system. The eluent fractions were collected and combined after thin-layer chromatography to obtain seven primary fractions Fr.1-Fr.7. Fr.3 in the primary fractions Fr.1-Fr.7 was separated by reverse-phase column chromatography using a MeOH-H2O solvent system as the eluent for gradient elution. Each gradient elution consisted of 3-6 column volumes. The fractions were then collected, and identical components were combined to obtain 9 secondary fractions Fr.3.1-Fr.3.9. The Fr.3.3 fraction in the secondary fractions Fr.3.1-Fr.3.9 was subjected to gel column chromatography with MeOH solvent as the eluent for gradient elution. The eluent fractions were collected, and similar fractions were combined after thin-layer chromatography to obtain five tertiary fractions Fr.3.3A-Fr.3.3E. The triterpenoid compound was obtained by separating Fr.3.3B and Fr.3.3C from the tertiary fractions Fr.3.3A-Fr.3.3E using high performance liquid chromatography.

[0007] Furthermore, the alcohol solvent is an ethanol solution with a volume concentration of 75% to 95%.

[0008] Furthermore, the alcohol solvent is an ethanol solution with a volume concentration of 90%.

[0009] Furthermore, the mass-to-volume ratio of the soil-planted tree to the ethanol solution is 1 kg: 2.6 L to 5 L.

[0010] Furthermore, when the extract is dispersed in water and extracted with petroleum ether, the volume ratio of water to petroleum ether is 1:1.

[0011] Furthermore, when using the petroleum ether-ethyl acetate solvent system as the eluent, gradient elution is performed sequentially using volume ratios of petroleum ether to ethyl acetate of 100:1, 85:15, 70:30, 55:45, 30:70, 15:85, and 1:100.

[0012] Furthermore, when using the MeOH-H2O solvent system as the eluent, gradient elution is performed by sequentially using MeOH:H2O with volume ratios of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10.

[0013] Furthermore, the gel in the gel column is Sephadex LH-20, and a semi-automatic receiver is used to collect one fraction every 20 mL.

[0014] Furthermore, the parameters for the high-performance liquid chromatography are as follows: the chromatographic column is an Agilent Zorbax Eclipse XDBC. 18 The chromatographic column was 9.4 mm × 250 mm with a diameter of 5 μm; the flow rate was 2 mL / min; and the mobile phase was a mixture of MeCN and H2O.

[0015] Furthermore, when R in the general formula of the triterpenoid compound is hydroxymethyl, the volume ratio of MeCN to H2O in the mobile phase of the high performance liquid chromatography is 35:65; when R in the general formula of the triterpenoid compound is carboxyl, the volume ratio of MeCN to H2O in the mobile phase of the high performance liquid chromatography is 30:70.

[0016] Furthermore, the leaves in the earthen jar are extracted 1 to 3 times, and each extraction takes 3 to 5 days.

[0017] Furthermore, when using the petroleum ether-ethyl acetate solvent system as the eluent for gradient elution, the fraction is collected every 500 mL.

[0018] Furthermore, when using MeOH solvent as the eluent, the fraction is collected every 20 mL.

[0019] A third aspect of the present invention provides the use of the above-described triterpenoid compounds in the preparation of medicaments for the prevention or treatment of tumors, wherein the tumor is cervical cancer, gastric cancer, liver cancer, or leukemia.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a triterpenoid compound, its extraction and separation method, and its application. Using *Echinochloa crus-galli* as raw material, the invention employs ethanol extraction to obtain a primary extract, followed by dispersion in pure water and petroleum ether extraction to obtain a petroleum ether fraction extract. This petroleum ether fraction extract is then subjected to silica gel column chromatography, reversed-phase column chromatography, gel column chromatography, and high-performance liquid chromatography (HPLC) for separation, resulting in the first extraction and separation of multiflorane-type triterpenoid compounds as shown in formulas (I) and (II). Pharmacological studies have shown that the triterpenoid compounds provided by this invention exhibit strong inhibitory activity against the proliferation of various tumor cells. Specifically, compound I, represented by formula (I), shows an IC50 inhibitory effect on gastric cancer cells (SGC-7901) and human gastric mucosal epithelial cells (GES-1). 50 The values ​​were 0.70µM±0.19µM and 0.47µM±0.09µM, respectively, which were stronger than the positive control drug (the positive control drug, doxorubicin hydrochloride, showed IC50 against gastric cancer cells and human gastric mucosal epithelial cells). 50The values ​​are 0.77µM±0.02µM and 0.53µM±0.01µM, respectively, and can be used to prepare drugs for the prevention or treatment of tumors. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is the crystal structure diagram of compound I.

