A triterpenoid alkaloid compound, and a preparation method and application thereof

Abstract

The present application belongs to the field of traditional Chinese medicine extract, and relates to a triterpenoid alkaloid compound and a preparation method and application thereof. The triterpenoid alkaloid compound is isolated from Buxus megazonta for the first time, and it is confirmed that the triterpenoid alkaloid compound has significant anti-inflammatory activity and anti-tumor activity, and has a good application prospect in preparation of anti-inflammatory drugs and anti-tumor drugs. In preparation, dry Buxus megazonta is used as raw material, and the raw material is extracted by heating reflux with methanol for multiple times, and then the alkaloid-rich part is extracted by acid extraction and alkali precipitation, and then the alkaloid-rich part is subjected to preliminary segmentation by means of normal phase silica gel column, amino silica gel column and gel column chromatography, and then the alkaloid-rich part is subjected to refined purification by means of thin layer chromatography and semi-preparative high performance liquid chromatograph. The preparation method has the advantages of easy availability of raw material, environmental protection, simple operation and suitability for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine extracts, and relates to a triterpenoid alkaloid compound, its preparation method, and its application. Background Technology

[0002] Inflammation is a highly complex physiological defense mechanism of the body in response to infection or injury, involving the synergistic effects of multiple immune cells and molecular pathways. Activated macrophages induce the excessive release of pro-inflammatory cytokines, while mediators such as endogenous free radicals and nitric oxide (NO) play important roles in the progression of various inflammatory diseases. NO levels are positively correlated with the degree of inflammation. In a lipopolysaccharide-stimulated macrophage model, upregulated expression of nitric oxide synthase (iNOS) and a significant increase in NO release can serve as a marker of inflammatory response. Drugs or compounds that can reduce NO levels usually indicate that they inhibit iNOS activity or expression, thereby exerting an anti-inflammatory effect. Excessive or persistent inflammatory responses disrupt tissue homeostasis and are closely related to the development and progression of various chronic diseases, such as rheumatoid arthritis and atherosclerosis. Therefore, anti-inflammatory drugs have become the foundation of clinical treatment for inflammatory diseases.

[0003] Boxwood (also known as Sparrow Tongue Boxwood) Buxu bodinieri Buxus macrocarpa is an evergreen shrub belonging to the genus Buxus in the family Buxaceae. It grows on plains or hillsides, and is mainly found in southern my country, such as Yunnan and Sichuan. Due to its lush foliage and unique leaf shape, Buxus macrocarpa is considered a highly ornamental plant and is often used for environmental beautification and air purification. The entire plant can be used medicinally, primarily for treating rheumatism and paralysis, and is believed to have the effects of promoting blood circulation, relieving pain, and clearing the meridians. It has significant medicinal value. Chemical analysis shows that Buxus macrocarpa contains abundant alkaloids, flavonoids, phenolic acids, tannins, and lignin compounds, with Buxus alkaloids being the characteristic and main active components. While many studies have shown that Buxus macrocarpa has anti-inflammatory effects, there are currently no reports of anti-inflammatory effects specifically targeting the triterpenoid alkaloids in Buxus macrocarpa. Summary of the Invention

[0004] The applicant discovered four novel natural active ingredients—triterpenoid alkaloids—with entirely new structures in the traditional Chinese medicinal herb *Buxus microphylla*. These triterpenoid alkaloids exhibit significant anti-inflammatory and antitumor activities, demonstrating great application potential. Based on this, the present invention provides a triterpenoid alkaloid compound, its preparation method, and its applications.

[0005] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides a triterpenoid alkaloid compound, wherein the triterpenoid alkaloid compound is at least one of compounds (1) to (4): .

[0006] Secondly, the present invention provides a method for preparing triterpenoid alkaloid compounds, comprising: Take dried Buxus chinensis branches and leaves, crush them, and extract them multiple times by heating and reflux with alcohol. Combine the extracts and recover the solvent under reduced pressure to obtain the total extract. The total extract was adjusted to pH 3.0 ± 0.5 with acid, defatted with water-saturated ethyl acetate, and the lower aqueous phase was adjusted to pH 10.0 ± 0.5 with alkali. It was then extracted with dichloromethane, and the extracts were combined and concentrated under reduced pressure to obtain a mixture of alkaline components. The mixture of alkaline components was sequentially separated by silica gel column chromatography, amino silica gel column chromatography, and dextran gel column chromatography. The target fraction was collected and then purified by thin-layer chromatography on silica gel plates and semi-preparative high-performance liquid chromatography to obtain at least one of compounds (1) to (4); wherein the structural formulas of compounds (1) to (4) are shown below: .

