A triterpenoid compound, and a preparation method and application thereof

Abstract

The application discloses a triterpenoid compound, and the structure is (3β, 6α, 10α, 12α, 16β, 24E)-4-methylol-6, 12-bis(acetyloxy)-3, 10, 16-trihydroxy-9, 19-cyclo-9, 10-secolanosta-9(11), 24-dien-26-yl β-D-glucopyranoside. The application obtains a new monomer compound with anticancer activity by separating and screening monomer components in leaf of Astragalus membranaceus Bge. var. mongholicus (Regel) H. The application provides a theoretical basis for further in-depth research on components and medicinal activities of the leaf of Astragalus membranaceus Bge. var. mongholicus (Regel) H, and also expands natural plant resources of anticancer drugs.

Description

Technical Field

[0001] This invention relates to the field of natural product chemistry, and specifically to a novel triterpenoid compound. Background Technology

[0002] Colorectal cancer is a common gastrointestinal malignancy characterized by the storage, fermentation, absorption, secretion, and motility of the colon, primarily occurring at the junction of the rectum and sigmoid colon. Currently, treatment for colorectal cancer mainly relies on surgery, chemotherapy, radiotherapy, and biological and targeted therapies. In recent years, the research and development of traditional Chinese medicine chemical components with anti-cancer activity has become a new avenue for developing effective treatments, attracting widespread attention due to its fewer side effects, multiple targets, and low likelihood of inducing drug resistance. The selection of natural, low-toxicity, and safe traditional Chinese medicines for cancer prevention and treatment is highly attractive and will provide new pathways and methods for the research and prevention of the increasingly serious disease of colorectal cancer.

[0003] Astragalus membranaceus (Fish.) Bge., a legume, is a traditional Chinese medicine. It is the dried root of the plant and is used to invigorate qi and raise yang, strengthen the body's defensive qi, promote diuresis and reduce swelling, and promote tissue regeneration. It is widely cultivated and grown in my country. The triterpenoid saponins and flavonoids it contains are widely recognized as the effective components of Astragalus membranaceus. Currently, there is limited research on the chemical composition and pharmacological effects of Astragalus membranaceus leaves. Existing studies indicate that the saponin composition in Astragalus membranaceus leaves is similar to that in the root, with the total saponin content being 5.6 times that of the root. Astragalus membranaceus leaves possess anti-inflammatory, hepatoprotective, and anti-aging effects.

[0004] However, existing activity studies mostly focus on the activity of total extracts of triterpenoid saponins, with very few studies on the activity of monomeric compounds. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and expand the range of drugs with anticancer activity by studying the monomeric components in Astragalus membranaceus.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A triterpenoid compound with the following structural formula:

[0008]

[0009] The present invention also provides a method for preparing the above-mentioned triterpenoids, wherein the triterpenoids are obtained by extraction and separation from the leaves of Astragalus membranaceus.

[0010] Further, in some embodiments, as a preferred embodiment, the preparation method of the triterpenoid compounds of the present invention includes the following steps: extracting Astragalus membranaceus leaves by heating and reflux with pure methanol, filtering, and concentrating the extract under reduced pressure to obtain an extract; dissolving the extract in water suspension, and extracting it sequentially with petroleum ether and water-saturated n-butanol, and concentrating the n-butanol extract under reduced pressure to obtain an n-butanol layer; dissolving the n-butanol layer in water-saturated n-butanol, adjusting the pH to 12 with 0.1% NaOH aqueous solution, extracting with equal volumes, and concentrating the n-butanol layer under reduced pressure to obtain an extract, thereby obtaining an n-butanol base layer; subjecting the n-butanol base layer to normal-phase silica gel column chromatography, eluting with a gradient of dichloromethane-methanol solvent system, analyzing by thin-layer chromatography (TLC) and HPLC, and combining the same fractions; then separating by reverse-phase ODS column chromatography, eluting with a gradient of methanol-water system, repeatedly separating by preparative HPLC column chromatography, eluting with methanol-water at a ratio of 54:46, to obtain the triterpenoid compounds.

[0011] This invention also provides the application of the above-mentioned triterpenoids in the preparation of cancer treatment drugs.

