New oxoisoaporphine alkaloids in Bauhinia championii and extraction and separation method and application thereof

Abstract

This invention belongs to the field of pharmaceutical technology, specifically concerning novel oxidized isoapofenzyme alkaloids from *Ipomoea aquatica*, their extraction and separation methods, and their applications. The structural formula of the oxidized isoapofenzyme alkaloids is selected from one of the following formulas: In this invention, dried rhizomes of *Ipomoea aquatica* are pulverized, extracted with ethanol under reflux, and the extract is concentrated to obtain an extract. The extract is then subjected to a series of chromatographic analyses and gradient elutions using an acid-soluble, alkali-precipitated, and organic extraction process to obtain compounds 1-3. In vitro cell experiments show that compounds 1-3 can significantly inhibit LPS-induced NO release from RAW264.7, exhibiting anti-inflammatory medicinal properties and broad application prospects in the anti-inflammatory field.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and relates to new oxoaporphine alkaloids from Menispermum dauricum, and their extraction, separation methods and applications. Background Art

[0002] Menispermum dauricum Menispermum dauricum DC.): a plant of the genus Menispermum in the family Menispermaceae. The medicinal part is its dried rhizome, and the drug name is Beidougen. Beidougen is bitter, cold in nature, slightly poisonous, and belongs to the lung, stomach and large intestine meridians. It has the effects of clearing heat and detoxifying, regulating qi and dampness, dispelling wind and relieving pain, and is clinically commonly used to treat symptoms such as heat-toxin diarrhea, sore throat, and rheumatic arthralgia.

[0003] Alkaloid components are the most characteristic active components of Menispermum dauricum. Among them, oxoaporphine alkaloids are a class of natural compounds with unique structures and few numbers, belonging to a rare subclass of aporphine alkaloids. At present, only 33 oxoaporphine alkaloids have been found in nature, mainly existing in plants of the family Menispermaceae, and the most in Menispermum dauricum.

[0004] Inflammation is a complex pathological reaction mainly for defense against damage caused by various inflammatory factors in the body. It is an important defense mechanism of the body's immune system and usually causes symptoms such as swelling, heat and pain. In recent years, the incidence and prevalence of inflammation-related diseases have shown a significant upward trend globally, posing a severe public health challenge. At present, the conventional drugs for the clinical treatment of inflammatory diseases are mainly glucocorticoids and non-steroidal anti-inflammatory drugs. However, long-term use is likely to cause serious side effects, and their clinical applications are limited. Therefore, there is an urgent practical need and important scientific significance to develop new anti-inflammatory drugs with high efficiency and safety.

[0005] Therefore, how to find oxoaporphine alkaloids with anti-inflammatory activity from Menispermum dauricum is the key to solving the limited clinical application of current anti-inflammatory drugs. Summary of the Invention

[0006] The main purpose of the present invention is to provide an oxoaporphine alkaloid extracted and separated from the rhizome of Menispermum dauricum, and its extraction, separation methods and applications, so as to solve the problem of limited clinical application of current anti-inflammatory drugs.

[0007] To achieve the above purpose, the technical solution of the present invention is as follows: The first aspect of the present invention is an oxoaporphine alkaloid, the structural formula of which is selected from one of the following formulas: .

[0008] Further, the oxoaporphine alkaloid is extracted and separated from the rhizome of Menispermum dauricum.

[0009] The second aspect of the present invention is a method for preparing the oxidized isopofiphene-type alkaloids described in the first aspect, comprising the following steps: Step 1: Take dried rhizomes of *Pueraria lobata*, crush them, extract them by reflux with ethanol, combine the extracts and concentrate them to obtain an extract. The extract is obtained by the method of "acid dissolution, alkali precipitation, and organic extraction" to obtain the dichloromethane fraction of total alkaloids. The obtained fraction is subjected to silica gel column chromatography and then eluted with a dichloromethane-methanol system to obtain 28 fractions. Step 2: The obtained fractions were subjected to ODS column chromatography and then eluted with an ethanol-water system. The fractions were then combined by HPLC analysis to obtain 8 fractions Fr.A-Fr.H. Step 3: The obtained fraction Fr.F was subjected to silica gel column chromatography and gradient elution with a petroleum ether-ethyl acetate system. The fractions were then cross-merged by thin-layer chromatography and analytical HPLC to obtain 8 fractions Fr.1-8. Step 4: Fraction Fr.5 was separated using a preparative HPLC system with a methanol-water system to obtain nine fractions Fr.5.1-Fr.5.9; Fraction Fr.5.4 was eluted using a semi-preparative HPLC system with an acetonitrile-water system to obtain compound 3; Step 5: Fraction Fr.6 was separated using a preparative HPLC system with a methanol-water system to obtain four fractions Fr.6.1-Fr.6.4; Fraction Fr.6.3 was eluted using a semi-preparative HPLC system with an acetonitrile-water system to obtain compound 1; Step 6: Fraction Fr.8 was separated using a preparative HPLC system with a methanol-water system to obtain five fractions Fr.8.1-Fr.8.5; fraction Fr.8.3 was eluted using a semi-preparative HPLC system with an acetonitrile-water system to obtain compound 2.