[0007] In conjunction with the second aspect of the invention, some embodiments include: The silica gel column chromatography sequentially used petroleum ether-ethyl acetate mixed solvents with volume ratios of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 1:4, and 0:1 as eluents; and / or, The amino silica gel column chromatography sequentially used petroleum ether-ethyl acetate mixed solvents with volume ratios of 50:1, 30:1, 20:1, 10:1, 6:1, 2:1, 1:1, 1:4, and 0:1 as eluents; and / or, The dextran gel column chromatography used methanol as the eluent; and / or... The thin-layer chromatography silica gel plate uses a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 3:1 as the developing solvent; and / or, The mobile phase of the semi-preparative high-performance liquid chromatography uses an acetonitrile aqueous solution with a volume fraction of 70%–75%, a flow rate of 3 mL / min, and a detection wavelength of 210 nm; and / or, The petroleum ether-ethyl acetate mixed solvent contains 1 vol‰ of diethylamine.

[0008] In conjunction with the second aspect of the present invention, in some embodiments: the heating reflux extraction is performed 2 to 3 times, and each extraction takes 1 to 3 hours.

[0009] In conjunction with the second aspect of the present invention, in some embodiments: the water-saturated ethyl acetate and dichloromethane are extracted 3 to 5 times each.

[0010] In conjunction with the second aspect of the present invention, some embodiments include: separating the alkaline component mixture sequentially by silica gel column chromatography, amino silica gel column chromatography, and dextran gel column chromatography, collecting the target fraction, and then purifying it by thin-layer chromatography on silica gel plates and semi-preparative high-performance liquid chromatography, including: Silica gel column chromatography was used to separate a mixture of basic components, and the fraction with characteristic bands and Rf values ​​ranging from 0.67 to 0.70 (Fra.B) was collected. Fra.B was separated by amino silica gel column chromatography, and the fractions FB2 with Rf values ​​ranging from 0.62 to 0.66 and / or FB3 with Rf values ​​ranging from 0.52 to 0.58 were taken from the characteristic bands. FB2 was separated by dextran gel column chromatography, and fractions FB2c (with an Rf value range of 0.57–0.60) and FB2d (with an Rf value range of 0.48–0.55) were taken from the characteristic band portion; and / or, FB3 was separated by dextran gel column chromatography, and fraction FB3b (with an Rf value range of 0.46–0.55) was taken from the characteristic band portion. Thin-layer chromatography using silica gel plates separates FB2c, and the fraction with the characteristic band and an Rf value ranging from 0.55 to 0.62 (FB2c-2) is selected; and / or, thin-layer chromatography using silica gel plates separates FB2d, and the fraction with the characteristic band and an Rf value ranging from 0.55 to 0.63 (FB2d-1) is selected; and / or, thin-layer chromatography using silica gel plates separates FB3b, and the fraction with the characteristic band and an Rf value ranging from 0.50 to 0.58 (FB3b-1) is selected. FB2c-2 was separated by semi-preparative high-performance liquid chromatography (HPLC) to obtain compound 1 and compound 2 in sequence; and / or, FB2d-1 was separated by semi-preparative HPLC to obtain compound 4; and / or, FB3b-1 was separated by semi-preparative HPLC to obtain compound 3.

[0011] Thirdly, the present invention provides the use of any one of compounds (1) to (4) in the preparation of a medicament for anti-inflammatory purposes.

[0012] Fourthly, the present invention provides the use of any one of compounds (1) to (4) in the preparation of a medicament for treating tumors.

[0013] In conjunction with the third or fourth aspect of the present invention, in some embodiments: the dosage form of the drug is at least one of tablets, capsules, oral liquids, ointments, injections, and suspensions.

[0014] In conjunction with the fourth aspect of the present invention, in some embodiments: the tumor is colorectal cancer.

[0015] Compared with the prior art, the present invention has at least the following beneficial effects: This invention isolates four novel triterpenoid alkaloid compounds from Buxus microphylla and confirms that they have significant anti-inflammatory and antitumor activities, providing a new source of natural active molecules for the development of anti-inflammatory and antitumor drugs. Attached Figure Description

[0016] Figure 1 HRMS plot of compound 1 (i.e., Buxubodinoid A).

[0017] Figure 2 Compound 1 (i.e., Buxubodinoid A) 1 H-NMR (400 MHz, CDCl3) image.

[0018] Figure 3 Compound 1 (i.e., Buxubodinoid A) 13 C-NMR (CDCl3, ppm, 100 MHz) plot.

[0019] Figure 4 : HSQC diagram of compound 1 (i.e., Buxubodinoid A).

[0020] Figure 5 HMBC diagram of compound 1 (i.e., Buxubodinoid A).

[0021] Figure 6 Compound 1 (i.e., Buxubodinoid A) 1 H- 1 H COSY diagram.

[0022] Figure 7 : ROESY diagram of compound 1 (i.e., Buxubodinoid A).