[0012] In some embodiments, as a preferred embodiment, the above-mentioned triterpenoid compounds are used in the preparation of drugs for treating colon cancer.

[0013] The present invention has the following advantages over the prior art:

[0014] This invention, through the isolation and activity screening of monomeric components in Astragalus membranaceus leaves, obtained new monomeric compounds with anticancer activity, providing a theoretical basis for further in-depth research on the components and medicinal activities of Astragalus membranaceus, and also expanding the natural plant resources for anticancer drugs. Attached Figure Description

[0015] Figure 1 The structural formula of the triterpenoid compound of this invention is shown below;

[0016] Figure 2 This is the mass spectrum of the triterpenoid compounds of this invention;

[0017] Figure 3 The hydrogen spectrum of the triterpenoid compounds of this invention;

[0018] Figure 4 The carbon spectra of the triterpenoid compounds of this invention are shown below.

[0019] Figure 5 The HSQC spectra of the triterpenoid compounds of this invention are shown below.

[0020] Figure 6 The HMBC spectrum of the triterpenoid compounds of this invention is shown below.

[0021] Figure 7 The triterpenoid compounds of this invention 1 H-1 H COSY spectrum;

[0022] Figure 8 The NOESY spectrum of the triterpenoid compounds of this invention is shown below.

[0023] Figure 9 This is the structural formula of the compound huangqiyenin G. Detailed Implementation

[0024] The present invention will now be described in detail with reference to specific embodiments.

[0025] Example 1

[0026] Preparation of triterpenoids in this invention

[0027] 5.00 kg of Astragalus membranaceus leaves were extracted three times by reflux with an appropriate amount of pure methanol for 2 hours each time. The extracts were filtered through gauze, and all extracts were combined and concentrated under reduced pressure to obtain an extract (500.00 g). The extract was dissolved in 10 L of water and extracted successively with petroleum ether and water-saturated n-butanol. The petroleum ether layer (171.57 g) and the n-butanol layer (296.35 g) were concentrated under reduced pressure respectively. The n-butanol layer was dissolved in 4 L of water-saturated n-butanol, and the pH was adjusted to 12 with 0.1% NaOH aqueous solution. The extracts were extracted three times with equal volumes. The aqueous and n-butanol layers were combined. The n-butanol layer was concentrated under reduced pressure to obtain an extract (105.00 g), named the n-butanol base layer. The combined aqueous layer was adjusted to pH 2-3 with 2% HCl aqueous solution and extracted three times with water-saturated n-butanol. The n-butanol layer was concentrated under reduced pressure to obtain an extract (153.00 g), named the n-butanol acid layer. 86.00 g of the n-butanol base layer was subjected to normal-phase silica gel column chromatography, eluted with a gradient of dichloromethane-methanol (20:1-10:1-5:1-3:1-2:1-1:1-0:1) solvent system. Thin-layer chromatography (TLC) and HPLC were performed, and fractions of the same type were combined to obtain Fr.1 to Fr.11, a total of 11 fractions. Fr.7 (12.40 g) was separated by reverse-phase ODS column chromatography, eluted with a methanol-water system (40%-50%-60%-70%-85%), and repeatedly injected into a preparative HPLC column, eluted with methanol-water (54:46) to obtain compound astramembraoside J (15.00 mg), a white amorphous powder, which is the triterpenoid compound of this invention.

[0028] Example 2

[0029] Compound structure identification

[0030] The structure of the compound obtained in Example 1 was identified, and the spectral data are as follows:

[0031] Table 1 1H-NMR and 13 C-NMR data(δin ppm)of astramembraoside J(C5D5N)

[0032]

[0033]

