Euphorbia kansui-type triterpenoids extracted from Chinaberry, their preparation method and applications

Abstract

This invention discloses a method for preparing and applying euphorbia triterpenoids extracted from *Melia azedarach*, belonging to the field of natural compound extraction technology. The preparation method involves soaking dried *Melia azedarach* in ethyl acetate to obtain an extract, eluting the extract by reverse-phase column chromatography, and then recrystallizing to obtain the final product. This method is simple, yields a high number of euphorbia triterpenoid components, and achieves a higher yield. Furthermore, the obtained euphorbia triterpenoids exhibit high anti-inflammatory activity, showing significant potential for development and application as anticancer and macrophage anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of natural compound extraction technology, specifically to a kansui-type triterpenoid extracted from Sichuan chinaberry, its preparation method, and its application. Background Technology

[0002] Melia toosendan Sieb. et Zucc. is a traditional medicinal plant in the genus Melia, with a long history of medicinal use. Both the fruit and bark of Melia toosendan can be used medicinally. It is cold in nature and bitter in taste, and enters the liver, stomach, small intestine, and bladder meridians. Currently, there is considerable research on Melia toosendan fruit both domestically and internationally, but reports on the chemical composition of Melia toosendan bark are relatively scarce.

[0003] Phellochin F, a triterpenoid of the euphorbia type, is a class of compounds with a tetracyclic triterpenoid system. It exhibits certain activity against KB cells and is relatively rare in plant distribution, primarily found in Rutaceae, Simaroubaceae, Euphorbiaceae, Anacardiaceae, Trifoliaceae, Rhizophoraceae, and Meliaceae species. Triterpenoids are characteristic components of Meliaceae plants, with approximately 200 reported results. However, their preparation methods are generally cumbersome, requiring extraction with methanol or ethanol followed by repeated separation using silica gel column chromatography, reversed-phase chromatography, and gel electrophoresis. For example, publication number CN117024491A discloses a euphorbia-type triterpenoid compound extracted from a species of *Gnaphalium affine* and its preparation method; publication number CN106117294A discloses a euphorbia-type triterpenoid compound Phellochin F extracted from the fruit of *Phellodendron chinense* and its application; and publication number CN111471079A discloses a new euphorbia-type triterpenoid compound from the pericarp of *Toona sinensis* and its extraction, separation method, and application. The aforementioned technical solutions all involve extraction with methanol or ethanol followed by repeated chromatography and elution, resulting in cumbersome preparation methods and low yields. Furthermore, no existing technology exists for extracting euphorbia-type triterpenes from *Melia azedarach*. Therefore, a simple extraction method for extracting euphorbia-type triterpenes from *Melia azedarach* is of great significance. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for preparing and applying a euphorbia triterpenoid extracted from neem. The triterpenoid is obtained by soaking in ethyl acetate and then reversing the phase and recrystallizing the extract. The preparation method is simple and quick, and yields a wider range of triterpenoid structures with broader applications.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0006] A triterpenoid of the euphorbia type extracted from neem has the following chemical structural formula:

[0007]

[0008] In formula (I), R1 is any one of =O, α-OH, β-OH; R2 is =O or -H; R3 is β-OH; R4 is any one of -CH3, -COOCH3, -CH2OH;

[0009] In formula (II), R is either =O or β-OH;

[0010] In formula (III), R is α-OH or β-OH.

[0011] This invention also discloses a method for preparing euphorbia kansui-type triterpenes extracted from chinaberry, comprising the following steps:

[0012] (1) After drying the bark of the Sichuan pepper, it is crushed and extracted with 95% ethanol under hot reflux for 1-3 times, each time for 1-3 hours. The ethanol solvent is recovered by vacuum concentration. The resulting black-brown extract is suspended in water and extracted with ethyl acetate and n-butanol 2-5 times in sequence. The extraction solvent is recovered to obtain the ethyl acetate extract of the Sichuan pepper bark.

[0013] (2) The ethyl acetate extract of Sichuan pepper bark was mixed with C18 polyamide in a 1:1 ratio as a sample. The extract was coarsely fractionated by C18 reversed-phase column chromatography. The extract was eluted with methanol-water in a gradient ratio of 40:60→100:0. The fractions were detected and analyzed by TLC thin-layer chromatography. The components that fluoresce under ultraviolet light and / or the colorimetric components of 5% sulfuric acid ethanol were collected to obtain extract Fr.2.