[0023] Figure 2 For compound I 1 H-NMR spectrum (CDCl3).

[0024] Figure 3 For compound I 13 C-NMR spectrum (CDCl3).

[0025] Figure 4 The image shows the DEPT (135°) spectrum (CDCl3) of compound I.

[0026] Figure 5 The image shows the HSQC spectrum (CDCl3) of compound I.

[0027] Figure 6 The image shows the HMBC spectrum (CDCl3) of compound I.

[0028] Figure 7 For compound I 1 H- 1 H COSY spectrum (CDCl3).

[0029] Figure 8 The NOESY spectrum (CDCl3) of compound I is shown.

[0030] Figure 9 This is the HRESIMS spectrum of compound I.

[0031] Figure 10 For compound II 1 H-NMR spectrum (CDCl3).

[0032] Figure 11 For compound II 13 C-NMR spectrum (CDCl3).

[0033] Figure 12The DEPT (135°) spectrum of compound II (CDCl3) is shown.

[0034] Figure 13 The HSQC spectrum (CDCl3) of compound II is shown.

[0035] Figure 14 The HMBC spectrum (CDCl3) of compound II is shown.

[0036] Figure 15 For compound II 1 H- 1 H COSY spectrum (CDCl3).

[0037] Figure 16 The NOESY spectrum (CDCl3) of compound II.

[0038] Figure 17 The image shows the HRESIMS spectrum of compound II. Detailed Implementation

[0039] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0040] Example 1: A method for separating triterpenoid compounds The general structural formula of this triterpenoid compound is: , where R is selected from either hydroxymethyl or carboxyl.

[0041] When R is hydroxymethyl, this compound is labeled as compound I, with the structural formula shown in formula (I); when R is carboxyl, this compound is labeled as compound II, with the structural formula shown in formula (II). .

[0042] A method for isolating triterpenoids includes the following steps: (1) After crushing 5.0 kg of leaves of the Tutan Tree, soak them in an ethanol solution with a volume concentration of 90% for 4 days. Repeat the soaking process 3 times. Combine the concentrated extracts to obtain 420 g of extract.

[0043] (2) Disperse the extract from step (1) in water, extract with petroleum ether, concentrate to obtain petroleum ether extract fraction, concentrate under reduced pressure to obtain 27.5 g of petroleum ether extract fraction.

[0044] (3) The petroleum ether fraction obtained in step (2) was subjected to silica gel column chromatography and gradient elution was performed using a petroleum ether-ethyl acetate solvent system. The fractions were collected every 500 mL, concentrated, and detected by thin-layer chromatography (TLC). The fractions containing the same components were combined to obtain 7 components Fr.1-Fr.7 with increasing polarity. Gradient elution was performed using a gradient of petroleum ether to ethyl acetate with volume ratios of 100:1, 85:15, 70:30, 55:45, 30:70, 15:85, and 1:100.

[0045] (4) The combined Fr.3 from step (3) was separated by reversed-phase silica gel column chromatography. The eluent was MeOH-H2O solvent. Gradient elution was performed according to the volume ratio of MeOH:H2O of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:10. After eluting for 5 column volumes for each gradient, the fractions were collected. After combining the same components, 9 components Fr.3.1-Fr.3.9 with polarity from large to small were obtained.

[0046] (5) The Fr.3.3 fractions combined in step (4) were subjected to gel column chromatography (Sephadex LH-20) with MeOH solvent as the eluent. The fractions were collected every 20 mL using a semi-automatic receiver. The fractions were analyzed by TLC thin layer analysis. Similar fractions were combined to obtain five fractions Fr.3.3A-Fr.3.3E with molecular weights from largest to smallest.

[0047] (6) The combined components Fr.3.3B and Fr.3.3C from step (5) were separated by high performance liquid chromatography (HPLC) to obtain the target extracts multiflorane-type triterpenoids I (28.8 mg) and II (35.8 mg). The parameters for high-performance liquid chromatography (HPLC) were as follows: Column: Agilent Zorbax Eclipse XDB C 18 The chromatographic column specifications were 9.4 mm × 250 mm, 5 μm; the flow rate was 2 mL / min; and the mobile phase was a mixed solution of MeCN and H2O. The volume ratio of MeCN to H2O in the mobile phase for compound I was 35:65, and the volume ratio of MeCN to H2O in the mobile phase for compound II was 30:70.

[0048] Using CDCl3 as a solvent, the structures of compounds I and II were identified, yielding the results for both compounds. 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) data, the specific results are shown in Tables 1 and 2.