[0034] Combination Figures 2 to 7 ,HRESIMS m / z 813.6094[M+HCOO] - The calcd value (813.4273) indicates that the compound has 9 degrees of unsaturation. IR spectroscopy reveals the presence of hydroxyl groups (3423 cm⁻¹). -1 ), carbonyl (1709cm) -1 ) and double bonds (1640cm) -1 Seven methyl signals δ can be observed from the proton NMR spectrum. H 1.03(3H,d,J=6.7Hz,H-21),1.30(3H,s,H-18),1.37(3H,s,H-30),1.79(3H,s,H-28),1.80(3H,s,H-27),2.06(3H,s,6- C H3CO) and 2.17(3H,s,12- C H3CO); two olefinic proton signals were observed at 5.37 (1H, br.s, H-11) and 5.73 (m, H-24). Forty carbon signals were observed in the carbon spectrum, including seven methyl δ-protons. C 12.2, 14.0, 14.6, 18.8, 22.1, 22.2, 28.7; 10 methylene δ C 26.2, 29.5, 33.1, 37.3, 43.5, 47.4, 63.2, 71.0, 75.6; 15 methine δ C 30.6, 44.3, 57.0, 58.3, 71.0, 72.1, 75.6, 77.3, 77.5, 77.7, 78.8, 78.9, 103.8, 126.1, 129.8; 8 quaternary carbon δ C 41.5, 49.2, 50.3, 72.8, 132.2, 140.8, 170.7, 171.1. No H atom signal was observed on the cyclopropane, indicating that this compound is a tetracyclic triterpene. A glucose terminal H proton signal H-1' (δ) can also be seen in the carbon spectrum. H (4.89, d, J = 7.7 Hz), the coupling constant indicates that glucose is in the β configuration. From H-19 α (δH 2.17-2.27, 1H, m) to C-8 (δ) C 44.3), C-5(δ) C 58.3) and C-10(δ C The HMBC correlation of 72.8) indicates that the CH2-19 group is located between C-9 and C-10. These data suggest that this compound is a novel cycloaromatic triterpene, whose cycloheptane ring is formed by the cleavage of the 9,10 bond of the cycloaromatic ring. H-1' (δ) can also be seen in the HMBC spectrum. H 4.89, 1H, d, J = 7.7 Hz) and C-26 (δ C 75.6) is related, indicating that glucose is linked to C-26. This compound is related to compound huangqiyenin G (the structural formula of which is shown below). Figure 9 The results are basically similar to those shown, except that the CH3 at the C-4 position is replaced by CH2OH, thus C-29 (δ) C The chemical shift at 71.0) is a low-field shift. This can be seen in the HMBC spectrum. C H3CO(δ H 2.17) and 12-CH3 C O(δ C 171.1) related, 11-H(δ) H 5.37) and 12-CH3 C O(δ C Based on the correlation with 171.1), it can be inferred that -CH3COO is connected to C-12, and 6- C H3CO(δ H 2.06) and 6-CH3 C O(δ C 170.7), C-6(δ) C Based on the correlation with 77.3), it can be inferred that -CH3COO is connected to C-6.

[0035] Meanwhile, the NOESY spectrum of the compound of the present invention was compared with the NMR data of huangqiyenin G. 13 The C-NMR data are basically the same, indicating that this compound and huangqiyenin G have exactly the same side chain, which means that the C(24)=C(25) bond is an E bond, according to HC(3) in δ HThe coupling constant at 3.67 (dd, J = 10.3, 3.9) can be assigned a value of β for the configuration of OH- in C-3. From the NOESY spectrum, we can observe HC(3) / Me(28), HC(3) / HC(29), HC(29) / HC(5), Me(30) / HC(16), HC(16) / HC(17), HC(17) / HC(12), HC(17) / Me-21, Me(18) / HC(20), HC(8) / Me(18), HC(8) / HC(6), HC(6) / H β -C(19), HC(30) / H β -C(19) indicates that HC(3),CH2OH-29, HC(5), HC(12), Me(28), HC(16), HC(17), and Me(21) are α-configurations, while Me(30), HC(6), HC(8), and Me(18) are β-configurations. Furthermore, since HC(30) / H β -C(19), therefore it can be deduced that CH2-19 is located at the β position of ring A, so OH-C(10) is the α configuration. The structure of this compound is (3β,6α,10α,12α,16β,24E)-4-methylol-6,12-bis(acetyloxy)-3,10,16-trihydroxy-9,19-cyclo-9,10-secolanosta-9(11),24-dien-26-ylβ-D-glucopyranoside, as Figure 1 As shown, it is named astramembraoside J.

[0036] Example 3

[0037] Pharmacological activity

[0038] I. Experimental Methods

[0039] 1. Cell plating:

[0040] ① Remove the cell culture flask from the incubator and observe its condition under a microscope.