[0014] (3) The Fr.2 extract was subjected to normal phase silica gel column chromatography, followed by gradient elution with petroleum ether / ethyl acetate, and then fractionated by YMC-ODS reverse phase column chromatography with methanol-water as solvent to obtain Fr.2D;

[0015] (4) Fr.2D was fractionated by gradient elution using YMC-ODS reversed-phase column chromatography with methanol-water as solvent to obtain Fr.2D3; the gradient of the solvent methanol-water was 70%→100% methanol content;

[0016] (5) The Fr.2D3 segment was separated by elution with a gradient of petroleum ether / acetone on a silica gel column, and then purified by semi-preparative HPLC. The mobile phase was eluted in a mixed solvent with a volume ratio of acetonitrile / water of 80:20-95:5. The peak was collected at a retention time of 20-30 minutes to obtain compound (I).

[0017] Furthermore, in the compound of formula (I), R1 is α-OH or β-OH, R2 is =O; R3 is β-OH; and R4 is -COOCH3.

[0018] Furthermore, in step (3), the gradient of petroleum ether / ethyl acetate is 20:1 → 10:1 → 5:1 → 1:0; the gradient of methanol-water in the YMC-ODS reversed-phase column chromatography gradient elution is 70% → 100% methanol content.

[0019] Furthermore, in step (5), the gradient of petroleum ether / acetone is 30:1 → 1:1.

[0020] This invention also discloses another method for preparing euphorbia kansui-type triterpenes extracted from chinaberry, comprising the following steps:

[0021] (1) After drying the bark of the Sichuan pepper, it is crushed and extracted with 95% ethanol under hot reflux for 1-3 times, each time for 1-3 hours. The ethanol solvent is recovered by vacuum concentration. The resulting black-brown extract is suspended in water and extracted with ethyl acetate and n-butanol 2-5 times in sequence. The extraction solvent is recovered to obtain the ethyl acetate extract of the Sichuan pepper bark.

[0022] (2) The ethyl acetate extract of Sichuan pepper bark was mixed with reversed-phase material at a ratio of 1:1. The extract was coarsely fractionated by C18 reversed-phase column chromatography. The extract was eluted with methanol-water at a gradient ratio of 40:60→100:0. The fractions were detected and analyzed by TLC thin-layer chromatography. The fluorescent components under ultraviolet light and / or the colorimetric components of 5% sulfuric acid ethanol were collected to obtain extract Fr.3.

[0023] (3) Fr.3 was separated by silica gel column elution with petroleum ether / acetone gradient (100:1→1:1) to obtain Fr.3B;

[0024] (4) Fr.3B was separated or precipitated as a white solid by elution, and then further purified by semi-preparative HPLC. It was then eluted in a mixed solvent with a mobile phase of acetonitrile / water volume ratio of 80:20-95:5. The peaks were collected at retention times of 15-25 minutes to separate compounds of formula (I), and / or formula (II), and / or formula (III).

[0025] Furthermore, in the compound of formula (I), R1 is =O or β-OH; R2 is =O or -H; R3 is β-OH; R4 is -CH3, -CH2OH, or -COOCH3;

[0026] In the compound of formula (II), R is either =O or β-OH;

[0027] In the compound of formula (III), R is α-OH or β-OH.

[0028] Furthermore, in step (3), the ratio gradient of petroleum ether / acetone is 100:1 → 1:1.

[0029] Furthermore, in step (4), the elution and separation of Fr.3B is as follows: Fr.3B is first eluted by gradient elution on a C18 reversed-phase column, and then separated by gradient elution on a silica gel column; the gradient ratio of petroleum ether / acetone is 100:1→10:1.

[0030] This invention also discloses the application of euphorbia triterpenoids extracted from Sichuan pepper, used alone or in addition to anticancer or anti-inflammatory drugs.

[0031] The present invention relates to the euphorbia kansui-type triterpenoids extracted from *Melia azedarach*, their preparation method, and their applications. The beneficial effects are as follows:

[0032] (1) In this invention, dried Sichuan pepper is soaked in ethyl acetate to obtain an extract. The extract is then eluted by reverse-phase column chromatography and recrystallized to obtain the final product. The method is simple and yields more euphorbia triterpenoid components.

[0033] (2) The euphorbia triterpenoids prepared by the method of the present invention have high anti-inflammatory activity and are of great potential for development and application as anti-cancer and macrophage anti-inflammatory drugs. Attached Figure Description

[0034] 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.