[0010] Furthermore, in step 1, the volume ratio of dichloromethane to methanol is 50:1 to 1:3.

[0011] Furthermore, in step 2, the volume ratio of ethanol to water is 40:60-100:0.

[0012] Furthermore, in step 3, the volume ratio of petroleum ether to ethyl acetate is 50:1 to 1:3.

[0013] Furthermore, in step 4, the volume ratio of methanol to water is 82:18, and the volume ratio of acetonitrile to water is 49:51.

[0014] Furthermore, in step 5, the volume ratio of methanol to water is 70:30, and the volume ratio of acetonitrile to water is 58:42.

[0015] Furthermore, in step 6, the volume ratio of methanol to water is 69:31, and the volume ratio of acetonitrile to water is 50:50.

[0016] A third aspect of the invention is a pharmaceutical composition comprising the isoaporphine-type alkaloids described in the first aspect and pharmaceutically acceptable excipients.

[0017] Furthermore, the dosage form of the pharmaceutical composition is an oral dosage form or a non-oral dosage form, wherein the oral dosage form is a tablet, capsule, powder or granule, and the non-oral dosage form includes suppositories or injections.

[0018] The fourth aspect of the present invention is the use of the isopofifen-type alkaloids described in the first aspect, or the pharmaceutical composition described in the third aspect, in the preparation of anti-inflammatory drugs.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention isolated and extracted three novel isoaporphine-type alkaloids from the dried rhizome of *Phyllostachys edulis*, a plant belonging to the Menispermaceae family. The protective effects of these alkaloids against an in vitro inflammation model constructed using LPS-induced RAW264.7 macrophages were investigated. In vitro cell experiments showed that compounds 1-3 significantly inhibited LPS-induced NO release from RAW264.7 macrophages. Therefore, the isoaporphine-type alkaloids extracted and isolated in this invention have anti-inflammatory medicinal applications. Attached Figure Description

[0020] Figure 1 UV spectrum of compound 1; Figure 2 HRESIMS spectrum of compound 1; Figure 3 Compound 1 1 H-NMR spectrum; Figure 4 Compound 1 13 C-NMR spectrum; Figure 5 HSQC spectrum of compound 1 (600 MHz, CDCl3); Figure 6 HMBC spectrum of compound 1 (600 MHz, CDCl3); Figure 7 Compound 1 1 H- 1 H COSY spectrum (600 MHz, CDCl3); Figure 8 X-ray single-crystal diffraction structure of compound 1; Figure 9 UV spectrum of compound 2; Figure 10 HRESIMS spectrum of compound 2; Figure 11 Compound 2 1 H-NMR spectrum; Figure 12 Compound 2 13 C-NMR spectrum; Figure 13 HSQC spectrum of compound 2 (600 MHz, CDCl3); Figure 14 HMBC spectrum of compound 2 (600 MHz, CDCl3); Figure 15 Compound 2 1 H- 1 H COSY spectrum (600 MHz, CDCl3); Figure 16 X-ray single-crystal diffraction structure of compound 2; Figure 17 UV spectrum of compound 3; Figure 18 HRESIMS spectrum of compound 3; Figure 19 Compound 3 1 H-NMR spectrum; Figure 20 Compound 3 13 C-NMR spectrum; Figure 21 HSQC spectrum of compound 3 (600 MHz, CDCl3); Figure 22 HMBC spectrum of compound 3 (600 MHz, CDCl3); Figure 23 Compound 3 1 H- 1 H COSY spectrum (600 MHz, CDCl3); Figure 24 Compound 1 1 H- 1 H COSY, HMBC related diagrams; Figure 25 Compound 2 1 H- 1 H COSY, HMBC related diagrams; Figure 26 Compound 3 1 H- 1 H COSY, HMBC related diagrams; Figure 27Effects of compounds 1-3 on the viability of normal RAW264.7 cells (n=3); Figure 28 Effects of compounds 1-3 on NO release from RAW264.7 cells stimulated by LPS. Detailed Implementation

