Neocadinane type sesquiterpene compound fisduterpene and application thereof in preparation of anti-inflammatory drugs

Abstract

The invention provides a neocadinane type sesquiterpene compound fisduterpene and application thereof in preparation of anti-inflammatory drugs, the compound is extracted from dry stems of fissistigma oldhamii, the structural formula of the compound is determined through spectrum analysis, the molecular formula of the compound is C15H22O3, and the compound belongs to neosesquiterpenes. The preparation method comprises the following steps: extracting stems with 60-80% ethanol, carrying out vacuum concentration, extracting with petroleum ether and ethyl acetate, and carrying out silica gel column chromatography, Sephadex LH-20 column chromatography and high performance liquid chromatography purification. Pharmacological experiments show that the compound has a remarkable inhibiting effect on LPS-induced BV2 cell NO release, IC50 is 5.63 + / -0.22 mu M, the activity is superior to that of indometacin, and the compound can be used as a candidate component of anti-inflammatory drugs and provides a material basis and an application prospect for research and development of novel anti-inflammatory drugs.

Description

Technical Field

[0001] This invention relates to the field of phytochemistry, and in particular to a novel juniperane-type sesquiterpene compound, fisduterpene, and its application in the preparation of anti-inflammatory drugs. Background Technology

[0002] Inflammation is a complex defense response of the body to external stimuli or damage. Moderate inflammation helps tissue repair, but excessive or chronic inflammation may cause a variety of diseases such as rheumatoid arthritis and neurodegenerative diseases. Therefore, the development of highly effective and low-toxicity anti-inflammatory drugs is of great clinical significance.

[0003] Natural products are an important source of anti-inflammatory drugs, among which sesquiterpenes have attracted much attention due to their diverse structures and broad biological activities. Juniperane-type sesquiterpenes, as an important branch of the sesquiterpene family, have been reported to have anti-inflammatory, antibacterial, and antitumor activities, but their distribution in natural plants and their specific mechanisms of action still need to be further explored.

[0004] Paradise Melon Tree ( Fissistigma tientangense Tsiang &P. T. Li) is a plant belonging to the genus *Calamus* in the family Annonaceae. Modern pharmacological studies have shown that it is rich in alkaloids, sesquiterpenes, and phenolic acids, possessing potential medicinal value. However, current research on this plant mainly focuses on alkaloid components, and systematic studies on its juniperane-type sesquiterpenes and their anti-inflammatory activities have not been reported in depth. The structural identification and pharmacological mechanisms of action of related compounds remain a gap. Summary of the Invention

[0005] Therefore, this invention isolates a new juniperane-type sesquiterpene compound from *Cinnamomum camphora* and clarifies its anti-inflammatory activity, providing a scientific basis for expanding the medicinal value of this plant and developing new anti-inflammatory drugs.

[0006] The technical solution of this invention is achieved as follows: a novel juniperane-type sesquiterpene compound, fisduterpene, has the following structural formula: .

[0007] Furthermore, a method for preparing a novel juniperane-type sesquiterpene compound, fisduterpene, includes the following steps: (a) Extract the dried stems of the Heavenly Melon Tree with 60-80% v / v ethanol to obtain an extract; (b) Concentrate the extract under reduced pressure to form a paste-like extract; (c) Dilute the above-mentioned melon extract with distilled water to form a suspension, and extract with petroleum ether and ethyl acetate in sequence; (d) Concentrate the ethyl acetate extract to obtain an extract; (e) Silica gel column chromatography of the extract: the first gradient uses petroleum ether-ethyl acetate mixed solvent, the second gradient uses ethyl acetate-methanol mixed solvent for silica gel column chromatography with increasing polarity, the fractions are collected and combined by TLC detection to obtain 9 components, numbered as Fr.1-Fr.9; (f) Fr.3 is columned on 300-400 mesh silica gel, eluted with petroleum ether solution containing 20-40% v / v ethyl acetate, and subjected to thin layer chromatography with chloroform solution containing 8-12% v / v methanol as the developing agent, and 5 components Fr.3-1, Fr.3-2, Fr.3-3, Fr.3-4 and Fr.3-5 are obtained by combining the fractions according to the chromatographic effect; (g) Fr.3-4 is subjected to Sephadex LH-20 column chromatography with methanol solution containing 40-60% v / v chloroform as the eluent to remove pigments to obtain component Fr.3-4-1; (h) Fr.3-4-1 is subjected to high performance liquid chromatography preparation with eluent of 30-40:60-70 v / v acetonitrile and water, and the target peak is collected to obtain fisduterpene.

