Cucurbitane triterpenoid glycosides, and preparation method and application thereof

Abstract

The application discloses cucurbitane triterpene glycosides and a preparation method and application thereof, and relates to the technical field of medicines. The application takes mature fruits of Citrullus colocynthis as raw materials, and adopts petroleum ether, ethyl acetate and n-butanol for fractional extraction of a methanol-extracted extractive, and separates the cucurbitane triterpene glycosides shown in formula I-II through MCI column chromatography, reverse-phase silica gel column chromatography, Sephadex gel column chromatography and semi-preparative liquid chromatography. The application also provides application of the cucurbitane triterpene glycosides in preparation of anticancer drugs. The application efficiently and highly purifies two cucurbitane triterpene glycosides with high content from traditional Chinese medicine, Citrullus colocynthis, finds that the two cucurbitane triterpene glycosides have inhibitory effects on various cancer cell lines, and can be applied to preparation of anticancer drugs, especially anticancer drugs for lung cancer, and can be used as a potential lead compound for treatment of human non-small cell lung cancer.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a cucurbitane-type triterpenoid glycoside compound, its preparation method, and its application. Background Technology

[0002] Medicinal watermelon is a plant belonging to the genus *Citrus* of the Cucurbitaceae family. Citrullus colocynthis The ripe fruit of *L.) Schrad., also known as bitter apple, is a vine widely distributed in desert regions. In traditional medicine, medicinal watermelon has a long history of use and is a commonly used clinical medicine by Uyghur doctors, widely applied in the treatment of diabetes, antibacterial diseases, asthma, bronchitis, and gastrointestinal disorders. Systematic studies of its chemical composition have shown that the main components of medicinal watermelon include cucurbitacin, alkaloids, flavonoids, and steroidal saponins, among which cucurbitacin compounds are the main active substances exerting its pharmacological effects. Modern pharmacological studies have shown that cucurbitacin possesses a wide range of biological activities, including cytotoxicity, anti-inflammatory, anti-diabetic, and antibacterial effects. Therefore, the efficient isolation of pharmacologically active cucurbitane-type triterpenoid compounds from medicinal watermelon is of great significance.

[0003] Malignant tumors are a serious threat to human health, ranking among the leading causes of death worldwide and becoming a major public health problem for countries around the world. Statistics show that tens of millions of new cancer patients are diagnosed globally each year, and the incidence rate continues to rise, placing enormous pressure on medical resources and socioeconomic development in various countries. The core pathological mechanism of malignant tumors lies in the accumulation of mutations in the cell genome, leading to abnormal cell cycle regulation, inhibited apoptosis, and the acquisition of unlimited proliferation capacity, thereby forming a malignant phenotype characterized by invasive growth and distant metastasis, destroying normal tissue structure and organ function. Clinical manifestations vary depending on the type, location, and stage of the tumor. Common symptoms include local mass, pain, weight loss, fatigue, and corresponding organ dysfunction. Late-stage patients often experience multiple organ failure, severely threatening their quality of life and life expectancy. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a cucurbitane-type triterpenoid glycoside compound, its preparation method and application, specifically involving the extraction and separation of two cucurbitane-type triterpenoid glycoside compounds rich in watermelon, a medicinal plant, and their preparation method and application.

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a cucurbitane-type triterpenoid glycoside compound having one of the following structural formulas: Formula I; Formula II.

[0006] Secondly, the present invention provides a method for preparing the aforementioned cucurbitane-type triterpenoid glycosides, comprising the following steps: S1. Soak ripe and dried watermelon fruit in methanol, combine the extracts and concentrate to obtain an extract. S2. The extract was sequentially fractionally extracted with petroleum ether and ethyl acetate to obtain the ethyl acetate extract. S3. The extract from the ethyl acetate fraction is separated by MCI column chromatography to obtain a first fraction; the first fraction is separated by reversed-phase silica gel column chromatography to obtain a second fraction; the second fraction is separated by Sephadex gel column chromatography to obtain a third fraction; the third fraction is separated by semi-preparative high-performance liquid chromatography to obtain the cucurbitane-type triterpenoid glycosides.