[0021] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0022] Example 1: Preparation of compounds 1-3 200 kg of dried rhizomes of *Ipomoea quamoclit* were pulverized and extracted three times by reflux with 95% ethanol for 3 hours each time. 30 kg of crude ethanol extract was obtained. A portion of the extract was taken, and an appropriate amount of 2% tartaric acid solution was added (to adjust the pH to 2) to completely dissolve the extract. All tartaric acid-soluble filtrates were combined, and ammonia was slowly added dropwise while stirring continuously to adjust the pH to 9-10, yielding a total alkaloid extract. The extract was repeatedly extracted with dichloromethane. All dichloromethane extracts were combined and concentrated under reduced pressure to obtain 1.0 kg of the dichloromethane fraction of total alkaloids from *Ipomoea quamoclit*. The fractions obtained from the dichloromethane extraction were subjected to silica gel column chromatography with gradient elution using a dichloromethane-methanol system (50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 1:1, 1:2, 1:3), collecting a total of 28 fractions. The obtained fractions were subjected to ODS column chromatography followed by gradient elution with an ethanol-water system (40:60, 60:40, 70:30, 80:20, 90:10, 100:0), and then combined by HPLC analysis to obtain a total of 8 fractions Fr.A-Fr.H. Fraction Fr.F was subjected to silica gel column chromatography with gradient elution with a petroleum ether-ethyl acetate system (50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 1:1, 1:2, 1:3), and then cross-combined by thin-layer chromatography and analytical HPLC to obtain a total of 8 fractions Fr.1-8. Fraction Fr.5 was separated using preparative HPLC with a methanol-water system of 82:18 to obtain nine fractions Fr.5.1-Fr.5.9; fraction Fr.6 was separated using preparative HPLC with a methanol-water system of 70:30 to obtain four fractions Fr.6.1-Fr.6.2; fraction Fr.8 was separated using preparative HPLC with a methanol-water system of 69:31 to obtain five fractions Fr.8.1-Fr.8.5; fraction Fr.5.4 was eluted using semi-preparative HPLC with an acetonitrile-water system of 49:51 to obtain compound 3 (7.3 mg); fraction Fr.6.3 was eluted using semi-preparative HPLC with an acetonitrile-water system of 58:42 to obtain compound 1 (4.6 mg); and fraction Fr.8.3 was eluted using semi-preparative HPLC with an acetonitrile-water system of 50:50 to obtain compound 2 (3.5 mg).

[0023] The results of systematic structural identification of compounds 1-3 are as follows: The planar structures of compounds 1-3 were identified using ultraviolet spectroscopy, high-resolution mass spectrometry, and one-dimensional and two-dimensional NMR techniques. The structures of compounds 1-2 were further determined using X-ray single-crystal diffraction.

[0024] Compound 1: Yellow needle-like crystals (methanol), readily soluble in dichloromethane. A positive Dragendorff colorimetric reaction indicates it is an alkaloid. UV (MeOH) λmax (logε): 260nm (0.23), suggesting the presence of a conjugated system in the compound. Figure 1 HRESIMS ( Figure 2 The quasi-molecular ion peak is given at 306.1133 [M+H]. + (calcd for C) 19 H 15 NO3, 306.1130), the molecular weight of this compound was determined to be 305, and the molecular formula is C. 19 H 15 NO3, the calculated degree of unsaturation is 13. 1 In the H-NMR (600 MHz, CDCl3) spectrum ( Figure 3 The image shows 15 hydrogen signals, including 2 methoxy proton signals. δ H 3.99 (3H, s, OCH3-9), δ H 4.05 (3H, s, OCH3-5); 1 methyl proton signal δ H 2.95 (3H, s, CH3-6); 2 cis-olefin proton signals δ H 8.65 (1H, d, J = 5.5 Hz, H-2), δ H 7.58 (1H, d, J = 5.5 Hz, H-3); δ H 7.85 (1H, d, J = 2.8 Hz, H-8), δ H 7.34 (1H, m, H-10), δ H 8.79 (1H, d, J = 8.8 Hz, H-11) The presence of 1, 2, 4 trisubstituted benzene ring hydrogen signals suggests that the compound has a basic skeleton of oxidized isoapofenine alkaloids, with 2 methoxy substitutions and 1 methyl substitution. 13 C-NMR (150 Hz, CDCl3) spectrum ( Figure 4 A total of 19 carbon signals were displayed, including one carbonyl carbon signal. δ C185.7 (C-7), 15 aromatic carbon signals, 2 methoxy carbon signals, and 1 methyl carbon signal. According to HSQC spectra (… Figure 5 It was assigned a direct carbon-hydrogen correlation. In the HMBC spectrum ( Figure 6 The correlations between H-2 and C-3a / 11b, H-3 and C-3b / 4, H-4 and C-3b / 6a, H-8 and C-7 / 10 / 11a, H-10 and C-8 / 9 / 11a, and H-11 and C-7a / 9 / 11b suggest that the compound possesses a basic parent nucleus of oxidized isopoflavone-type alkaloids. In HMBC, δ H 2.95 is associated with C-5 / 6 / 6a, suggesting that the methyl group is attached to C-6; furthermore, δ H 4.05 is associated with C-5, suggesting that the methoxy group is attached to C-5; δ H 3.99 is associated with C-9, suggesting that the methoxy group is linked to C-9. (This is followed by a continuation of the previous sentence, which is incomplete and requires further context.) 1 H- 1 H COSY ( Figure 7 The planar structure of the compound was determined. Figure 24 Compound 1 was obtained as a single crystal in methanol, and its structure was further determined by X-ray single-crystal diffraction using a Bruker D8 VENTURE PHOTON II diffractometer (CuKα), as shown below. Figure 8 .