[0008] Further, the step (a) is extracted for 4-6 times, 6-8 days each time, and the solid-liquid ratio g / mL of the stem of Feijoa sellowiana Betron ex Berg and ethanol is 1:5-10 during each extraction.

[0009] Further, the temperature of the step (b) is 40-60°C, and the pressure is -0.01~-0.10 MPa.

[0010] Further, the mass-volume ratio g / L of the Feijoa sellowiana Betron ex Berg extract and distilled water in the step (c) is 1000-1200:4.

[0011] Further, the mass-volume ratio g / L of the Feijoa sellowiana Betron ex Berg extract and petroleum ether in the step (c) is 1000-1200:4, and the extraction is performed 8-10 times; the mass-volume ratio g / L of the Feijoa sellowiana Betron ex Berg extract and ethyl acetate is 1000-1200:4, and the extraction is performed 8-10 times.

[0012] Further, the extraction temperature in the step (c) is 20-60°C, and the extraction time is 60-90 minutes.

[0013] Further, the concentration temperature in the step (d) is 40-60°C, and the negative pressure is -0.01~-0.10 MPa.

[0014] Furthermore, in step (e), the volume ratio of the petroleum ether-ethyl acetate mixed solvent is 1-100:1-100, and the volume ratio of the ethyl acetate-methanol mixed solvent is 1-100:1-100.

[0015] Furthermore, the application of the novel juniperane-type sesquiterpene compound fisduterpene in the preparation of anti-inflammatory drugs.

[0016] Compared with the prior art, the beneficial effects of the present invention are: A novel juniperane-type sesquiterpene compound, fisduterpene, was discovered. Its structural formula was confirmed through spectroscopic analysis, enriching the variety of sesquiterpenes and providing a new material basis for natural product chemistry research. An efficient and feasible method for preparing this compound was provided. Starting from the dried stems of *Fisduterpene spp.*, the target compound can be stably obtained through ethanol extraction, extraction, and chromatographic purification. The operation is standardized and reproducible, providing a guarantee for its subsequent research and application.

[0017] Pharmacological experiments confirmed that this compound has significant anti-inflammatory activity and a marked inhibitory effect on LPS-induced NO release from BV2 cells, with an IC50 value of [missing information]. 50 The concentration was 5.63±0.22 μM, showing superior activity compared to the positive control indomethacin. This provides a high-quality candidate ingredient for the development of novel anti-inflammatory drugs, possessing significant medicinal value and application prospects. This invention fully utilizes the plant resource of *Calamus sieboldii*, exploring its active ingredients and expanding the plant's medicinal development potential, which is of positive significance for the comprehensive utilization of natural drug resources. Attached Figure Description

[0018] Figure 1 The hydrogen spectrum of the compound fisduterpene.

[0019] Figure 2 This is the carbon spectrum of the compound fisduterpene. Detailed Implementation