[0035] Figure 1 It is compounds 1–6 1 H- 1 H COSY and HMBC spectra;

[0036] Figure 2 These are the NOESY spectra of compounds 1-2, 4, and 6;

[0037] Figure 3 It is the single crystal structure of compounds 1 and 5;

[0038] Figure 4 These are the ECD spectra of compounds 1–4 and 6;

[0039] Figure 5 The effect of the compound on the viability of RAW 264.7 cells;

[0040] Figure 6 This is a schematic diagram showing the inhibitory effect of the compound on LPS-induced NO production in RAW264.7 mouse macrophages (6B (n=3)). ### P<0.001 vs. blank; ***P<0.001 vs. LPS);

[0041] Figure 7 This study investigated the effect of compound 1 on the LPS-induced NF-κB signaling pathway in RAW264.7 cells. Figures A-C show the inhibition of LPS-induced iNOS and COX-2 in macrophages by different concentrations of compound 1; Figures D-E show the inhibition of LPS-induced IκBα / P-IκBα protein expression in macrophages by different concentrations of compound 1. Data are presented as mean ± standard deviation (n=3). ### P<0.001, compared with the control group. *P<0.05, **P<0.01, ***P<0.001 (relative to the LPS group);

[0042] Figure 8 It is compound 1 1 H NMR spectrum;

[0043] Figure 9 It is compound 1 13 C NMR spectrum;

[0044] Figure 10 It is compound 2. 1 H NMR spectrum;

[0045] Figure 11 It is compound 2. 13 C NMR spectrum;

[0046] Figure 12 It is compound 3. 1 H NMR spectrum;

[0047] Figure 13 It is compound 3. 13 C NMR spectrum;

[0048] Figure 14 It is compound 4. 1 H NMR spectrum;

[0049] Figure 15 It is compound 4. 13 C NMR spectrum;

[0050] Figure 16 It is compound 5. 1 H NMR spectrum;

[0051] Figure 17 It is compound 5. 13 C NMR spectrum;

[0052] Figure 18 It is compound 6. 1 H NMR spectrum;

[0053] Figure 19 It is compound 6. 13 C10 NMR spectrum. Detailed Implementation

[0054] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all 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.

[0055] A triterpenoid of the euphorbia type extracted from *Melia azedarach*, characterized by the following chemical structural formula:

[0056]

[0057] In formula (I), R1 is any one of =O, α-OH, β-OH; R2 is =O or -H; R3 is β-OH; R4 is any one of -CH3, -COOCH3, -CH2OH;

[0058] In formula (II), R is either =O or β-OH;

[0059] In formula (III), R is α-OH or β-OH.

[0060] As a compound 1, it has the structure of formula (I), where R1 is =O; R2 is =O; R3 is β-OH; and R4 is -CH3.

[0061] As a compound 2, it has the structure of formula (I), where R1 is α-OH; R2 is =O; R3 is β-OH; and R4 is -COOCH3.

[0062] As a compound 3, it has the structure of formula (II), and R in formula (II) is =O.

[0063] As a compound 4, it has the structure of formula (II), where R in formula (II) is β-OH.

[0064] As a compound 5, it has the structure of formula (III), where R in formula (III) is β-OH.

[0065] As a compound 6, it has the structure of formula (III), where R in formula (III) is α-OH.

[0066] As a compound 7, it has the structure of formula (I), where R1 is β-OH; R2 is -H; R3 is β-OH; and R4 is -COOCH3.

[0067] As a compound 8, it has the structure of formula (I), where R1 is β-OH; R2 is -H; R3 is β-OH; and R4 is -CH2OH.

[0068] As a compound 9, it has the structure of formula (I), where R1 is β-OH; R2 is =O; R3 is β-OH; and R4 is -COOCH3.

[0069] Example 1

[0070] A method for preparing euphorbia kansui-type triterpenes extracted from Sichuan chinaberry includes the following steps:

[0071] (1) After drying the bark of Sichuan pepper (23.0 kg), it was crushed and extracted three times by hot reflux with 95% ethanol (3×150 L) for 2.5 h each time. The ethanol solvent was recovered by vacuum concentration. The resulting dark brown extract (1.6 kg) was suspended in water and extracted four times each with ethyl acetate and n-butanol. The extraction solvent was recovered to obtain ethyl acetate extract of Sichuan pepper (800 g).

[0072] (2) The ethyl acetate extract of *Melia azedarach* bark was mixed with an appropriate amount of C18 polyamide in a 1:1 ratio as a sample. The extract was coarsely fractionated using C18 reversed-phase column chromatography with a methanol / water gradient elution. The gradient elution consisted of a first elution with a 40% methanol-water solution followed by a second elution with 100% methanol. The fractions were analyzed by TLC thin-layer chromatography, and the fluorescence under UV light and the color development in 5% sulfuric acid ethanol were observed. Similar fractions were combined to obtain four extract components: Fr.1, Fr.2, Fr.3, and Fr.4.

