Pterosin sesquiterpenes, methods of making and uses thereof

Abstract

The application discloses a pterosin sesquiterpenoid compound and a preparation method and application thereof, belongs to the technical field of biological medicines, aims at the problems of a research blank of pterosin sesquiterpenoid compounds in Anemone raddeana and a lack of structure-confirmed anti-inflammatory active monomers, and provides a pterosin sesquiterpenoid compound with a structural formula as shown in the following.The compound preparation needs to first crush dried whole grass of Anemone raddeana, extract by heating and refluxing with an organic solution, concentrate under reduced pressure to obtain total extract; then, the extract is extracted by petroleum ether, ethyl acetate and n-butanol step by step, the ethyl acetate part extract is enriched, and the high-purity product is prepared through multi-step purification of a silica gel column, a C-18 reverse phase column, a gel column and semi-preparative liquid chromatography.In addition, the compound can dose-dependently inhibit the NO release of LPS-induced macrophages, and can be used for preparing anti-inflammatory drugs for treating or preventing inflammatory diseases related to excessive production of nitric oxide, fills the research blank, and provides a high-quality candidate component for anti-inflammatory drug research and development.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a sesquiterpene compound, its preparation method, and its application. Background Technology

[0002] *Pteris vittata* is a commonly used traditional Chinese medicine with various medicinal effects according to literature. However, systematic research on its known chemical components is still insufficient. In particular, regarding the pteridin sesquiterpenoids it may contain, current techniques lack reports of isolating compounds with well-defined structures and specific skeletons from this plant. Due to the complex chemical composition of plant extracts, the large number of structurally similar congeners, and the potentially low concentration of target compounds, the targeted discovery, isolation, and purification of novel single pteridin sesquiterpenoid compounds from *Pteris vittata* presents significant challenges. This research gap limits a deeper understanding of the medicinal material basis of *Pteris vittata* and the further development of its potential value. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide a pteridin sesquiterpene compound that, as the first structurally defined chemical entity isolated from the traditional Chinese medicine *Pistacia chinensis*, fills a research gap in such components of this plant and provides candidate compounds for the development of anti-inflammatory drugs.

[0005] To achieve these objectives and other advantages of the present invention, a pteridin sesquiterpene compound is provided, the structural formula of which is shown below: .

[0006] A method for preparing a sesquiterpene compound, comprising the following steps: S1: Pulverize the dried whole herb of *Haloxylon ammodendron* and mix it with a 60-95% (v / v) ethanol or methanol solution. Heat and reflux at 70-85 °C for 1-5 times, each extraction lasting 2-5 h. Combine the extracts and concentrate under reduced pressure to obtain the total extract. S2: The total extract is suspended in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. After each extraction, the mixture is allowed to stand to achieve phase separation. The solvent is recovered from the ethyl acetate fraction to obtain the ethyl acetate fraction extract. S3: The ethyl acetate fraction extract was subjected to silica gel column chromatography with an eluent gradient of dichloromethane to a mixed solvent of dichloromethane and methanol or ethanol, and then to methanol or ethanol, to obtain 8 fractions Fr.s1 to Fr.s8; S4: The Frs5 fraction was separated by medium-low pressure C-18 reversed-phase column chromatography, using gradient elution with methanol or ethanol at a volume fraction of 10% to 100% to obtain 10 fractions Fr.s5-1 to Fr.s5-10. S5: The Frs5-5 fraction was separated by silica gel column chromatography using petroleum ether, a mixed solvent of petroleum ether and ethyl acetate or acetone, and ethyl acetate or acetone as gradient elution to obtain three fractions Fr.s5-5-1 to Fr.s5-5-3. S6: Separate the Frs5-5-2 fraction by gel column chromatography using a mixed solvent of dichloromethane, methanol and petroleum ether in a volume ratio of 1:1:1, or by elution with methanol, to obtain three fractions Fr.s5-5-2-1 to Fr.s5-5-2-3; S7: The Frs5-5-2-2 fraction was separated by semi-preparative liquid chromatography using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as the mobile phase; wherein the volume fraction of methanol or acetonitrile was 20-70%, and the fern sesquiterpene compound was obtained by elution.