[0020] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0021] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0022] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0023] Example 1: Preparation of sesquiterpenoids 9.5 kg of dried stems of *Gnaphalium affine* were extracted five times with 80% v / v ethanol for seven days each time, yielding an extract. The extract was concentrated under reduced pressure to a paste, yielding 1150 g of *Gnaphalium affine* extract. The extract was diluted with distilled water (4 L) to form a suspension, and then extracted sequentially with petroleum ether (4 L × 10 times) and ethyl acetate (4 L × 10 times). The ethyl acetate extract was concentrated to a paste (approximately 195 g) and subjected to silica gel column chromatography. Column chromatography was performed with a petroleum ether-ethyl acetate mixture (1-100:1-100, v / v) and ethyl acetate-methanol (1-100:1-100, v / v) in increasing polarity, collecting fractions of approximately 1000 mL each time. TLC analysis separated similar fractions into nine groups, Fr. 1–9. Fr.3 was eluted using a 300-400 mesh silica gel column with a 30% (v / v) ethyl acetate-petroleum ether mixture as the eluent. Thin-layer chromatography (TLC) was performed using a 10% (v / v) chloroform-methanol mixture as the developing solvent. Based on the chromatographic results, the fractions were combined to obtain five fractions: Fr.3-1, Fr.3-2, Fr.3-3, Fr.3-4, and Fr.3-5. Fr.3-4 was further purified by Sephadex LH-20 molecular sieve column chromatography with a 50% (v / v) chloroform-methanol mixture as the eluent to remove pigments, yielding fraction Fr.3-4-1. Fr.3-4-1 was then analyzed by high-performance liquid chromatography (HPLC) with acetonitrile and water in a 35:65 volume ratio to obtain the juniperane-type sesquiterpene compound fisduterpene.

[0024] The structural identification results of fisduterpene, a juniperane-type sesquiterpene compound, are as follows: Fisduterpene: a colorless paste.

[0025] No dark spots were observed at 254 nm UV; a purplish-red color was observed with 5% sulfuric acid-vanillin. The optical rotation was [insert value here]. The molecular formula of this compound was determined to be C15H22O3 by high-resolution HR-ESI-MS m / z 249.1495 [MH]-, with a calculated degree of unsaturation of 5. ¹H-NMR and DEPT revealed four methyl signals [δH 2.26 (3H,s), 1.14 (3H,s), 1.00 (3H,d,J=6.8Hz), and 0.46 (3H,d,J=6.8Hz)]. ¹³C-NMR and DEPT indicated six benzene ring carbons, three oxygen-linked sp³ hybrid carbons, one methylene group, one sp³ hybridized methine carbon, and four methyl carbons. Therefore, the compound fisduterpene is a sesquiterpene.

[0026] It can be seen that the 1H-1HCOSY correlation of H-2 / H-3 and H-8 / H-9 with HMBC spectra, H-14 with C-1 / C-2 / C-10, H-2 with C-1 / C-3 / C-4 / C-10, H-11 with C-3 / C-4 / C-5, and H-15 with C-6 / C-7 / C-8 respectively, confirms the planar structure of the compound as shown in the figure above. The relative configuration of the compound was determined by one-dimensional NOE. When irradiated with the hydrogen at position 2, there was no gain for either the methyl group at position 14 or the hydrogen at position 11, indicating that the 2-OH and 1 / 4-OH are not on the same side. Therefore, this compound is identified as a novel sesquiterpene compound of the juniperane type.

[0027] Table 1. 1H-NMR spectra of compound fisduterpene and 13 C-NMR data

[0028] Example 2: Pharmacological Activity Experiment A neuroinflammation model was established using lipopolysaccharide (LPS)-induced BV2 cells, and NO release from BV2 cells was measured using the Griess method. Specifically, BV2 cells were seeded at a density of 1×10⁵ cells / mL into 96-well plates, treated with LPS for 2 hours, and then incubated with a 40 µmol / mL compound for 24 hours. 50 µL of cell supernatant was collected, and 50 µL / well of Griess I reagent was added, followed by an equal volume of Griess II reagent. After mixing, the absorbance (A) was measured at 540 nm. Each sample was repeated three times. The NO release inhibition rate and IC50 were calculated. 50 .

[0029] Experimental results Experimental results showed that fisduterpene, a juniperane-type sesquiterpene, possessed significant NO release inhibitory activity, and its activity was superior to that of the positive control indomethacin, with an IC50 value of [missing information]. 50 The concentration is 5.63 ± 0.22 μM, which can be used for the preparation of anti-inflammatory drugs.