[0023] Figure 8 : Schematic diagram of the relationship between the flow branches in S4~S12 in Example 1. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0025] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range. Similarly, any upper limit can be combined with any other upper limit to form an undefined range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit, combined with any other point or individual value, or combined with other lower or upper limits to form an undefined range.

[0026] It should be noted that, in this description, unless otherwise stated, "above" and "below" include the number itself, and "multiple" in "one or more" means two or more. Relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0027] In the description of this specification, the references to terms such as "any embodiment / mode," "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0028] The above description of the invention is not intended to describe every disclosed embodiment or implementation. Instead, exemplary embodiments are described in more detail below. These embodiments can be used in various combinations. The examples listed are representative only and should not be construed as exhaustive.

[0029] In this paper, the term "fraction" refers to components with similar migration behavior that elute from a chromatographic column or plate under specific elution conditions (such as solvent ratio, flow rate, etc.) during chromatographic separation. These components are usually mixtures and require further separation through subsequent purification steps (such as thin-layer chromatography, semi-preparative high-performance liquid chromatography, etc.) to obtain a single compound. For example, "fraction Fra.B" collected after silica gel column chromatography and "fraction FB2" after amino silica gel column chromatography are intermediate products that need to be purified to obtain the target compounds (1) to (4).

[0030] The naming and numbering of fractions and compounds in this article are for distinction only and do not represent limitations on their components or structures.

[0031] Currently, for mild inflammation, nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen and aspirin or topical corticosteroids are commonly used in clinical practice. Although these drugs can relieve symptoms, long-term use can easily cause adverse reactions such as gastrointestinal damage and skin atrophy. Moderate to severe inflammation requires the use of immunosuppressants (such as oral corticosteroids and TNF-α inhibitors), but hormone therapy can lead to osteoporosis and metabolic disorders.

[0032] While there are existing Chinese herbal medicines that can be used to reduce inflammation, these medicines may contain inactive ingredients, toxic impurities, or allergens (such as tannins and proteins). Extraction and purification can remove these components, reducing the risk of allergies and the metabolic burden on the liver and kidneys.

[0033] To address this technical problem, this invention provides a natural triterpenoid alkaloid compound extracted from the traditional Chinese medicine Buxus microphylla, which possesses a unique atunane-type skeleton and multiple substituent modifications, making it suitable for the preparation of anti-inflammatory drugs. The extracted active ingredients (such as monomeric compounds) have clearly defined chemical structures, allowing for precise quantification using modern technology. This avoids interference from complex components in traditional Chinese medicine formulas, ensuring a stable correlation between dosage and efficacy, and reducing fluctuations in efficacy caused by factors such as origin and harvesting time.

[0034] The triterpenoid alkaloid compounds provided by this invention are at least one of compounds (1) to (4): .

[0035] These four triterpenoid alkaloids were discovered for the first time and exhibit significant anti-inflammatory and anti-tumor activities. As natural active ingredients from Buxus microphylla, their long-term natural evolution and biological screening, coupled with the plant's long history of use in traditional Chinese medicine, further confirm the biocompatibility advantages of these components with the human body, effectively reducing the risk of side effects and demonstrating great application potential in the field of anti-inflammatory drug development.

[0036] The method for preparing triterpenoid alkaloids provided by this invention includes: Take dried Buxus chinensis branches and leaves, crush them, and extract them multiple times by heating and reflux with alcohol. Combine the extracts and recover the solvent under reduced pressure to obtain the total extract. The total extract was adjusted to pH 3.0 ± 0.5 with acid, defatted with water-saturated ethyl acetate, and the lower aqueous phase was adjusted to pH 10.0 ± 0.5 with alkali. It was then extracted with dichloromethane, and the extracts were combined and concentrated under reduced pressure to obtain a mixture of alkaline components. The mixture of alkaline components was sequentially separated by silica gel column chromatography, amino silica gel column chromatography, and dextran gel column chromatography. The target fraction was collected and then purified by thin-layer chromatography on silica gel plates and semi-preparative high-performance liquid chromatography to obtain at least one of compounds (1) to (4); wherein the structural formulas of compounds (1) to (4) are shown below: .

[0037] This invention marks the first successful isolation of four triterpenoid alkaloid monomers with anti-inflammatory and antitumor activities from Buxus microphylla. This method allows for the targeted extraction of high-value components from low-content Buxus microphylla, improving resource utilization and avoiding toxic impurities or irrelevant components found in the plant. The method is simple and uses low-cost raw materials.