[0025] Compound 2: Yellow needle-like crystals (methanol), readily soluble in dichloromethane. A positive Dragendorff colorimetric reaction indicates it is an alkaloid. λmax (logε): 255 nm (0.22), suggesting the presence of a conjugated system in the compound. Figure 9 HRESIMS ( Figure 10 The quasi-molecular ion peak is given at 307.1087 [M+H]. + (calcd for C) 18 H 14 N₂O₃ (307.1083), the molecular weight of this compound was determined to be 306, and the molecular formula is C₂O₃. 18 H 14 N₂O₃, its degree of unsaturation is calculated to be 13. 1 H-NMR (600MHz, CDCl3) spectrum ( Figure 11 The data shows 14 hydrogen signals, including 2 methoxy proton signals. δ H 4.01 (3H, s, OCH3-9), δ H4.08 (3H, s, OCH3-5); two active hydrogen signals of the amino group. δ H 10.66, 6.44 (each 1H, s, NH2-6); 3 cis-olefin proton signals δ H 8.64 (1H, d, J = 5.2 Hz, H-2), δ H 7.50 (1H, d, J = 5.2Hz, H-3), δ H 7.34 (1H, m, H-4); 1 aromatic proton signal δ H 7.12 (1H, s, H-4); δ H 7.99 (1H,d, J = 2.8 Hz, H-8), δ H 7.38 (1H, d, J = 8.9, 2.8 Hz, H-10), δ H 8.95 (1H, d, J = 8.9Hz, H-11) The presence of 1, 2, 4 trisubstituted benzene ring hydrogen signals suggests that the compound has a basic skeleton of oxidized isoapofenine alkaloids, with 2 methoxy substitutions and 1 amino substitution. 13 C-NMR (150 Hz, CDCl3) spectrum ( Figure 12 A total of 18 carbon signals were displayed, including one carbonyl carbon signal. δ C 183.7 (C-7); 15 aromatic carbon signals and 2 methoxy carbon signals. These data suggest that compound 2 is an oxidized isopofenline alkaloid. Based on HSQC spectra (… Figure 13 It was assigned a direct carbon-hydrogen correlation. In the HMBC spectrum ( Figure 14 In the HMBC, H-2 is associated with C-3a / 11b, H-3 with C-3b / 4, H-4 with C-3b, H-8 with C-7 / 10 / 11a, H-10 with C-11a, and H-11 with C-7a / 9 / 11b, suggesting that the compound possesses a basic parent nucleus of oxidized isopofiphene-type alkaloids. δ H 6.40 and δ C A value of 105.3 suggests that the amino group is attached to C-6; furthermore, δ H 4.01 and δ C 55.8 is relevant, suggesting that the methoxy group is linked to C-5; δ H 4.08 and δ C A value of 148.4 suggests that the methoxy group is linked to C-5. (This is related to the previous sentence.) 1 H- 1 H COSY ( Figure 15 The planar structure of the compound was determined, such as... Figure 25 Compound 2 was obtained as a single crystal in methanol, and its structure was further determined by X-ray single-crystal diffraction using a Bruker D8 VENTURE PHOTON II diffractometer (Cu Kα), as shown below. Figure 16 .