[0007] Optionally, in S1, the mature and dried fruit of the medicinal watermelon is extracted with 100% methanol at room temperature 3 to 5 times, each time for 6 to 7 days, and the ratio of the mature and dried fruit of the medicinal watermelon to 100% methanol is 4 g: 10 to 14 mL. In S2, each solvent is extracted 4 to 6 times during fractional extraction, and the extracts are concentrated under reduced pressure to obtain the corresponding extracts; the ratio of mature dried watermelon fruit to petroleum ether and ethyl acetate is 40 g: 4~8 mL: 4~8 mL. In step S3, the extract from the ethyl acetate fraction was subjected to gradient elution using MCI column chromatography to obtain eight fractions, Fr.A to Fr.H, with the first fraction being Fr.E. The Fr.E fraction was then subjected to gradient elution using reversed-phase silica gel column chromatography to obtain nine fractions, Fr.E12a to Fr.E12i, with the second fraction being Fr.E12g. The Fr.E12g fraction was purified using Sephadex gel column chromatography to obtain the third fraction, Fr.E12g1B. The third fraction, Fr.E12g1B, was then separated using semi-preparative high-performance liquid chromatography to obtain the cucurbitane-type triterpenoid glycosides.

[0008] Optionally, in S3, The MCI column chromatography method uses MCI small-pore resin as the packing material and methanol-water rapid gradient elution at a volume concentration of 20% to 100%. Reversed-phase column chromatography was performed using reversed-phase C18 silica gel as the packing material, with elution via a methanol-water system at a gradient concentration of 40%–100% by volume. Sephadex gel column chromatography, using methanol as the eluent for isocratic elution.

[0009] Optionally, in S3, The semi-preparative HPLC used a YMC-Pack ODS-A column with a particle size of 5 μm and a specification of 250 × 10 mm, with isocratic elution using 69% methanol-water as the mobile phase and a flow rate of 3 mL / min.

[0010] Optionally, in S3, the retention time of the cucurbitane-type triterpenoid glycosides represented by formulas I and II is... t R The maximum UV absorption λ values ​​for cucurbitane-type triterpenoid glycosides represented by Formulas I and II are 16.6 min and 19.4 min, respectively. max They are 292nm and 298nm respectively.

[0011] Thirdly, the present invention provides the application of the cucurbitane-type triterpenoid glycosides or the cucurbitane-type triterpenoid glycosides obtained by the preparation method described above in the preparation of anticancer drugs.

[0012] Optionally, the anticancer drug includes an anti-lung cancer drug.

[0013] Optionally, the anti-lung cancer drug includes drugs for treating human non-small cell lung cancer.

[0014] Optionally, the application is the use of cucurbitane-type triterpenoid glycosides of Formula II in the preparation of anti-lung cancer drugs, wherein the cucurbitane-type triterpenoid glycosides of Formula II have an IC50 effect on the A549 cancer cell line. 50 It was 9.84 ± 0.26 μmol / L.

[0015] Optionally, the high-performance liquid chromatography (HPLC) detection uses a Hitachi Primaide HPLC system equipped with a 1210 quaternary solvent management system, a 1220 autosampler, a 1230 column temperature management unit, and a 1430 diode array detection system.

[0016] This invention has at least one of the following beneficial effects: 1. This invention provides a highly efficient and pure method for isolating and preparing two abundant cucurbitane-type triterpenoid glycosides from the traditional Uyghur medicinal watermelon. The compounds were obtained from the crude extract of mature watermelon fruit after methanol extraction using Sephadex gel column chromatography, ODS column chromatography, MCI column chromatography, and semi-preparative liquid chromatography. Identification confirmed that the compounds possess the structures shown in Formulas I and II, classifying them as cucurbitane-type triterpenoids. Specifically, this invention systematically separated and purified eight fractions (Fr.A to Fr.H) obtained from the ethyl acetate extract after gradient elution using MCI column chromatography. During the separation of the Fr.E fraction, two monomeric compounds with relatively high content were obtained. These compounds were then further purified by semi-preparative high-performance liquid chromatography (HPLC), and their structures were confirmed by spectroscopic methods to be cucurbitane-type triterpenoid glycosides. Therefore, this invention obtains cucurbitane-type triterpenoid glycosides by separating and purifying each fraction individually during the separation of the Fr.E fraction, rather than by selecting the Fr.E fraction through prior screening and comparison.