[0038] The separation conditions of this invention are not intended to be limiting. In some embodiments of this invention, the separation conditions used to separate four triterpenoid alkaloids from Buxus microphylla are as follows: The silica gel column chromatography sequentially used petroleum ether-ethyl acetate mixed solvents with volume ratios of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 1:4, and 0:1 as eluents; and / or, The amino silica gel column chromatography sequentially used petroleum ether-ethyl acetate mixed solvents with volume ratios of 50:1, 30:1, 20:1, 10:1, 6:1, 2:1, 1:1, 1:4, and 0:1 as eluents; and / or, The dextran gel column chromatography used methanol as the eluent; and / or... The thin-layer chromatography silica gel plate uses a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 3:1 as the developing solvent; and / or, The mobile phase of the semi-preparative high-performance liquid chromatography uses an acetonitrile aqueous solution with a volume fraction of 70%–75%, a flow rate of 3 mL / min, and a detection wavelength of 210 nm; and / or, The petroleum ether-ethyl acetate mixed solvent contains 1 vol‰ of diethylamine.

[0039] This invention is not intended to limit the extraction conditions. In some embodiments of the invention, the number of heating and reflux extractions is 2 to 3 times, and the extraction time for each extraction is 1 to 3 hours.

[0040] In some embodiments of the present invention, the alcohol solution is methanol or ethanol or an aqueous solution thereof; in some embodiments, the alcohol solution is a methanol aqueous solution of 75 vol% or more, preferably a methanol aqueous solution of 95 vol%.

[0041] This invention is not intended to limit the extraction conditions. In some embodiments of the invention, the water-saturated ethyl acetate and dichloromethane are extracted 3 to 5 times.

[0042] Unintentionally limiting the separation process of this invention, in some embodiments of this invention, the alkaline component mixture is sequentially separated by silica gel column chromatography, amino silica gel column chromatography, and dextran gel column chromatography, the target fraction is collected, and then purified by thin-layer chromatography on silica gel plates and semi-preparative high-performance liquid chromatography, including: Silica gel column chromatography was used to separate a mixture of basic components, and the fraction with characteristic bands and Rf values ​​ranging from 0.67 to 0.70 (Fra.B) was collected. Fra.B was separated by amino silica gel column chromatography, and the fractions FB2 with Rf values ​​ranging from 0.62 to 0.66 and / or FB3 with Rf values ​​ranging from 0.52 to 0.58 were taken from the characteristic bands. FB2 was separated by dextran gel column chromatography, and fractions FB2c (with an Rf value range of 0.57–0.60) and FB2d (with an Rf value range of 0.48–0.55) were taken from the characteristic band portion; and / or, FB3 was separated by dextran gel column chromatography, and fraction FB3b (with an Rf value range of 0.46–0.55) was taken from the characteristic band portion. Thin-layer chromatography using silica gel plates separates FB2c, and the fraction with the characteristic band and an Rf value ranging from 0.55 to 0.62 (FB2c-2) is selected; and / or, thin-layer chromatography using silica gel plates separates FB2d, and the fraction with the characteristic band and an Rf value ranging from 0.55 to 0.63 (FB2d-1) is selected; and / or, thin-layer chromatography using silica gel plates separates FB3b, and the fraction with the characteristic band and an Rf value ranging from 0.50 to 0.58 (FB3b-1) is selected. FB2c-2 was separated by semi-preparative high-performance liquid chromatography (HPLC) to obtain compound 1 and compound 2 in sequence; and / or, FB2d-1 was separated by semi-preparative HPLC to obtain compound 4; and / or, FB3b-1 was separated by semi-preparative HPLC to obtain compound 3.

[0043] This invention evaluates the anti-inflammatory activity of compounds (1) to (4) using a lipopolysaccharide-activated macrophage model and finds that compounds (1) to (4) have inhibitory activity on lipopolysaccharide-induced NO production in RAW264.7 cells. Therefore, it provides the application of any one of compounds (1) to (4) in the preparation of drugs for anti-inflammatory purposes.

[0044] This invention verifies the in vitro cytotoxic activity of triterpenoid alkaloids and finds that any one of compounds (1) to (4) has significant growth inhibition or killing ability against three tumor cell lines (HCT-116, HT-29, and SW480). Therefore, it provides the application of any one of compounds (1) to (4) in the preparation of a drug for treating tumors. The drug for treating tumors is a drug for treating colorectal cancer.

[0045] In some embodiments of the present invention, the dosage form of the drug used for anti-inflammatory or tumor treatment may be at least one of tablets, capsules, oral liquids, ointments, injections, and suspensions.

[0046] All four compounds can be isolated from Buxus microphylla using conventional column chromatography. Unless otherwise specified, all materials and reagents used in the following examples are commercially available. The developing solvent for the thin-layer chromatography used in the following examples is a petroleum ether-ethyl acetate mixture (containing 1 vol‰ diethylamine) with a volume ratio ranging from 5:1 to 3:1. The requirements for thin-layer chromatography tracking and detection are as follows: after drying the developed thin-layer plate by blowing or air-drying, irradiate it with a 254 nm ultraviolet lamp or wet it with a colorimetric reagent (5% sulfuric acid ethanol solution) and then heat it for color development.