[0073] (3) Fr.2 (160g) was subjected to normal phase silica gel column chromatography (200-300 mesh) with a gradient elution of petroleum ether / ethyl acetate. The gradient elution was performed by first elution with a mixed solution of petroleum ether and ethyl acetate in a mass ratio of 20:1, then with a mixed solution of petroleum ether and ethyl acetate in a mass ratio of 10:1, then with a mixed solution of petroleum ether and ethyl acetate in a mass ratio of 5:1, and finally with petroleum ether. TLC analysis combined similar fractions and separated them into 6 components (Fr.2A, Fr.2B, Fr.2C, Fr.2D, Fr.2E, Fr.2F).

[0074] (4) Fr.2D (12.7g) was fractionated by gradient elution using YMC-ODS reversed-phase column chromatography with methanol / water as solvent. The gradient elution was performed by first elution with a 70% methanol aqueous solution and then elution with 100% methanol. After elution, four fractions were obtained: Fr.2D1, Fr.2D2, Fr.2D3, and Fr.2D4.

[0075] (5) The Fr.2D3 fraction was separated by gradient elution with petroleum ether / acetone (30:1→1:1) on a silica gel column. The gradient elution was performed by first elution with a mixed solution of petroleum ether and acetone in a mass ratio of 30:1, and then by a mixed solution of petroleum ether and acetone in a mass ratio of 1:1. After elution, the fraction was purified by semi-preparative HPLC with a mobile phase of acetonitrile / water in a volume ratio of 85:15. The peaks were collected at retention times of 20 minutes and 28 minutes, respectively, to obtain compound 2 (5.3 mg) and compound 9 (49.0 mg).

[0076] (6) Fr.3 (170g) was separated by silica gel column elution with a gradient of petroleum ether / acetone (100:1→1:1). The gradient elution was performed by first elution with a mixed solution of petroleum ether and acetone at a mass ratio of 100:1, and then by a mixed solution of petroleum ether and acetone at a mass ratio of 1:1. After elution, 11 fractions were obtained, namely Fr.3A, Fr.3B, Fr.3C, Fr.3D, Fr.3E, Fr.3F, Fr.3G, Fr.3H, Fr.3I, Fr.3J, and Fr.3K.

[0077] (7) The mother liquor of Fr.3B (27.7g) was first eluted by gradient elution on a C18 reversed-phase column with methanol / water as the eluent. The gradient elution was performed by first elution with a 75% methanol aqueous solution and then by a second elution with 100% methanol. After elution, the solution was further separated by gradient elution on a silica gel column with petroleum ether / acetone as the eluent. The petroleum ether / acetone gradient elution was performed by first elution with a mixed solution of petroleum ether and acetone in a mass ratio of 100:1 and then by a mixed solution of petroleum ether and acetone in a mass ratio of 10:1. After elution, the solution was further purified by semi-preparative HPLC with a mobile phase of acetonitrile / water in a volume ratio of 80:20. The peaks were collected at a retention time of 16 minutes to obtain compound 3 (8.0mg).

[0078] (8) Fr.3F (5.5 g) precipitated as a white solid, which was purified by semi-preparative HPLC with a mobile phase of acetonitrile / water mixed solvent of 88:22. Compound 8 (6.6 mg) was obtained after a retention time of 20 minutes.

[0079] (9) Separation was performed by Fr.3F silica gel column chromatography with gradient elution of petroleum ether and acetone (30:1→1:1). Six fractions were obtained after elution, namely Fr.3F1, Fr.3F2, Fr.3F3, Fr.3F4, Fr.3F5, and Fr.3F6. Fr.3F5 (1.82 g) showed only one purple main spot by TLC thin-layer chromatography. After elution with a mixed solvent of acetonitrile / water at a volume ratio of 87:23 in a semi-preparative HPLC, compound 1 (15.0 mg), compound 7 (6.6 mg), and compound 4 (6.3 mg) were obtained after retention times of 19, 21, and 25 minutes, respectively.

[0080] (10) Fr.3K (6.5 g) was eluted by Sephadex LH-20 gel column chromatography (chloroform:methanol 1:1) to obtain 5 fractions (Fr.3K1, Fr.3K2, Fr.3K3, Fr.3K4, Fr.3K5); Fr.3K3 (1.7 g) was purified by repeated positive silica gel separation and then prepared by HPLC. The eluent was a mixed solvent of acetonitrile / water with a volume ratio of 95:5. The retention times were 16 and 20 minutes, respectively, to separate compound 5 (9.0 mg) and compound 6 (5.3 mg).