[0017] 2. Subsequent in vitro activity screening revealed that the two compounds exhibited certain inhibitory activity against non-small cell lung cancer cells. Specifically, the cucurbitane-type triterpenoids extracted in this invention, as shown in Formulas I and II, showed inhibitory effects on three cancer cell lines: A549, A375, and HepG2. Among them, the cucurbitane-type triterpenoid glycosides shown in Formula II showed an inhibitory effect on the IC50 of the A549 cancer cell line. 50 The concentration was 9.84 ± 0.26 μmol / L, which was superior to the inhibitory effect of doxorubicin. Therefore, the cucurbitane-type triterpenoids isolated from the ethyl acetate extract of the methanol extract of watermelon can be used to prepare anticancer drugs, especially anti-lung cancer drugs, and can serve as a potential lead compound for the treatment of human non-small cell lung cancer. Attached Figure Description

[0018] Figure 1 This is a liquid chromatogram of compounds I-II extracted at the optimal wavelength during preparation in Example 1 of this invention; Figure 2 This is the high-resolution mass spectrum of compound I in Example 1 of the present invention; Figure 3 This is the high-resolution mass spectrum of compound II in Example 1 of the present invention; Figure 4 Compound I in one embodiment of the present invention 1 1H NMR spectrum (400 MHz, DMSO- d 6); Figure 5 Compound I in Example 1 of this invention 13 C10 NMR spectrum (100 MHz, DMSO-) d6); Figure 6 Compound I in Example 1 of this invention 1 H- 1 H COSY spectrum; Figure 7 The HSQC spectrum of compound I in Example 1 of this invention; Figure 8 The HMBC spectrum of compound I in Example 1 of this invention; Figure 9 The NOESY spectrum of compound I in Example 1 of this invention; Figure 10 Compound II in Example 1 of this invention 1 1H NMR spectrum (400 MHz, DMSO- d 6); Figure 11 Compound II in Example 1 of this invention 13 C10 NMR spectrum (100 MHz, DMSO-) d 6) Figure 12 Compound II in Example 1 of this invention 1 H- 1 H COSY spectrum; Figure 13 The HSQC spectrum of compound II in Example 1 of this invention; Figure 14 The HMBC spectrum of compound II in Example 1 of this invention; Figure 15 The NOESY spectrum of compound II in Example 1 of this invention; Figure 16 This invention relates to the detection of the anti-inflammatory activity of compounds I and II in Example 1 of the present invention. Figure 17 This is the chromatogram of compound I in Example 1 of the present invention for high performance liquid chromatography purity analysis; Figure 18 This is a high-performance liquid chromatography (HPLC) chromatogram of compound II in Example 1 of the present invention for purity analysis. Detailed Implementation

[0019] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and 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.

[0020] Example 1: Extraction and separation of socotroside and deoxocucurbitoside B The dried fruit of the medicinal watermelon (4 kg) was crushed and repeatedly extracted with 12 L of 100% methanol at room temperature 4 times, each extraction lasting 7 days. After the solvent was concentrated under reduced pressure, an extract of about 343.8 g was obtained.

[0021] The extract was dispersed in 1 L of water to form a suspension, and then extracted sequentially with 0.6 L of petroleum ether, 0.6 L of ethyl acetate, and 0.6 L of n-butanol. Specifically: First, 0.6 L of petroleum ether was added, shaken thoroughly, and allowed to stand. The petroleum ether layer (upper layer, removing non-polar lipid-soluble components) was separated. Second, the lower aqueous phase was retained, and 0.6 L of ethyl acetate was added, shaken, and allowed to stand. The ethyl acetate layer (upper layer, depending on density) was separated. Third, the lower aqueous phase was retained again, and finally 0.6 L of n-butanol was added, shaken, and allowed to stand. The n-butanol layer (upper layer) was separated. The extract was concentrated under reduced pressure to obtain the corresponding extract fraction.