[0049] Table 1 Table 2 is a continuation of Table 1. Compounds I and II were detected, and the results are as follows: Figures 1-17 .in Figure 1 Here is the crystal structure diagram of compound I; Figure 2 For compound I 1 H-NMR spectrum (CDCl3); Figure 3 For compound I 13 C-NMR spectrum (CDCl3); Figure 4 The DEPT (135°) spectrum (CDCl3) of compound I is shown. Figure 5 The HSQC spectrum (CDCl3) of compound I is shown. Figure 6 The HMBC spectrum (CDCl3) of compound I is shown. Figure 7 For compound I 1 H- 1 H COSY spectrum (CDCl3); Figure 8 The NOESY spectrum (CDCl3) of compound I; Figure 9 The HRESIMS spectrum of compound I; Figure 10 For compound II 1 H-NMR spectrum (CDCl3); Figure 11 For compound II 13 C-NMR spectrum (CDCl3); Figure 12 The DEPT (135°) spectrum of compound II (CDCl3); Figure 13 The HSQC spectrum (CDCl3) of compound II is shown. Figure 14 The HMBC spectrum (CDCl3) of compound II is shown. Figure 15 For compound II 1 H- 1 H COSY spectrum (CDCl3); Figure 16 The NOESY spectrum (CDCl3) of compound II is shown. Figure 17 The image shows the HRESIMS spectrum of compound II.

[0050] The structural identification results of compound I are as follows: Compound I is a colorless crystal, readily soluble in chloroform and methanol. High-resolution mass spectrometry (HR-ESI-MS) was used to determine its structure. m / z 443.3893 [M+H] + (calcd for C 30 H 50 O2H +(443.3889), its molecular formula is determined to be C 30 H 50 O2; according to 1 H, 13 Its structure was determined using C and two-dimensional NMR data, and its absolute configuration was determined to be 8 by single-crystal diffraction. E ,3 R 5 R 10 S ,13 S ,14 S 17 S 18 S Compound I was identified as a multiflorane-type triterpenoid and named 3α-hydroxymultiflora-8-en-17-hydroxymethyl. 1 H and 13 The attribution of C NMR data is shown in Tables 1 and 2.

[0051] The structural identification results of compound II are as follows: Compound II is a colorless oily substance, readily soluble in methanol. High-resolution mass spectrometry (HR-ESI-MS) was used to identify its structure. m / z 479.3490 [M+Na] + (calcd for C 30 H 48 O3Na + (479.3496), its molecular formula was determined to be C 30 H 48 O3; according to 1 H, 13 Its structure was determined by C10 and two-dimensional NMR data, classifying it as a multiflorane-type triterpene, and it was named 3α-hydroxymultiflora-8-en-17-oic acid. 1 H and 13 The C NMR data attribution is shown in Tables 1 and 2.

[0052] Experimental Example 1: Evaluation of the in vitro antitumor activity of compounds I and II 1. Experimental Materials Cells: Human liver cancer cells (Hep G2), human liver cancer cells (BEL-7402), human gastric cancer cells (SGC-7901), human chronic myeloid leukemia cells (K562), human lung cancer cells (A549), human cervical cancer cells (Hela), GES-1 (human gastric mucosal cells), B16 (mouse melanoma cells).

[0053] Reagents: DMEM basic (1X) gibco culture medium, fetal bovine serum (Sijiqing), trypsin (solarbio).

[0054] 2. Experimental Methods The inhibitory activity of the compound against tumor cell growth was determined using the MTT assay. 100 µL of a 1×10⁻⁶ compound was added to each well of a 96-well plate. 4 Cells were cultured at 37°C, 5% CO2, and above 90% humidity for 24 h, with a cell density of 1 / mL. Then, 100 μL of culture medium containing the test compound (doxorubicin hydrochloride was used as a positive control) was added. The cells were cultured for another 48 h. Afterward, 15 μL of 5 mg / mL MTT solution was added to each well, and the reaction was carried out at 37°C for 4 h. The supernatant was then aspirated, and 100 μL of LDMSO was added to each well to dissolve the cells completely. The OD value of each well was measured at 490 nm. The formula for calculating the cell growth inhibition rate is as follows:

[0055] Inhibition rate (%) = (1 - B / B0) × 100%.

[0056] In the formula, B0 and B are the absorbance values ​​of the blank group and the experimental group measured at a wavelength of 490 nm, respectively. The compound to be tested was serially diluted sigmatrix, and the inhibition rate at each concentration was determined according to the above steps. The half-maximal inhibitory concentration (IC50) of the compound was obtained by processing the data using GraphPad Prism software. 50 value).