[0081] I. Identification of Compound Structure

[0082] The structures of known compounds were determined by NMR spectroscopy analysis combined with comparison of their NMR data. The known compounds isolated and identified from *Melia azedarach* are as follows:

[0083] 3β,16β-Dihydroxyeupha-7,24-dien-21-oic acid methyl ester (compound 7);

[0084] (3S,5R,9R,10R,13S,14S,16S,17S,20S)-3,16,21-trihydroxy-tirucalla-7,24-diene (compound 8);

[0085] Toosendine E (compound 9).

[0086] Compound 1

[0087] Compound 1 is a colorless, transparent, blocky crystal, according to HR-ESIMS spectra ( Figure 14 The quasi-molecular ion peak in positive ion mode is 477.3329 m / z ([M+Na)). + calcd for C 30 H 46 O3Na, 477.3339), corresponding to the molecular formula C 30 H46 O3 has eight degrees of unsaturation. From 1 The H NMR spectrum showed seven singlet methyl signals and one doublet methyl signal. 13 Thirty carbon signals were inferred from the C10 NMR and HSQC two-dimensional spectra, including eight methyl groups, seven methylene groups, seven methine groups, and eight quaternary carbons (as shown in Tables 1 and 2).

[0088] The above data indicates that compound 1 is a tetracyclic triterpenoid, and its structure is very similar to that of kulinone, the only difference being that the C-6 position on ring B is oxidized to a ketone. HMBC spectra show that H-5 to C-6 (δ) C The relevant signals of 198.4) and H-7 to C-6 (δ) C The relevant signals from 198.4 further confirm this. In addition, the HMBC-related signals from H-15 to C-14 and C-20, combined with... 1 H- 1 The H-15 / H-16 / H-17 fragments in the HCl COSY spectrum show that the hydroxyl group is located at C-16. The configurations of H-5 and H-9 in this structure are determined by NOESY spectroscopy (…). Figure 2 The correlation signals between H3-28 and H-5, and H-5 and H-9 were determined to be α-orientation; secondly, the correlations between H3-18 and H-16, H-15α and H-16, and H3-18 and H-20 proved that H-16 and H-20 are in the same plane of the structure and are α-orientation; while the configurations of H-17 and H3-21 were determined to be β-orientation by the correlation signals between H3-30 and H-17 / H-15β, and H-17 and H3-21; thus, the relative configuration of compound 1 was determined. Finally, bulk crystals precipitated in a methanol:water (7:3) solvent, and the results were determined by X-ray single crystal analysis (Cu target) (CCDC number 2291263). Figure 3 The Flack constant was -0.01(5), and its absolute configuration was determined to be 5R,9R,10R,13S,14S,16S,17S,20S. It was identified as a new compound by a search of the SciFinder database and named meliasanine A.

[0089] Compound 2

[0090] Compound 2 is a white amorphous powder with the molecular formula C2. 31 H 48 O5, obtained from high-resolution mass spectrometry at m / z 523.3393 ([M+Na)). + ,calcd for 523.3393,C 31 H 48O5Na) yields an unsaturation degree of 8. Analysis of one-dimensional and two-dimensional NMR data shows that compound 2 has a structure similar to the known compound toosendine E, differing only in the 3-OH substitution. Based on the C-H data δ in compound 2... H 3.35,δ C 76.8; Hydrocarbon data δ in toosendine E H 3.21,δ C 79.2. Toosendine E and C-3 are epimers. (Based on the NOESY spectrum) Figure 2 The correlation between H-3 / H3-29 determined that the hydroxyl group at the C-3 position is in the α configuration. Finally, through quantum computational chemistry, the experimental values ​​were compared with the ECD curve of compound 1. Figure 3 The absolute configuration of compound 2 was determined to be 3R,5R,9R,10R,13S,14S,16S,17S,20S. A search of the SciFinder database identified it as a new compound, which was named meliasanine B.