[0022] The ethyl acetate extract (85.4 g) was subjected to MCI small-pore resin column chromatography with a particle size of 75–150 μm. Gradient elution was performed using a methanol-water system (v:v, 20%–100%), and the eluent was collected fractionally in 250 mL Erlenmeyer flasks. Each fraction was analyzed by TLC (developing solvent: DCM:MeOH = 10:1, v:v), and the fractions were combined. f Similar fractions with similar values ​​yielded a total of 8 fractions (Fr.A-Fr.H).

[0023] Eight fractions, Fr.A to Fr.H, obtained from the ethyl acetate extract by gradient elution using MCI column chromatography, were systematically separated and purified sequentially. During the separation of the Fr.E fraction, two relatively abundant monomeric compounds were obtained, which were further purified by semi-preparative high-performance liquid chromatography (HPLC) and further purified by spectroscopic methods (such as ¹H NMR). 13 C-NMR, HRESIMS, etc. confirmed that its structure is a cucurbitane-type triterpenoid glycoside compound. The specific separation method is as follows: The Fr.E fractions were graded eluted using ODS (methanol-water, v:v, 40%-100%, particle size 50 μm), and the eluent was collected in 20 mL test tubes. Each fraction was analyzed by HPLC using a 250 × 4.6 mm, 5 μm YMC-Pack ODS-A column (analytical method: MeOH:H2O, v:v, 60:40-80:20, 15 min, 80:20-100:0, 5 min, 100:0-100:0, 6 min, 0.75 mL / min). Fractions with similar retention times were combined to obtain nine fractions, Fr.E12a-Fr.E12i. These nine fractions were then subjected to systematic separation and purification studies. The results showed that Fr.E12i contained cucurbitane-type triterpenoid glycosides. The specific separation method is as follows: The fractions obtained from Fr. E12g by purification and separation via Sephadex LH-20 gel column chromatography (methanol) were collected using an automated collector. Each fraction was analyzed by HPLC using a 250 × 4.6 mm, 5 μm YMC-Pack ODS-A column (analytical method: MeOH:H2O, v:v, 60:40-80:20, 15 min, 80:20-100:0, 5 min, 100:0-100:0, 6 min, 0.75 mL / min). Fractions with similar retention times were combined to obtain fraction Fr. E12g1B, which was then separated from Fr. E12g1B by semi-preparative liquid chromatography (69% methanol-water) to obtain compound I (70.2 mg). t R =16.6 min) and compound II (84 mg, t R =19.4 min) Figure 1 The semi-preparative HPLC used a YMC-Pack ODS-A column with a particle size of 5 μm and a specification of 250 × 10 mm, with 69% methanol-water as the mobile phase for isocratic elution at a flow rate of 3 mL / min.

[0024] Example 2: Spectral data of socotroside and deoxocucurbitoside B The structures of compounds I and II were determined by combining high-resolution mass spectrometry data with NMR data from relevant literature.

[0025] Compound I was identified as socotroside, and compound II was identified as deoxocucurbitoside B. The structural formulas of the isolated socotroside and deoxocucurbitoside B are shown below: Compound I and Compound II 1 H / 13 C NMR (400 MHz, DMSO-) d 6) The data are shown in Table 1 below, and the corresponding 1D / 2D NMR spectra are shown in Table 1 below. Figures 4-15 The chromatograms for high performance liquid chromatography purity analysis of compounds I and II are shown below. Figures 17-18 .