[0047] Example 1: Preparation of triterpenoid alkaloids S1. Take the branches and leaves of Buxus microphylla from Lijiang, Yunnan in August, dry and crush them to 0.5 cm~1 cm, extract them with 95% methanol under reflux for 2~3 times, combine the extracts, and recover the solvent under reduced pressure to obtain the total extract.

[0048] S2. Acidify the total extract to pH 3 with 1M hydrochloric acid, then extract multiple times with water-saturated ethyl acetate. Adjust the pH of the lower aqueous fraction to 10 with 1M sodium hydroxide solution, and then extract 3-5 times with dichloromethane. Combine the extracts to obtain the alkaloid fraction extract.

[0049] S3. The above-mentioned alkaloid extracts were separated by silica gel column chromatography: gradient elution was performed sequentially with a petroleum ether-ethyl acetate mixed solvent (containing 1 vol‰ diethylamine) at volume ratios of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 1:4, and 0:1. After thin-layer chromatography, five fractions were collected, concentrated under reduced pressure, and numbered in elution order as Fra.A, Fra.B, Fra.C, Fra.D, and Fra.E. The Rf values ​​of the characteristic bands ranged from 0.73 to 0.75, 0.67 to 0.70, 0.59 to 0.64, 0.48 to 0.54, and 0.35 to 0.42, respectively.

[0050] S4. Fraction Fra.B was separated using amino silica gel column chromatography: gradient elution was performed sequentially with a petroleum ether-ethyl acetate mixed solvent at volume ratios of 50:1, 30:1, 20:1, 10:1, 6:1, 2:1, 1:1, 1:4, and 0:1. Thin-layer chromatography was used for identification, and four fractions were collected. After concentration under reduced pressure, they were numbered FB1, FB2, FB3, and FB4 in elution order. The Rf values ​​of the characteristic bands ranged from 0.69 to 0.75, 0.62 to 0.66, 0.52 to 0.58, and 0.40 to 0.48, respectively.

[0051] S5. Separation of fraction FB2 using dextran gel column chromatography: Methanol was used as the eluent, and five fractions were collected by thin-layer chromatography. After concentration under reduced pressure, the fractions were numbered in elution order as FB2a, FB2b, FB2c, FB2d, and FB2e. The Rf values ​​of the characteristic bands were 0.69~0.72, 0.63~0.66, 0.57~0.60, 0.48~0.55, and 0.38~0.45, respectively.

[0052] S6. Separate fraction FB2c using thin-layer chromatography silica gel plate: Use a petroleum ether-ethyl acetate mixed solvent (containing 1 vol‰ diethylamine) at a volume ratio of 3:1 as the developing solvent, and collect fractions FB2c-1, FB2c-2 and FB2c-3 in sequence. The Rf values ​​of the characteristic bands range from 0.69 to 0.75, 0.55 to 0.62 and 0.45 to 0.50, respectively.

[0053] S7. The fraction FB2c-2 was separated using a semi-preparative high performance liquid chromatograph: the mobile phase was an aqueous solution of acetonitrile with a volume fraction of 75%, the flow rate was set to 3 mL per minute, the detection wavelength was 210 nm, and compound 1 (retention time 15.5 min) and compound 2 (retention time 18.9 min) were collected.

[0054] S8. The fraction FB2d was separated using a thin-layer chromatography silica gel plate. A petroleum ether-ethyl acetate mixed solvent (containing 1 vol‰ diethylamine) with a volume ratio of 3:1 was used as the developing solvent. Fractions FB2d-1 and FB2d-2 were collected sequentially. The Rf values ​​of the characteristic bands ranged from 0.55 to 0.63 and from 0.45 to 0.52, respectively.

[0055] S9. The fraction FB2d-1 was separated using a semi-preparative high-performance liquid chromatograph: the mobile phase was 70% acetonitrile, the flow rate was set to 3 mL per minute, the detection wavelength was set to 210 nm, and compound 4 was collected at a retention time of 21.0 min.

[0056] S10. Separate fraction FB3 using dextran gel column chromatography: using methanol as the eluent, and based on thin-layer chromatography tracking and identification, collect three fractions sequentially and number them FB3a, FB3b, and FB3c. Concentrate them under reduced pressure, and the Rf values ​​of the characteristic bands are 0.58~0.68, 0.46~0.55, and 0.36~0.44, respectively.

[0057] S11. The fraction FB3b was separated using a thin-layer chromatography silica gel plate. A petroleum ether-ethyl acetate mixed solvent (containing 1 vol‰ diethylamine) with a volume ratio of 3:1 was used as the developing solvent. Two fractions were collected sequentially and numbered as FB3b-1 and FB3b-2. The Rf values ​​of the characteristic bands were 0.50~0.58 and 0.38~0.47, respectively.