[0041] ② Place the cells in a clean bench and wash twice with 3 ml of PBS.

[0042] ③ Add 1.5 mL of trypsin to digest the cells and observe them under a microscope. When the cells are no longer spindle-shaped and a small amount are suspended, stop the digestion with 10% culture medium containing antibiotics and acetic acid. Pipe the cells 20 times and place them in a 15 mL centrifuge tube.

[0043] ④ Centrifuge at 1000 rpm for 5 min, discard the supernatant, add 2 mL of 10% culture medium with antibiotics and mix by pipetting, then add the culture medium to 10 mL and mix well.

[0044] ⑤ Take 10 μL of culture medium, add it to a cell counting plate, and count the cells.

[0045] ⑥ Plate the cells, with approximately 3000 cells per well, and add 200 μL of PBS around the perimeter of each well.

[0046] 2. Cell-based drug delivery:

[0047] ① Prepare the stock solution of the drug and dilute it with culture medium to the required concentration.

[0048] ② Aspirate the liquid from the 96-well plate and wash twice with 300 μL PBS.

[0049] ③Group C: Add 200μL of 10% antibiotic-free cell culture medium to each well;

[0050] In the drug administration group, 200 μL of drug solutions of different gradients were added to each well.

[0051] ④ Add 200 μL of PBS to each well around the perimeter.

[0052] 3. MTT experiment:

[0053] ① Take 4.8 mg of MTT powder and prepare 0.96 mL of MTT 5 mg / mL solution. Filter through a membrane and store in the dark. ② Wash three times with 300 μL of PBS.

[0054] ③ Add 180 μL DMEM and 20 μL MTT solution to each well.

[0055] ④ Add 200 μL of PBS to each well around the perimeter.

[0056] ⑤ Incubate in an incubator for 4 hours.

[0057] ⑥ After 4 hours, remove all liquid and reconstitute with 150 μL DMSO.

[0058] ⑦ Shake in a shaker for 10 minutes in a dark environment.

[0059] ⑧ Measure the absorbance at 562 nm using an ELISA reader.

[0060] II. Experimental Conclusions

[0061] 1. Activity data are shown in the table below:

[0062] Table 2

[0063]

[0064] The present invention relates to the inhibitory effect of the triterpenoid compound astramembraoside J on human colon cancer RKO cells and HT-29 cells. Compared with the positive control drug 5-FU, astramembraoside J showed better inhibitory effect on human colon cancer RKO cells and moderate inhibitory effect on HT-29 cells. Its IC50 value was [not specified in the original text]. 50 The values ​​were 73.8 and 87.5 μmol / L, respectively. This suggests the potential for development into drugs or health products for the prevention and treatment of colon cancer, providing important scientific evidence for the full utilization of the abundant natural plant resources of Astragalus membranaceus leaves.

Claims

1. A triterpenoid compound with the following structural formula: 。 2. The method for preparing the triterpenoid compound according to claim 1, wherein the triterpenoid compound is obtained by extraction and isolation from Astragalus membranaceus leaves, comprises the following steps: The leaves of Astragalus membranaceus were extracted by heating and refluxing with pure methanol, filtered, and the extract was concentrated under reduced pressure to obtain the extract. The extract was dissolved in water suspension and extracted sequentially with petroleum ether and water-saturated n-butanol. The n-butanol extract was concentrated under reduced pressure to obtain the n-butanol layer. The n-butanol layer was dissolved in water-saturated n-butanol, and the pH was adjusted to 12 with 0.1% NaOH aqueous solution. The extract was extracted by equal volume extraction, and the n-butanol layer was concentrated under reduced pressure to obtain the extract, yielding the n-butanol base layer. The n-butanol base layer was subjected to normal-phase silica gel column chromatography with gradient elution using a dichloromethane-methanol solvent system. The fractions were analyzed by thin-layer chromatography (TLC) and HPLC, and the same fractions were combined. The fractions were then separated by reverse-phase ODS column chromatography with gradient elution using a methanol-water system. The separation was repeated by preparative HPLC column chromatography with methanol-water at a ratio of 54:46 to obtain the triterpenoid compounds.

3. The use of the triterpenoid compound of claim 1 in the preparation of a drug for treating colon cancer.

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