[0091] Compound 3

[0092] Compound 3 is a white amorphous powder with the molecular formula C3. 31 H 46 O4, obtained from high-resolution mass spectrometry at m / z 505.3281 ([M+Na)). + ,calcd for 505.3281,C 31 H 46 O4Na) yielded an unsaturation degree of 9. Its NMR data (Tables 1 and 2) show 7 methyl groups [δ] H 1.68(s), 1.59(s), 1.34(s), 1.31(s), 1.28(s), 0.87(s) and 0.77(s), 3H], and methoxy[δ] H 3.72(s); δ C 51.7] 2 sets of double bond signals [δ H 5.56(dd,J=6.0,3.0Hz),5.83(d,J=6.0Hz),1H; δ C [115.8, 118.4], the above signals suggest that compound 3 is similar to the known compound 3β,16β-Dihydroxyeupha-7,24-dien-21-oic acid methyl ester (7), suggesting that compound 3 is also a euphorbia triterpenoid. The structural difference from the known compound is the absence of a hydroxyl group signal and the presence of an additional carbonyl group signal, as indicated by HMBC ( Figure 1 H3-28(δ) H 1.31) / C-3(δ C214.9) is related. Furthermore, H3-19 / H3-28 is associated with C-5. 1 H- 1 H COSY spectrum ( Figure 1 H-6 / H-7 correlation was used to determine the B-ring exocyclic diene substitution in compound 3. Its relative configuration was determined using NOESY (…). Figure 2 The correlation between H3-18 / H-16 / H-20 confirms that H-16 and H-20 are in the α configuration. Finally, through quantum computational chemistry, the ECD curves of the experimental and calculated values ​​are compared. Figure 4 The absolute configuration of compound 3 was determined to be 9R,10R,13S,14S,16S,12R,17S,20S. A search of the SciFinder database revealed it to be a new compound, which was named meliasanine C.

[0093] Compound 4

[0094] Compound 4 is a white amorphous powder with the molecular formula C4. 31 H 48 O4, obtained from high-resolution mass spectrometry at m / z 507.3442 ([M+Na)). + ,calcd for 505.3281,C 31 H 46 O4Na) yielded an unsaturation degree of 9. Careful analysis of the NMR spectra of compounds 4 and 3 (Tables 1 and 2) revealed a striking similarity, suggesting that compounds 4 and 3 are analogs. The difference lies in the hydroxyl substitution at C-3 in compound 4, rather than a carbonyl group. This is confirmed by the correlation between H3-28 / 29 and C-3 in their HMBC spectra. The relative configuration of the C-3 hydroxyl group in compound 4 is NOESY ( Figure 2 It was determined that H-3 is related to H3-28, and C-3 is a hydroxyl group with a β configuration. Finally, through quantum computational chemistry, the ECD curves of the experimental and calculated values ​​were compared. Figure 4 The absolute configuration of compound 4 was determined to be 3S,9R,10R,13S,14S,16S,12R,17S,20S. Figure 1 The compound was identified as a new compound by the Scifinder database and named meliasanine D.

[0095] Compound 5

[0096] Compound 5 is a colorless crystal with the molecular formula C. 30 H 50 O3 was determined by high-resolution mass spectrometry at m / z 481.3647 ([M+Na)). + ,calcdfor 481.3652,C 30 H 50According to O3Na, its degree of unsaturation is 6. Combined with... 1 H NMR and 13 C10 NMR (Tables 1 and 2) and comparison show that it has a similar planar structure to indicalilacol B, except that compound 7 has an additional set of trisubstituted olefin double bonds, which are located at H-5 and H3-19 and Sp. 2 Quaternary carbon (δ C 145.0) and the HMBC related signals of H3-18 and C-12, combined with the H-11 and H2-12 1 H- 1 H COSY is associated with bit C-9 ( Figure 1 The lower-field shift of the chemical shift at H-23 indicates the presence of an ester carbonyl group at C-21. This, combined with the COSY correlation between H-17 / H-20 / H2-22 / H-23 and the H2-22 and ester carbonyl carbons (δ...), further supports this finding. C The HMBC cross signal peak at 178.1 further confirms the presence of an ester carbonyl group at C-21.

[0097] In the NOESY spectrum, due to the presence of the three angular methyl groups H3-28, H3-29, and H3-19... 1 The chemical shift values ​​in the 1H NMR spectrum are very close, making it difficult to determine the chiral center of 3-OH from the spectrum. However, the chiral center of 3-OH can be determined by the coupling constant of H-3 (δ). H 3.46, brs) determined that the hydroxyl group at the C-3 position is in the α configuration. The NOESY correlation signals of H-20 and H-23 in this structure indicate that they are coplanar, but the relative configuration of OH-24 on the side chain cannot be elucidated. Finally, suitable crystals of compound 5 were obtained from a methanol-water (9:1) solvent, and the results were determined by X-ray single crystallography (Cu target) (CCDC number 2291266). Figure 3 The Flack constant was 0.18(13), and its absolute configuration was determined to be 3R,5R,17S,20S,23R,24R. It was identified as a new compound by a search of the SciFinder database and named meliasanine E.