[0026] Compound 3: Yellow powder (methanol), readily soluble in dichloromethane. Dragendorff colorimetric reaction is positive, indicating an alkaloid. UV (MeOH) λmax (logε): 255 nm (0.20), suggesting the presence of a conjugated system in the compound. Figure 17 HRESIMS ( Figure 18 The quasi-molecular ion peak is given at 366.1343 [M+H]. + (calcd for C) 21 H 19 NO5, 366.1341), the molecular weight of this compound was determined to be 365, and the molecular formula is C. 21 H 19 NO5, its degree of unsaturation is calculated to be 13. 1 H-NMR (600 MHz, CDCl3) spectrum ( Figure 19 The data shows 19 hydrogen signals, including 3 methoxy proton signals. δ H 3.98 (3H, s, J = 5.5 Hz, OCH3-9), δ H 4.05 (3H, s, OCH3-5), δ H 4.26 (3H, s, OCH3-5); 1 methyl proton signal δ H 1.62 (3H, t, J = 7.5 Hz, CH3-2'); 1 methylene proton signal δ H 4.35 (2H,q,J = 7.0 Hz, CH2-1'); 2 cis-olefin proton signals δ H 8.68 (1H, d, J = 5.5 Hz, H-2), δ H 7.95 (1H, d, J = 5.5 Hz, H-3); δ H 7.88 (1H, d, J = 2.8 Hz, H-8), δ H 7.32 (1H, m, H-10), δ H 8.81(1H, d, J = 8.7 Hz, H-11) The presence of 1, 2, 4 trisubstituted benzene ring hydrogen signals suggests that the compound has a basic skeleton of oxidized isoapofen alkaloids, with 3 methoxy substitutions and 1 ethoxy substitution. 13 C-NMR (150 Hz, CDCl3) spectrum ( Figure 20 A total of 21 carbon signals were displayed, including one carbonyl carbon signal. δ C 181.5 (C-7), 15 aromatic carbon signals, 3 methoxy carbon signals, and 1 ethoxy carbon signal. According to HSQC spectra (… Figure 21 It was assigned a direct carbon-hydrogen correlation. In the HMBC spectrum ( Figure 22 In the ), H-2 is associated with C-3a / 11b, H-3 with C-3b / 4, H-8 with C-7 / 10 / 11a, H-10 with C-11a, and H-11 with C-7a / 9 / 11b, suggesting that the compound possesses a basic parent nucleus of oxidized isopofiphene-type alkaloids. In HMBC, from δ H 1.62 and δ C 70.9 related, δ H 4.36 is associated with C-6, suggesting that the ethoxy group is attached to C-6; furthermore, δ H 3.98 is associated with C-9, suggesting that the methoxy group is linked to C-9; δ H 4.05 is associated with C-5, suggesting that the methoxy group is attached to C-5; δ H 4.26 is related to C-4, suggesting that the methoxy group is attached to C-4. (This is in conjunction with...) 1 H- 1H COSY ( Figure 23 The planar structure of the compound was determined, such as... Figure 26 .

[0027]

[0028]

[0029] Example 2: Investigation of the anti-inflammatory activity of compounds 1-3 The MTT assay was used to determine the viability of RAW264.7 cells and the highest safe and effective concentration of the compound. The anti-inflammatory effect of the compound on lipopolysaccharide (LPS)-induced mouse macrophage RAW264.7 cells was determined using a NO assay kit, with indomethacin as a positive control. RAW264.7 cells in the logarithmic growth phase were collected, gently pipetted to prepare a homogeneous cell suspension, counted using a cell counting chamber, and then the cell density was adjusted to 4 × 10⁶ cells / cells. 5 Cells were seeded at a rate of 500 μL per well in a 48-well plate. After seeding, the 48-well plates were placed in a 37°C, 5% CO2 cell culture incubator for static culture. After 12 h of cell adhesion and growth, the original culture supernatant in each well was carefully discarded, and drug intervention was initiated. The experiment included a blank group, a model group (containing 1 μg / mL LPS), and a positive control group (containing 1 μg / mL LPS + 10 μM indomethacin). Simultaneously, for each test drug, three concentrations (high, medium, and low) were selected within the previously determined safe concentration range to establish a treatment group. After drug administration, the culture plate was incubated under the same conditions for 24 h. Then, the cell supernatant from each well was collected, gently centrifuged, and 50 μL of the supernatant was added to the corresponding well of a 96-well plate. First, 50 μL of Griess reagent I was added, and after 5 min in the dark, 50 μL of Griess reagent II was added. After thorough mixing, the absorbance of each well was measured at 540 nm using a microplate reader. The concentration of NO in the supernatant was calculated based on the standard curve, and analysis was performed using Graphad Prism 9.5. The results showed that ( Figure 27 The safe concentrations for each compound were: 25 μM for compound 1, 0.78 μM for compound 2, and 1.56 μM for compound 3. Compounds 1-3 all exhibited varying degrees of anti-inflammatory activity, with compound 2 showing the most significant anti-inflammatory activity (IC50). 50 The value was 2.12 ± 0.09 μM ( Figure 28 ).