[0058] S12. The fraction FB3b-1 was separated using a semi-preparative high-performance liquid chromatography system with a mobile phase of 72% acetonitrile, a flow rate of 3 mL / min, a detection wavelength of 210 nm, and compound 3 was collected at a retention time of 16.5 min.

[0059] Example 2 1. Identification of compound 1 (Buxubodinoid A): Compound 1 (i.e., Buxubodinoid A) extracted in Example 1 was subjected to high-resolution mass spectrometry and NMR spectroscopy for detection and structural analysis. The compound was analyzed using a Bruker AV-400 spectrometer. 1 H-NMR (400 MHz, CDCl3), 13 C-NMR (100 MHz, CDCl3), HSQC, HMBC and 1 H- 1 HRMS chromatogram of the compound was obtained by H COSY assay. 1 H-NMR (400 MHz, CDCl3), 13 C-NMR (CDCl3, ppm, 100 MHz), HSQC, HMBC, 1 H- 1 HCOSY and ROESY are respectively as follows: Figures 1-7 As shown, the obtained spectral data are shown in Table 1 below: Table 1

[0060] Buxubodinoid A is a white powder; HRESIMS data show a molecular ion peak. m / z 436.2810 [M + Na] + (C) 26 H39 The calculated value of NO3Na is 436.2828, which leads to the molecular formula of the compound being C. 26 H 39 NO3, the calculated degree of unsaturation is 8.

[0061] like Figures 2-3 One-dimensional spectra show that Buxubodinoid A contains a typical set of acetyl signals ( d H 2.04; d C (171.3 and 23.2), the remaining signals have four methyl groups [one of which is a doublet methyl signal located on a double bond]. d H 1.83(d, J = 7.5 Hz)], nine methylene groups [a characteristic cycloditin-type methylene signal] d H 0.61 (d, J = 4.4 Hz) and d H 0.41 (d, J = 4.4 Hz), a hydroxymethyl signal d H 3.35 and 3.01], three methine signals [1 azomethine ( d H 4.02; d C [51.4], five quaternary carbon signals, one pair of trisubstituted double bond signals, and one carbonyl carbon signal d C 206.6. By comparing the data with the common skeletons of Buxus alkaloids, it was determined that it is a cycloatunane-type 6 / 6 / 6 / 5 skeleton ring system parent nucleus.

[0062] pass Figures 4-6 Determine the position of the substituent. HMBC spectrum shows H3-29 ( d H 0.62) and H2-28 ( d H 3.35 and 3.01) and C-3 ( d C 51.4), C-4 ( d C 44.3) and C-5 ( d C 40.1) Related signals, H-3 ( d H 4.02) and acetyl carbonyl carbon ( d C 171.3), C-28 (d C 64.1) and C-29 ( d C 11.3) Related signals, combined with 1 H- 1 H COSY spectrum N a -H ( d H 5.39) / H-3 / H2-2 ( d H The connection fragments 1.67 and 1.61 suggest that there is an acetamino substitution at C-3 and a hydroxymethyl group at C-28. H2-15 ( d H 2.17 and 2.01) and C-16 ( d C 206.6) and C-17 ( d C 146.7) related signals, H3-21 ( d H 1.83) and C-17 and C-20 ( d C 130.0) related signals, H-20 ( d H 6.56) and C-13 ( d C Based on the C-16 related signals (42.4) and the relevant chemical shift values, the carbon-carbon double bond is assigned to the C-17 and C-20 positions, and the carbonyl group is assigned to the C-16 position.

[0063] like Figure 7 As shown, N a -H ( d H 5.39) Related signals to H3-29, H3-29 and H-19 b Related signals and H-19 b The H-8 correlation signal confirms that both the acetamino group at C-3 and the methyl group at C-29 are substituted. b - Oriented, thus the hydroxymethyl group is located in a - Oriented towards C-28, according to H3-21 ( d H 1.82) and H3-18 ( d H 1.32) Based on relevant signals and literature data analysis, the stereo configuration of Buxubodinoid A was determined.

[0064] 2. Identification of compound 2 (Buxubodinoid B): Buxubodinoid B is a white powder; HRESIMS data show a molecular ion peak. m / z 428.3155 [M + H] + (C) 27 H 42 The calculated value of NO3 is 428.3165, which leads to the molecular formula of the compound being C. 27 H 41 NO3 has a molecular weight 14 Daltons larger than Buxubodinoid A, and its calculated degree of unsaturation is 8.