[0030] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A novel juniperane-type sesquiterpene compound, fisduterpene, characterized in that, Its structural formula is as follows: 。 2. The method for preparing the novel juniperane-type sesquiterpene compound fisduterpene as described in claim 1, characterized in that, Includes the following steps: (a) Extract the dried stems of the Heavenly Melon Tree with 60-80% v / v ethanol to obtain an extract; (b) Concentrate the extract under reduced pressure to form a paste-like extract; (c) Dilute the above-mentioned melon extract with distilled water to form a suspension, and extract with petroleum ether and ethyl acetate in sequence; (d) Concentrate the ethyl acetate extract to obtain an extract; (e) Silica gel column chromatography was performed on the extract: the first gradient was performed using a petroleum ether-ethyl acetate mixed solvent, and the second gradient was performed using an ethyl acetate-methanol mixed solvent in increasing polarity. The fractions were collected and similar fractions were combined by TLC to separate them into 9 components, which were numbered Fr.1-Fr.

9. (f) Fr.3 was loaded onto a 300-400 mesh silica gel column and eluted with a petroleum ether solution containing 20-40% v / v ethyl acetate. Thin-layer chromatography was performed with a chloroform solution containing 8-12% v / v methanol as the developing solvent. Based on the chromatographic results, the fractions were combined to obtain five fractions: Fr.3-1, Fr.3-2, Fr.3-3, Fr.3-4, and Fr.3-5. (g) After removing the pigments from Fr.3-4 by column chromatography using a methanol solution containing 40-60% v / v chloroform as the eluent, fraction Fr.3-4-1 was obtained. (h) Fr.3-4-1 was prepared by high performance liquid chromatography with acetonitrile and water in a volume ratio of 30-40:60-70 and the target peak was collected to obtain fisduterpene, a juniperane-type sesquiterpene compound.

3. The method for preparing the novel juniperane-type sesquiterpene compound fisduterpene as described in claim 1, characterized in that, Step (a) involves extraction 4-6 times, each time for 6-8 days, with the solid-liquid ratio of the stem of the Heavenly Melon Tree to ethanol being 1:5-10 g / mL during each extraction.

4. The method for preparing the novel juniperane-type sesquiterpene compound fisduterpene as described in claim 1, characterized in that, The temperature of the vacuum concentration process in step (b) is 40-60℃ and the pressure is -0.01~-0.10MPa.

5. The method for preparing the novel juniperane-type sesquiterpene compound fisduterpene as described in claim 1, characterized in that, In step (c), the mass-to-volume ratio of the *Citrus aurantiacus* extract to distilled water is 1000-1200:4 (g / L).

6. The method for preparing the novel juniperane-type sesquiterpene compound fisduterpene as described in claim 1, characterized in that, In step (c), the mass-to-volume ratio of *Hymenochloa praecox* extract to petroleum ether is 1000-1200:4 (g / L), and the extraction is performed 8-10 times; the mass-to-volume ratio of *Hymenochloa praecox* extract to ethyl acetate is 1000-1200:4 (g / L), and the extraction is performed 8-10 times.

7. The method for preparing the novel juniperane-type sesquiterpene compound fisduterpene as described in claim 1, characterized in that, In step (c), the extraction temperature is 20-60℃ and the extraction time is 60-90 minutes each time.

8. The method for preparing the novel juniperane-type sesquiterpene compound fisduterpene as described in claim 1, characterized in that, The concentration temperature in step (d) is 40~60℃, and the negative pressure is -0.01~-0.10MPa.

9. The method for preparing the novel juniperane-type sesquiterpene compound fisduterpene as described in claim 1, characterized in that, In step (e), the volume ratio of the petroleum ether-ethyl acetate mixed solvent is 1-100:1-100, and the volume ratio of the ethyl acetate-methanol mixed solvent is 1-100:1-100.

10. The use of fisduterpene, a novel juniperane-type sesquiterpene compound according to claim 1, in the preparation of an anti-inflammatory drug.

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