[0007] This invention first achieves preliminary enrichment of the target compound in the ethyl acetate fraction through solvent extraction, laying a high-quality material foundation for subsequent purification. Then, employing a strategy combining normal-phase silica gel column chromatography, Sephadex LH-20 dextran gel chromatography, reversed-phase C-18 column chromatography, and semi-preparative liquid chromatography, and by switching separation mechanisms multiple times, the differences in adsorption and partition behavior of the compound in different chromatographic systems are utilized to greatly improve the selectivity of the separation, effectively removing structurally similar impurities. Ultimately, this invention successfully isolated the novel pteridin sesquiterpene monomer from the traditional Chinese medicine *Pinellia ternata* for the first time, filling a research gap. Furthermore, its excellent purification efficiency is demonstrated with a purity of up to 99.5% and a stable yield. Simultaneously, the entire process parameters are well-defined and reproducible, providing a reliable and sufficient material guarantee for subsequent pharmacological research and drug development of this active compound.

[0008] Preferably, in step S1, the vacuum concentration is carried out at a temperature of 40-60 ℃ and a pressure of 0.05-0.1 MPa until it is concentrated to 10-20% of the total volume of the original extract.

[0009] Preferably, after the vacuum concentration in step S1, the extract drying step is further included: the concentrated total extract is vacuum dried at a temperature of 30-45 ℃ and a pressure of 0.01-0.05 MPa for 4-8 h until the moisture content of the extract is 3-5%.

[0010] Preferably, in step S2, the extraction is carried out at a temperature of 15-25 °C, and the mixture is allowed to stand for 20-40 min after each extraction to achieve phase separation. The volume ratio of petroleum ether to water suspension is 1:1 to 1:2. The volume ratio of ethyl acetate to water suspension is 1:1 to 1:2; The volume ratio of n-butanol to water suspension is 1:1 to 1:2.

[0011] Preferably, in step S1, the whole herb of *Hedyotis diffusa* is pulverized into particles with a diameter of 0.5-2 mm, and then mixed with ethanol or methanol at a weight-to-volume ratio of 1:8-1:12.

[0012] This invention pulverizes the raw materials into particles of 0.5-2 mm, which significantly increases the solid-liquid contact area, promotes the penetration of the solvent into the cell and the diffusion of the target compound into the solvent; and uses ethanol or methanol with a volume fraction of 60%-95% and a material-liquid ratio of 1:8 to 1:12 to balance the solubility of the target compound and the co-solubility of coexisting impurities.

[0013] The application of the pteridin sesquiterpene compound in the preparation of anti-inflammatory drugs.

[0014] Preferably, the anti-inflammatory drug is a drug for treating or preventing inflammatory diseases associated with excessive nitric oxide production.

[0015] This invention offers at least the following advantages: First, it provides a novel pteridin sesquiterpene compound, whose chemical structure, confirmed by spectroscopic data, is (2S,3S)-acetyl-14-nor-pterosin C, filling a research gap in the isolation of this type of compound from *Pteris vittata*. Second, through optimized extraction and multi-step purification processes, the target compound can be stably and efficiently isolated from *Pteris vittata* with good process reproducibility. Furthermore, this compound exhibits dose-dependent inhibition of LPS-induced nitric oxide release from macrophages in in vitro experiments, suggesting its potential as an anti-inflammatory active ingredient and providing a new material basis for the development of related drugs.

[0016] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0017] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0018] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0019] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0020] Example 1 A method for preparing a sesquiterpene compound, comprising the following steps: 20 kg of dried whole herb of *Haloxylon ammodendron* was extracted three times with 95% ethanol under reflux at 75 °C for 2 h each time. The extracts were combined and concentrated under reduced pressure (at 50 °C and 0.075 MPa) to obtain 3.1 kg of extract. The total extract was suspended in an equal mass of water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol at 20 °C. After each extraction, the extract was allowed to stand for 30 min to achieve phase separation. When using petroleum ether for extraction, the volume ratio of petroleum ether to water suspension is 1:1. When extracting with ethyl acetate, the volume ratio of ethyl acetate to water suspension is 1:1. And when using n-butanol for extraction, the volume ratio of n-butanol to water suspension is 1:1; The solvent was recovered to obtain 465 g of petroleum ether extract, 263 g of ethyl acetate extract, 527 g of n-butanol extract, and 1750 g of water extract. 263 g of the ethyl acetate fraction was separated by silica gel column chromatography with the following eluent gradient: starting from dichloromethane, passing sequentially through a mixture of dichloromethane and methanol (volume ratios of 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, and 60:40), and finally to methanol, yielding eight fractions Fr.s1 to Fr.s8. Fr.s5 was separated by medium-low pressure C-18 reversed-phase column chromatography, and eluted with a methanol gradient of 10% to 100% (increasing by 10%) to separate it into 10 fractions Fr.s5-1 to Fr.s5-10. Fr.s5-5 was separated by silica gel column chromatography with an eluent gradient of: starting with petroleum ether, then successively through a mixed solvent of petroleum ether and ethyl acetate (volume ratios of 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, and 60:40), and finally to ethyl acetate, yielding three fractions Fr.s5-5-1 to Fr.s5-5-3; Fr.s5-5-2 was separated by gel column chromatography, with the elution system being a mixed solvent of dichloromethane, methanol, and petroleum ether; wherein the volume ratio of dichloromethane, methanol, and petroleum ether was 1:1:1, yielding three components Fr.s5-5-2-1 to Fr.s5-5-2-3; Fr.s5-5-2-2 was separated using semi-preparative liquid chromatography (methanol / water = 43 / 57). t R =106.5 min), to obtain the pteridin sesquiterpene compound. The yield of the compound was 0.00017%, and the purity was 99.5%.