[0065] Buxubodinoid B is a structural analog of Buxubodinoid A, the only difference being that Buxubodinoid B has an additional methyl signal, and the methyl group is located at the acetamide group. N Atomically. Based on the signal characteristics of Buxubodinoid B and Buxubodinoid A, it was determined that they have the same stereo configuration.

[0066] 3. Identification of compound 3 (Buxubodinoid C): Buxubodinoid C is a white powder; HRESIMS data show a molecular ion peak. m / z 475.3870 [M + H] + (C) 29 H 51 The calculated value of N2O3 is 475.3900, which leads to the derivation of the molecular formula of this compound as C. 29 H 50 N2O3, calculated degree of unsaturation is 6.

[0067] Buxubodinoid C differs from the previous two in the signal of its five-membered ring and the signal of its branch positions; Buxubodinoid C lacks the signals found in Buxubodinoid A and Buxubodinoid B. α , β The structure is an unsaturated carbonyl group, with an additional dimethylamino signal and a hydroxyl signal. Based on the signal characteristics of Buxubodinoid C, the stereostructure of Buxubodinoid C was determined.

[0068] 4. Identification of compound 4 (Buxubodinoid D): Buxubodinoid D is a white powder, and its HRESIMS data show a molecular ion peak. m / z 459.3947 [M +H] + (C) 29 H 51The calculated value of N2O2 is 459.3951, which leads to the derivation of the molecular formula of the compound as C. 29 H 50 N2O2, its degree of unsaturation is calculated to be 6.

[0069] Buxubodinoid D is a structural analogue of Buxubodinoid C, the only difference being that Buxubodinoid D lacks a hydroxyl group signal at position 16. Based on the signal characteristics of Buxubodinoid B and Buxubodinoid A, it is determined that they have the same stereoconfiguration.

[0070] Example 3: Performance Testing 1. Anti-inflammatory activity: The anti-inflammatory potential of compounds 1-4 obtained in Example 1 was evaluated by detecting their inhibitory effect on the release of nitric oxide (NO), an inflammatory factor, in a lipopolysaccharide-activated macrophage model. The experimental methods were as reported in the literature. The brief steps are as follows: Mouse mononuclear macrophage leukemia cell line RAW264.7 was cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells / well in 96-well plates and pre-incubated for 24 hours. Subsequently, either the test compound or dexamethasone (positive control, concentration range 0.31–40 μM) was added, followed by stimulation with lipopolysaccharide (1 μg / mL) for 18 hours. Finally, the NO concentration in the supernatant was determined using Griess' reagent. All cell culture procedures conformed to standardized cell culture protocols. Performance results are expressed as the mean ± standard error of three independent assays.

[0071] Table 2. Inhibitory activity against lipopolysaccharide-induced NO production in RAW 264.7 cells.

[0072] As shown in Table 2, compared with the positive control group dexamethasone (Dex) (IC50) 50 Compared to (12.4 μM), compounds 1-2 exhibited stronger NO generation inhibitory activity (IC50). 50 (Values ​​ranged from 6.1 to 7.5 μM). This result suggests that α,β-unsaturated ketone structural fragments at positions C-16, C-17, and C-20 may be more conducive to enhancing the anti-inflammatory activity of the compound.

[0073] 2. Antitumor activity: The in vitro cytotoxic activity of the compounds obtained in Example 1 against three human colon cancer cell lines (HCT-116, HT-29, and SW480) was detected using the CCK-8 assay. The experimental method was as described in the literature. The brief steps are as follows: Cells were cultured at 1 × 10⁶ cells per well. 4Inoculated at a density of [number] cells / wells in 96-well plates and incubated for 24 hours. Subsequently, different concentrations of the test compound were added to complete culture medium for 48 hours. After treatment, 10 μL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for another 90 minutes. The absorbance was measured at 450 nm. Cisplatin was used as a positive control. Performance results are expressed as the mean ± standard deviation of three independent replicates. Half-maximal inhibitory concentration (IC50) is also considered. 50 The measurement results are shown in Table 3.

[0074] Table 3. Half-maximal inhibitory concentration (IC50) 50 Measurement results (unit: μM)

[0075] As shown in Table 3, compound 2 exhibited significantly enhanced cytotoxicity against the HT-29 cell line, with an IC50 value of [missing information]. 50 The values ​​were 6.4 μM, compared to the positive control cisplatin (IC50). 50 The concentration (18.6 μM) was increased by 3-fold. Furthermore, compound 1 showed moderate cytotoxic activity against HCT-116 and HT-29 cells, with an IC50 of [missing value]. 50 The values ​​ranged from 17.2 to 24.1 μM, which was comparable to the positive control. These results suggest that compounds 1-4 have a strong killing ability against various colorectal cancer cells.