[0098] Compound 6

[0099] The HR-ESIMS data of compound 6 are identical to those of compound 5, confirming that they share the same molecular formula. Analysis of the 1D NMR data of compounds 8 and 7 reveals highly similar spectra (Tables 1 and 2), indicating they possess the same planar structure. However, significant differences exist in the chemical shifts of the carbon and hydrogen atoms at positions 23, 24, 25, 26, and 27 of the side chains (Tables 1 and 2). Therefore, it is speculated that compounds 6 and 5 may be C-24 epimers, based on the difference in the coupling constant between H and H (6:δ).H 3.67J = 3.6Hz; 5:δ H The existence of such epimers is proven by the result of 3.29 J = 9.6 Hz. Finally, through quantum computational chemistry, the ECD curves of the experimental and calculated values ​​are compared. Figure 4 The absolute configuration of compound 6 was determined to be 3R, 5R, 17S, 20S, 23R, 24S. A search of the SciFinder database revealed it to be a new compound, which was named meliasanine F.

[0100] Table 1. Compounds 1–6 1 H-NMR data (600MHz; δ) H in CDCl3)

[0101]

[0102] Table 2 Compounds 1–6 13 C-NMR data (600MHz; in CDCl3)

[0103]

[0104] II. Tests on the anti-inflammatory activity of the compounds

[0105] 2.1 Effect of the compound on RAW 264.7 cell viability as determined by MTT assay

[0106] After cell resuscitation and passage, when the cells are in the logarithmic growth phase, collect the cells and calculate their concentration. Adjust the cell suspension concentration and add 90 μL of cell suspension (5 × 10⁻⁶) to each well of a 96-well cell culture plate. 3 (cells / well). Incubate the cell culture plate at 37°C and 5% CO2 for 24 hours. Remove the plate and add 10 μM of the experimental sample to each well, with three replicates per group. Continue incubating for another 24 hours, observing cell status. Then, add 20 μL of MTT solution to each well and incubate for another 4 hours. Terminate the culture, discard the supernatant, and add 150 μL of DMSO solution to each well. Place the plate on a shaker and shake at low speed for 15 minutes. Measure the absorbance at 490 nm using a microplate reader and calculate the cell viability.

[0107] 2.2 Determination of NO concentration in cell culture medium using the Griess reagent method

[0108] Cells were loaded at 2.0 × 10 4Cells were seeded at a concentration of [number] cells / well in 96-well plates. Experimental groups included a control group, a model group, and an experimental group. Cells were incubated for 24 hours. After cell attachment, 10 μL of DMEM blank medium was added to the control group, and 10 μL of 1 μg / mL LPS solution was added to each of the remaining wells to stimulate cell inflammation for 1 hour. Different concentration gradients of sample solutions were added to the experimental groups, with three replicates per group. After incubation for 24 hours, cell supernatant was collected, and absorbance was measured at 540 nm according to the Griess kit instructions. Data were statistically analyzed using Prism and SPSS 20.0 software.

[0109] 2.3 Western Blot detection of the expression of proteins related to the NF-κB signaling pathway

[0110] After adjusting the cell suspension concentration according to the ratio, 1.5 ml of cell suspension was added to each well of a 6-well cell culture plate, and the plate was incubated overnight in a cell culture incubator. The experiment included one blank control group, one model group, and three experimental groups. The blank control group was supplemented with DMEM medium; the model group was supplemented with LPS solution at a final concentration of 1 μg / ml; and the experimental groups were prepared with three concentrations: each well was supplemented with LPS solution at a final concentration of 1 μg / ml and Compound 1 solutions at concentrations of 2, 5, and 10 μM, respectively. The plates were then incubated for another 24 hours. Total protein was extracted from the cells, and the expression levels of proteins related to the NF-κB signaling pathway were detected by Western blotting. The experiment was repeated in triplicate.

[0111] 2.4 Results of anti-inflammatory activity experiments

[0112] 2.4.1 Effects of the compound on the viability of RAW264.7 cells

[0113] To rule out that the inhibition of NO production in cells by the compound was due to its toxicity, the experiment first determined the effect of the monomeric compound on the viability of RAW 264.7 cells using the MTT assay. The cell viability results are as follows: Figure 5 As shown, the results indicate that compounds 1–9 were not cytotoxic at a concentration of 10 μM.

[0114] 2.4.2 Effects of the compound on LPS-induced NO production in RAW264.7 cells

[0115] The concentration of NO in RAW246.7 cell culture medium was detected using the Griess reagent method. At a concentration of 10 μM, compounds 1–9 significantly inhibited LPS-induced NO release from RAW246.7 cells compared to the model group. The results are as follows: Figure 6 show.