[0030] The above description is only for better explaining the embodiments of the present invention and is not intended to limit them. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention shall fall within the scope of the present invention.

Claims

1. An oxidized isoaporphine-type alkaloid, characterized in that, The structural formula is selected from one of the following formulas: 。 2. The isoaporphine-type alkaloid according to claim 1, characterized in that, The isoapofene alkaloids were extracted and isolated from the rhizome of *Pueraria lobata*.

3. The method for preparing oxidized isopofiphene-type alkaloids according to claim 1 or 2, characterized in that, Includes the following steps: Step 1: Take dried rhizomes of *Pueraria lobata*, crush them, extract them by reflux with ethanol, combine the extracts and concentrate them to obtain an extract. The extract is obtained by "acid dissolution, alkali precipitation, and organic extraction" to obtain the dichloromethane fraction of total alkaloids. The obtained fraction is subjected to silica gel column chromatography and then eluted with a dichloromethane-methanol system to obtain 28 fractions. Step 2: The obtained fractions were subjected to ODS column chromatography and then eluted with an ethanol-water system. The fractions were then combined by HPLC analysis to obtain 8 fractions Fr.A-Fr.H. Step 3: The obtained fraction Fr.F was subjected to silica gel column chromatography and gradient elution with a petroleum ether-ethyl acetate system. The fractions were then cross-merged by thin-layer chromatography and analytical HPLC to obtain 8 fractions Fr.1-8. Step 4: Fraction Fr.5 was separated using a preparative HPLC system with a methanol-water system to obtain nine fractions Fr.5.1-Fr.5.9; Fraction Fr.5.4 was eluted using a semi-preparative HPLC system with an acetonitrile-water system to obtain compound 3; Step 5: Fraction Fr.6 was separated using a preparative HPLC system with a methanol-water system to obtain four fractions Fr.6.1-Fr.6.4; Fraction Fr.6.3 was eluted using a semi-preparative HPLC system with an acetonitrile-water system to obtain compound 1; Step 6: Fraction Fr.8 was separated using a preparative HPLC system with a methanol-water system to obtain five fractions Fr.8.1-Fr.8.5; fraction Fr.8.3 was eluted using a semi-preparative HPLC system with an acetonitrile-water system to obtain compound 2.

4. The method for preparing oxidized isopofiphene-type alkaloids according to claim 3, characterized in that, In step 1, the volume ratio of dichloromethane to methanol is 50:1 to 1:3; in step 2, the volume ratio of ethanol to water is 40:60 to 100:

0.

5. The method for preparing oxidized isopofiphene-type alkaloids according to claim 3, characterized in that, In step 3, the volume ratio of petroleum ether to ethyl acetate is 50:1 to 1:

3.

6. The method for preparing oxidized isoapofen-type alkaloids according to claim 3, characterized in that, In step 4, the volume ratio of methanol to water is 82:18, and the volume ratio of acetonitrile to water is 49:

51.

7. The method for preparing oxidized isopofiphene-type alkaloids according to claim 3, characterized in that, In step 5, the volume ratio of methanol to water is 70:30, and the volume ratio of acetonitrile to water is 58:42; in step 6, the volume ratio of methanol to water is 69:31, and the volume ratio of acetonitrile to water is 50:

50.

8. A pharmaceutical composition, characterized in that, It comprises the oxidized isoapofene alkaloids as described in claim 1 or 2, and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, The dosage form of the pharmaceutical composition is an oral dosage form or a non-oral dosage form, wherein the oral dosage form is a tablet, capsule, powder or granule, and the non-oral dosage form includes suppositories or injections.

10. The use of the isoapofene alkaloid of claim 1 or 2, or the pharmaceutical composition of claim 8 or 9, in the preparation of an anti-inflammatory drug.

Images