[0076] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A triterpenoid alkaloid compound, characterized in that, The triterpenoid alkaloid compound is at least one of compounds (1) to (4): 。 2. A method for preparing a triterpenoid alkaloid compound, characterized in that, Includes the following steps: Take dried Buxus chinensis branches and leaves, crush them, and extract them multiple times by heating and reflux with alcohol. Combine the extracts and recover the solvent under reduced pressure to obtain the total extract. The total extract was adjusted to pH 3.0 ± 0.5 with acid, defatted with water-saturated ethyl acetate, and the lower aqueous phase was adjusted to pH 10.0 ± 0.5 with alkali. It was then extracted with dichloromethane, and the extracts were combined and concentrated under reduced pressure to obtain a mixture of alkaline components. The mixture of alkaline components was sequentially separated by silica gel column chromatography, amino silica gel column chromatography, and dextran gel column chromatography. The target fraction was collected and then purified by thin-layer chromatography on silica gel plates and semi-preparative high-performance liquid chromatography to obtain at least one of compounds (1) to (4); wherein the structural formulas of compounds (1) to (4) are shown below: 。 3. The method for preparing triterpenoid alkaloids according to claim 2, characterized in that: The silica gel column chromatography sequentially used petroleum ether-ethyl acetate mixed solvents with volume ratios of 50:1, 30:1, 15:1, 8:1, 4:1, 1:1, 1:4, and 0:1 as eluents; and / or, The amino silica gel column chromatography sequentially used petroleum ether-ethyl acetate mixed solvents with volume ratios of 50:1, 30:1, 20:1, 10:1, 6:1, 2:1, 1:1, 1:4, and 0:1 as eluents; and / or, The dextran gel column chromatography used methanol as the eluent; and / or... The thin-layer chromatography silica gel plate uses a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 3:1 as the developing solvent; and / or, The mobile phase of the semi-preparative high-performance liquid chromatography is an acetonitrile aqueous solution with a volume fraction of 70%–75%, a flow rate of 3 mL / min, and a detection wavelength of 210 nm; and / or, The petroleum ether-ethyl acetate mixed solvent contains 1 vol‰ of diethylamine.

4. The method for preparing triterpenoid alkaloids according to claim 2, characterized in that: The heating and reflux extraction is performed 2 to 3 times, with each extraction lasting 1 to 3 hours.

5. The method for preparing triterpenoid alkaloids according to claim 2, characterized in that: The extraction times for both water-saturated ethyl acetate and dichloromethane are 3 to 5 times.

6. The method for preparing triterpenoid alkaloids according to claim 3, characterized in that: The mixture of alkaline components was sequentially separated by silica gel column chromatography, amino silica gel column chromatography, and dextran gel column chromatography. The target fraction was collected and then purified by thin-layer chromatography on silica gel plates and semi-preparative high-performance liquid chromatography, including: Silica gel column chromatography was used to separate a mixture of basic components, and the fraction with characteristic bands and Rf values ​​ranging from 0.67 to 0.70 (Fra.B) was collected. Fra.B was separated by amino silica gel column chromatography, and the fractions FB2 with Rf values ​​ranging from 0.62 to 0.66 and / or FB3 with Rf values ​​ranging from 0.52 to 0.58 were taken from the characteristic bands. FB2 was separated by dextran gel column chromatography, and fractions FB2c (with an Rf value range of 0.57–0.60) and FB2d (with an Rf value range of 0.48–0.55) were taken from the characteristic band portion; and / or, FB3 was separated by dextran gel column chromatography, and fraction FB3b (with an Rf value range of 0.46–0.55) was taken from the characteristic band portion. Thin-layer chromatography using silica gel plates separates FB2c, and the fraction with the characteristic band and an Rf value ranging from 0.55 to 0.62 (FB2c-2) is selected; and / or, thin-layer chromatography using silica gel plates separates FB2d, and the fraction with the characteristic band and an Rf value ranging from 0.55 to 0.63 (FB2d-1) is selected; and / or, thin-layer chromatography using silica gel plates separates FB3b, and the fraction with the characteristic band and an Rf value ranging from 0.50 to 0.58 (FB3b-1) is selected. FB2c-2 was separated by semi-preparative high-performance liquid chromatography (HPLC) to obtain compound 1 and compound 2 in sequence; and / or, FB2d-1 was separated by semi-preparative HPLC to obtain compound 4; and / or, FB3b-1 was separated by semi-preparative HPLC to obtain compound 3.

7. The use of any one of the triterpenoid alkaloid compounds (1) to (4) as described in claim 1 in the preparation of a medicament for anti-inflammatory purposes.

8. Use of any one of the triterpenoid alkaloids (1) to (4) as described in claim 1 in the preparation of a medicament for treating colon cancer.

9. The application according to claim 7 or 8, characterized in that: The dosage form of the drug is at least one of tablets, capsules, oral liquids, ointments, injections, and suspensions.

Images