[0021] The structural identification data of the compound are as follows: 1H and 13C NMR data, Table 1; HRESIMS m / z 285.1099 [M + Na]+, calcd for 285.1097.

[0022] The NMR data (CD3OD) of the compounds of Formula I are shown in Table 1 below.

[0023] Table 1. NMR data of kaurane-type diterpenoids The structural formula of compound I is: .

[0024] Example 2 A method for preparing a sesquiterpene compound, which differs from Example 1, involves extraction with 60% methanol under reflux for 5 times, each time for 5 hours.

[0025] In this embodiment, the yield of the pteridin sesquiterpene compound was 0.00016%, and the purity was 99.3%.

[0026] Example 3 A method for preparing a sesquiterpene compound, differing from Example 1, involves separating the ethyl acetate fraction by silica gel column chromatography with an eluent gradient of: starting with dichloromethane, passing sequentially through a mixed solvent of dichloromethane and ethanol (volume ratios of 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30, and 60:40), and finally to ethanol, yielding eight fractions Fr.s1 to Fr.s8.

[0027] In this example, the yield of the pteridin sesquiterpene compound was 0.00018%, and the purity was 99.1%.

[0028] Example 4 A method for preparing a sesquiterpene compound, Fr.s5-5-2-2, differs from Example 1 in that it is separated using a semi-preparative liquid chromatography system (acetonitrile / water = 40 / 60). t R =89.5 min), to obtain the pteridin sesquiterpene compound.

[0029] In this example, the yield of the pteridin sesquiterpene compound was 0.00023%, and the purity was 93.4%.

[0030] Comparative Example 1 The difference between this comparative example and Example 1 is that the extraction solvent and method have been changed: 20 kg of dried whole plant of *Haloxylon ammodendron* was extracted three times by heating and reflux at 85 °C for 2 h each time, using pure water (0% ethanol by volume) as the extraction solvent. The remaining steps were exactly the same as in Example 1.

[0031] After processing, the final product was analyzed by high performance liquid chromatography. No characteristic peaks consistent with the retention time and ultraviolet characteristics of the target compound were detected, indicating that the target compound could not be effectively extracted under these conditions.

[0032] Comparative Example 2 The difference between this comparative example and Example 1 is that the extraction temperature was changed: 20 kg of dried whole plant of *Haloxylon ammodendron* was extracted three times at 50 °C using 95% ethanol as the extraction solvent, with each extraction lasting 2 h. The remaining steps were exactly the same as in Example 1.

[0033] The amount of the ethyl acetate extract obtained was 131 g, which was significantly less than that in Example 1.

[0034] In this example, the yield of the pteridin sesquiterpene compound was 0.00013%, and the purity was 98.4%.

[0035] Effect test RAW264.7 cells were seeded into 96-well plates and treated with 1... m Induction stimulation was performed using g / ml LPS, while the test compound (final concentration starting from 50 g / ml) was added. m M was initially diluted 2-fold, and the cells were treated with a drug-free group and an L-NMMA positive drug group as controls. After overnight culture, the culture medium was collected to detect NO production, and the absorbance was measured at 570 nm. MTS was added to the remaining culture medium to detect cell viability and rule out the toxic effects of the compound on the cells.

[0036] NO generation inhibition rate (%) = (OD570 nm of non-drug treatment group - OD570 nm of sample group) / OD570 nm of non-drug treatment group × 100%.

[0037] Table 2 NO generation inhibition rate As shown in Table 2, the inhibitory activity of the positive control L-NMMA exhibited a typical dose-dependent effect. Its inhibition rate gradually decreased from 54.02% at 50 μM to 9.55% at 3.15 μM. This trend validated the effectiveness of the experimental system and was consistent with its known pharmacological characteristics. Compound I of this invention also showed a significant dose-dependent anti-inflammatory effect. When the concentration decreased from 50 μM to 3.15 μM, its inhibition rate of NO release decreased from 81.01% to 6.25%, and its activity differed significantly from that of L-NMMA in different concentration ranges. In the high concentration range (50 μM, 25 μM), the anti-inflammatory activity of compound I was significantly better than that of the positive control, while in the low concentration range (12.5 μM and below), the inhibition rates of the two were similar. Based on the NO generation inhibition rate, the IC50 was calculated using the Reed & Muench method. 50 (50% inhibition concentration), the results are shown in Table 3.