[0026] Table 1 Key information for compounds I-II 1 H / 13 C NMR data Example 3: Anti-inflammatory activity of socotroside and deoxocucurbitoside B Given the traditional use of watermelon in treating inflammatory diseases, this invention, after obtaining compounds I and II, first conducted a preliminary investigation into their anti-inflammatory activity. To avoid false positives due to cytotoxicity interfering with the evaluation results of anti-inflammatory activity, the applicant first used the CCK-8 assay to determine the cytotoxicity of compounds I and II on RAW264.7 cells, as follows: First, CCK is used. The effects of compounds I and II on the viability of mouse macrophages RAW264.7 were assessed using an 8-method approach. RAW264.7 cells in good growth condition were harvested and subjected to approximately 2 × 10⁸ cells per cell. 4 Seeds were planted at a density of cells / well in 96-well plates and incubated at a constant temperature for 24 h. The supernatant was removed, and RAW264.7 cells were incubated for another 24 h with 40 μmol / L complete culture medium containing the sample. Six replicates were set up for each sample. 100 μL of medium containing 10% CCK was added. Continue incubation in complete culture medium 8 for 2–4 h, then measure the absorbance at 450 nm using a microplate reader. A The result is as follows: Figure 16 As shown, compounds I and II at 40 μmol / L exhibited significant cytotoxicity, with survival rates of 14.2% and 20.3% for RAW264.7 cells, respectively.

[0027] Therefore, the above results show that the survival rates of compounds I and II against RAW264.7 cells at a concentration of 40 μmol / L were only 14.2% and 20.3%, respectively, indicating that both compounds exhibited significant cytotoxicity at this concentration. Since excessive cytotoxicity would distort the results of subsequent anti-inflammatory experiments based on cell viability assays (such as the measurement of NO release and inflammatory factor secretion), further evaluation of anti-inflammatory activity was not conducted based on these results.

[0028] Given the significant cytotoxicity exhibited by compounds I-II, the applicant further speculated that they might have potential cytotoxicity-related pharmacological activities (such as antitumor activity), and thus turned to screening and evaluating the antitumor activity of compounds I-II. Specifically, relevant experiments were carried out on A549, A375, and HepG2 cells. The results showed that compound II had good inhibitory activity against non-small cell lung cancer cells.

[0029] Example 4: Anticancer activity of socotroside and deoxocucurbitoside B Given the significant cytotoxicity exhibited by compounds I-II, the applicant further speculated that they might possess potential cytotoxicity-related pharmacological activities (such as antitumor activity), and thus shifted to screening and evaluating the antitumor activity of compounds I-II. Specifically, relevant experiments were conducted on A549, A375, and HepG2 cells. The results showed that compound II had good inhibitory activity against non-small cell lung cancer cells. The specific methods are as follows: Given the significant cytotoxicity exhibited by compounds I-II in Example 3, the cytotoxicity of compounds I-II against three cancer cell lines, A549, A375, and HepG2, was evaluated using the CCK-8 assay.

[0030] Take A549, A375, and HepG2 cells in good growth condition, at a ratio of approximately 1 × 10⁻⁶. 4 Seeds were planted at a density of cells / well in 96-well plates and incubated at 37°C with 5% CO2 for 24 h. The supernatant was removed, and the test samples, serially diluted with different concentrations of culture medium, were added to A549, A375, and HepG2 cells for further incubation for 24 h. Six replicates were set for each sample. Afterwards, 100 μL of a solution containing 10% CCK was added. Continue incubation in complete culture medium 8 for 2–4 h, then measure the absorbance at 450 nm using a microplate reader. A The same method was used to set up a positive control.

[0031] As shown in Table 2, the results indicate that compound II exhibited superior inhibitory effects against A549 cells compared to doxorubicin, while the inhibitory effects of compounds I-II on A375 and HepG2 cells were weaker than those of the positive control. Based on these results, compound II, isolated from the ethyl acetate extract of the methanol extract of watermelon, can serve as a potential lead compound for the treatment of human non-small cell lung cancer.

[0032] Table 2. Inhibitory effects of compounds on the proliferation of A549, A375, and HepG2 cells. The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A cucurbitane-type triterpenoid glycoside compound, characterized in that, It has one of the following structural formulas: Equation I; Formula II.