[0057] The results are shown in Table 3. Compounds I and II exhibited cytotoxicity against various tumor cells, with compound I showing the greatest cytotoxicity against BEL-7402 (IC50). 50 The value is 1.81 μM ± 0.01 μM) and SGC-7901 (IC 50 Compound II (with a concentration of 0.70 μM ± 0.19 μM) exhibited inhibitory activity against cell proliferation comparable to the positive control. Furthermore, compound II showed good cytotoxic activity against various tumor cell types (IC50 ± 0.19 μM). 50 The values ​​range from 1.90 μM to 4.13 μM.

[0058] Table 3. Cytotoxicity assays of the compounds (μM) Note: a This represents the mean ± standard deviation of three parallel experiments. b This indicates a positive control (doxacin hydrochloride). It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0059] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A triterpenoid compound, characterized in that, The general structural formula of the triterpenoid compound is: ; R is selected from either hydroxymethyl or carboxyl groups.

2. A method for separating triterpenoid compounds according to claim 1, characterized in that, Includes the following steps: The leaves of the leaves of the *Tutan* tree were extracted using an alcohol solvent, and the resulting extract was concentrated to obtain a paste. The extract was dispersed in water, extracted with petroleum ether, and concentrated to obtain the petroleum ether extract fraction. The petroleum ether extract was subjected to silica gel column chromatography with a gradient elution using a petroleum ether-ethyl acetate solvent system. The eluent fractions were collected and combined after thin-layer chromatography to obtain seven primary fractions Fr.1-Fr.

7. Fr.3 in the primary fractions Fr.1-Fr.7 was separated by reverse-phase column chromatography using a MeOH-H2O solvent system as the eluent for gradient elution. Each gradient elution consisted of 3-6 column volumes. The fractions were then collected and combined with the same components to obtain 9 secondary fractions Fr.3.1-Fr.3.

9. The Fr.3.3 fraction in the secondary fractions Fr.3.1-Fr.3.9 was subjected to gel column chromatography with MeOH solvent as the eluent for gradient elution. The eluent fractions were collected, and similar fractions were combined after thin-layer chromatography to obtain five tertiary fractions Fr.3.3A-Fr.3.3E. The triterpenoid compound was obtained by separating Fr.3.3B and Fr.3.3C from the tertiary fractions Fr.3.3A-Fr.3.3E using high performance liquid chromatography.

3. The method for separating triterpenoid compounds according to claim 2, characterized in that, The alcohol solvent is an ethanol solution with a volume concentration of 75% to 95%; the mass-to-volume ratio of the soil tree to the ethanol solution is 1 kg: 2.6 L to 5 L.

4. The method for separating triterpenoid compounds according to claim 2, characterized in that, When the extract is dispersed in water and extracted with petroleum ether, the volume ratio of water to petroleum ether is 1:

1.

5. The method for separating triterpenoids according to claim 2, characterized in that, When using the petroleum ether-ethyl acetate solvent system as the eluent, gradient elution is performed sequentially using volume ratios of petroleum ether to ethyl acetate of 100:1, 85:15, 70:30, 55:45, 30:70, 15:85, and 1:

100.

6. The method for separating triterpenoid compounds according to claim 2, characterized in that, When using the MeOH-H2O solvent system as the eluent, gradient elution is performed sequentially using MeOH:H2O volume ratios of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, and 90:

10.

7. The method for separating triterpenoids according to claim 2, characterized in that, The gel column used Sephadex LH-20 gel, and a semi-automatic receiver was used to collect one fraction every 20 mL.

8. The method for separating triterpenoids according to claim 2, characterized in that, The parameters for the high-performance liquid chromatography (HPLC) were as follows: the chromatographic column was an Agilent Zorbax Eclipse XDB C. 18 The chromatographic column was 9.4 mm × 250 mm with a diameter of 5 μm; the flow rate was 2 mL / min; and the mobile phase was a mixture of MeCN and H2O.

9. The method for separating triterpenoids according to claim 8, characterized in that, When R in the general formula of a triterpenoid compound is hydroxymethyl, the volume ratio of MeCN to H2O in the mobile phase of the high-performance liquid chromatography is 35:65; when R in the general formula of a triterpenoid compound is a carboxyl group, the volume ratio of MeCN to H2O in the mobile phase of the high-performance liquid chromatography is 30:

70.

10. The use of the triterpenoid compound of claim 1 in the preparation of a medicament for the prevention or treatment of tumors, characterized in that, The tumor is cervical cancer, stomach cancer, liver cancer, or leukemia.

Images