[0038] Table 3. Inhibitory effects of compounds on LPS-induced NO release in RAW264.7 macrophages. a: Data are expressed as mean ± standard error. All experiments were repeated at least three times. b: L-NMMA was used as a positive control.

[0039] As can be seen from the results in Table 3, the sesquiterpenoid compound pteridin can inhibit NO production in a dose-dependent manner and has a good anti-inflammatory effect, suggesting that this compound may be the pharmacodynamic material basis for the anti-inflammatory effect of *Bambusa textilis*.

[0040] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A sesquiterpene compound, characterized in that, The structural formula of the compound is shown below: 。 2. A method for preparing the pteridin sesquiterpene compound as described in claim 1, characterized in that, Includes the following steps: S1: Pulverize the dried whole herb of *Haloxylon ammodendron* and mix it with a 60-95% (v / v) ethanol or methanol solution. Heat and reflux at 70-85℃ for 1-5 times, each extraction lasting 2-5 hours. Combine the extracts and concentrate under reduced pressure to obtain the total extract. S2: The total extract is suspended in water and extracted sequentially with petroleum ether, ethyl acetate and n-butanol. After each extraction, the mixture is allowed to stand to achieve phase separation. The solvent is recovered from the ethyl acetate fraction to obtain the ethyl acetate fraction extract. S3: The ethyl acetate fraction extract was subjected to silica gel column chromatography with an eluent gradient of dichloromethane to a mixed solvent of dichloromethane and methanol or ethanol, and then to methanol or ethanol, to obtain 8 fractions Fr.s1 to Fr.s8; S4: The Frs5 fraction was separated by medium-low pressure C-18 reversed-phase column chromatography, using gradient elution with methanol or ethanol at a volume fraction of 10% to 100% to obtain 10 fractions Fr.s5-1 to Fr.s5-10. S5: The Frs5-5 fraction was separated by silica gel column chromatography using petroleum ether, a mixed solvent of petroleum ether and ethyl acetate or acetone, and ethyl acetate or acetone as gradient elution to obtain three fractions Fr.s5-5-1 to Fr.s5-5-3. S6: Separate the Frs5-5-2 fraction by gel column chromatography using a mixed solvent of dichloromethane, methanol and petroleum ether in a volume ratio of 1:1:1, or by elution with methanol, to obtain three fractions Fr.s5-5-2-1 to Fr.s5-5-2-3; S7: The Frs5-5-2-2 fraction was separated by semi-preparative liquid chromatography using a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as the mobile phase; wherein the volume fraction of methanol or acetonitrile was 20-70%, and the fern sesquiterpene compound was obtained by elution.

3. The method for preparing the pteridin sesquiterpene compound according to claim 2, characterized in that, In step S1, the vacuum concentration is carried out at a temperature of 40-60 ℃ and a pressure of 0.05-0.1 MPa until the concentration is reduced to 10-20% of the total volume of the original extract.

4. The method for preparing the pteridin sesquiterpene compound according to claim 3, characterized in that, After the vacuum concentration in step S1, the extract drying step is also included: the concentrated total extract is vacuum dried for 4-8 hours at a temperature of 30-45 ℃ and a pressure of 0.01-0.05 MPa until the moisture content of the extract is 3-5%.

5. The method for preparing the pteridin sesquiterpene compound according to claim 2, characterized in that, In step S2, the extraction is carried out at a temperature of 15-25 °C, and the mixture is allowed to stand for 20-40 min after each extraction to achieve phase separation. The volume ratio of petroleum ether to water suspension is 1:1 to 1:

2. The volume ratio of ethyl acetate to water suspension is 1:1 to 1:2; The volume ratio of n-butanol to water suspension is 1:1 to 1:

2.

6. The method for preparing the pteridin sesquiterpene compound according to claim 2, characterized in that, In step S1, the whole herb of *Hedyotis diffusa* is crushed into particles with a diameter of 0.5-2 mm, and then mixed with ethanol or methanol at a weight-to-volume ratio of 1:8-1:

12.

7. The use of the pteridin sesquiterpene compound as described in claim 1 in the preparation of anti-inflammatory drugs.

8. The application as described in claim 7, characterized in that, The anti-inflammatory drug is a drug for treating or preventing inflammatory diseases associated with excessive nitric oxide production.

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