[0116] 2.4.3 Anti-inflammatory activity test results

[0117] IC50 of nitric oxide 50 The test results are shown in Table 3. The results show the IC50 values ​​of compounds 1, 7, and 9. 50 The values ​​were 2.47 μM, 4.09 μM, 1.35 μM, and 5.53 μM, respectively, and were similar to the positive control drug indomethacin (IC50). 50 Compared to 13.18 μM, it exhibited stronger inhibitory activity. Other compounds showed weak anti-inflammatory activity, with inhibition rates greater than 10 μM.

[0118] Table 3 shows the in vitro anti-inflammatory activity (IC50) of the compounds. 50 (μM)

[0119]

[0120] a Positive control group

[0121] 2.4.4 Anti-inflammatory mechanism of compound 1

[0122] NO inhibition rate and IC50 of the compound 50 Based on the test results, novel compound 1 was selected for further in vitro anti-inflammatory mechanism experiments. This experiment used Western blotting to detect the expression levels of iNOS, COX-2, IκBα, P-IκBα, and GAPDH proteins in cells. Figure 7 As shown in the Western blot results, LPS treatment significantly increased the expression of iNOS, COX-2, and P-IκBα in RAW 264.7 cells, while the addition of compound 1 significantly downregulated the expression of iNOS, COX-2, and P-IκBα in a concentration-dependent manner. This result indicates that compound 1 exerts its anti-inflammatory activity through the NF-κB signaling pathway, demonstrating that compound 1 has a significant anti-inflammatory effect.

[0123] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0124] Finally, it should be noted that the embodiments disclosed in this invention are merely preferred embodiments of this invention and are only used to illustrate the technical solutions of this invention, not to limit it. Although this invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this invention.

Claims

1. The application of a euphorbia triterpenoid extracted from *Melia azedarach* in the preparation of anti-inflammatory drugs, characterized in that: The chemical structural formula of the euphorbia triterpenoid is: ； In formula (I), R1 is =O; R2 is =O; R3 is β-OH; and R4 is -CH3.

2. A method for preparing euphorbia-type triterpenes extracted from *Melia azedarach*, characterized in that: Includes the following steps: (1) 23.0 kg of Sichuan pepper bark was dried and pulverized. It was extracted three times by hot reflux with 3×150 L of 95% ethanol for 2.5 h each time. The ethanol solvent was recovered by vacuum concentration. 1.6 kg of the resulting dark brown extract was suspended in water and extracted four times each with ethyl acetate and n-butanol. The extraction solvent was recovered to obtain 800 g of ethyl acetate extract of Sichuan pepper bark. (2) Select the ethyl acetate extract of Sichuan pepper bark and add an appropriate amount of C18 polyamide as a reverse phase material in a 1:1 ratio to prepare the sample; use C18 reverse phase column chromatography to perform coarse fractionation of the extract, and use methanol / water gradient elution. The gradient elution is to first use 40% methanol aqueous solution for the first elution, and then use 100% methanol for the second elution; the fractions are detected and analyzed by TLC thin layer chromatography, and the fluorescence of the fractions under ultraviolet light and the color development of 5% sulfuric acid ethanol are observed. Similar fractions are combined to obtain a total of 4 fractions of extract Fr.1, Fr.2, Fr.3 and Fr.

4. (3) Take 170 g of Fr.3 and elute it with a silica gel column using a gradient elution of petroleum ether / acetone from 100:1 to 1:

1. The gradient elution separation is to first elute with a mixed solution of petroleum ether and acetone at a mass ratio of 100:1, and then elute with a mixed solution of petroleum ether and acetone at a mass ratio of 1:

1. After elution separation, 11 fractions are obtained, namely Fr.3A, Fr.3B, Fr.3C, Fr.3D, Fr.3E, Fr.3F, Fr.3G, Fr.3H, Fr.3I, Fr.3J, and Fr.3K. (4) Separation by gradient elution of petroleum ether and acetone in a 30:1 to 1:1 ratio using Fr.3F silica gel column chromatography. After elution, six fractions were obtained, namely Fr.3F1, Fr.3F2, Fr.3F3, Fr.3F4, Fr.3F5, and Fr.3F6. 1.82 g of Fr.3F5 was analyzed by TLC thin-layer chromatography and found to have only one purple main spot. After elution by semi-preparative HPLC with a mixed solvent of acetonitrile / water in a volume ratio of 87:23, 15.0 mg of compound 1 was obtained after a retention time of 19 minutes. The chemical structural formula of compound 1, a euphorbia-type triterpene, is as follows: ； In formula (I), R1 is =O; R2 is =O; R3 is β-OH; and R4 is -CH3.

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