2. The method for preparing the cucurbitane-type triterpenoid glycoside compound according to claim 1, characterized in that, Includes the following steps: S1. Soak ripe and dried watermelon fruit in methanol, combine the extracts and concentrate to obtain an extract. S2. The extract was sequentially fractionally extracted with petroleum ether and ethyl acetate to obtain the ethyl acetate extract. S3. The extract from the ethyl acetate fraction is separated by MCI column chromatography to obtain a first fraction; the first fraction is separated by reversed-phase silica gel column chromatography to obtain a second fraction; the second fraction is separated by Sephadex gel column chromatography to obtain a third fraction; the third fraction is separated by semi-preparative high-performance liquid chromatography to obtain the cucurbitane-type triterpenoid glycosides.

3. The preparation method according to claim 2, characterized in that, In S1, the mature and dried fruit of the medicinal watermelon was extracted with 100% methanol at room temperature 3 to 5 times, each time for 6 to 7 days. The ratio of the mature and dried fruit of the medicinal watermelon to 100% methanol was 4 g: 10 to 14 mL. In S2, each solvent is extracted 4 to 6 times during fractional extraction, and the extracts are concentrated under reduced pressure to obtain the corresponding extracts; the ratio of mature dried watermelon fruit to petroleum ether and ethyl acetate is 40 g: 4~8 mL: 4~8 mL. In step S3, the extract from the ethyl acetate fraction was subjected to gradient elution using MCI column chromatography to obtain eight fractions, Fr.A to Fr.H, with the first fraction being Fr.E. The Fr.E fraction was then subjected to gradient elution using reversed-phase silica gel column chromatography to obtain nine fractions, Fr.E12a to Fr.E12i, with the second fraction being Fr.E12g. The Fr.E12g fraction was purified using Sephadex gel column chromatography to obtain the third fraction, Fr.E12g1B. The third fraction, Fr.E12g1B, was then separated using semi-preparative high-performance liquid chromatography to obtain the cucurbitane-type triterpenoid glycosides.

4. The preparation method according to claim 2, characterized in that, In S3, The MCI column chromatography method uses MCI small-pore resin as the packing material and methanol-water rapid gradient elution at a volume concentration of 20% to 100%. Reversed-phase column chromatography was performed using reversed-phase C18 silica gel as the packing material, with elution via a methanol-water system at a gradient concentration of 40%–100% by volume. Sephadex gel column chromatography, using methanol as the eluent for isocratic elution.

5. The preparation method according to claim 2, characterized in that, In S3, The semi-preparative HPLC used a YMC-Pack ODS-A column with a particle size of 5 μm and a specification of 250 × 10 mm, with isocratic elution using 69% methanol-water as the mobile phase and a flow rate of 3 mL / min.

6. The preparation method according to claim 5, characterized in that, In S3, the retention time of cucurbitane-type triterpenoid glycosides represented by formulas I and II t R The maximum UV absorption λ values ​​for the cucurbitane-type triterpenoid glycosides represented by Formulas I and II are 16.6 min and 19.4 min, respectively. max The corresponding nm values ​​are 292 nm and 298 nm, respectively.

7. The use of the cucurbitane-type triterpenoid glycoside compound according to claim 1 or the cucurbitane-type triterpenoid glycoside compound obtained by any of the preparation methods according to claims 2 to 6 in the preparation of anticancer drugs.

8. The application according to claim 7, characterized in that, The anticancer drugs include anti-lung cancer drugs.

9. The application according to claim 8, characterized in that, The anti-lung cancer drugs include those for treating human non-small cell lung cancer.

10. The application according to claim 7, characterized in that, The application refers to the use of cucurbitane-type triterpenoid glycosides represented by Formula II in the preparation of anti-lung cancer drugs. The cucurbitane-type triterpenoid glycosides represented by Formula II exhibit IC50 activity against the A549 cancer cell line. 50 It was 9.84 ± 0.26 μmol / L.

Images