A steroidal compound aglatestate A, a preparation method and application thereof

Abstract

The present application relates to a kind of steroid compound Aglatestate A, its preparation method and purposes, belong to medical field.The compound is extracted from the leaves of horse kidney fruit branch, preparation separation is simple.The present application has better inhibiting effect on the release of inflammatory factor of LPS-induced RAW264.7 cell, when in non-toxic concentration, the inhibiting effect of compound on the release of inflammatory factor NO of LPS-induced RAW264.7 cell is good, and the anti-inflammatory effect IC 50 =3.41±0.05 close to positive drug PDTC (NF-κB inhibitor / antioxidant), therefore, compound Aglatestate A has good development prospects of anti-inflammatory drug, and lay a solid foundation for the preparation of anti-inflammatory drug technology development.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a steroid compound, Aglatestate A, its preparation method, and its uses. Background Technology

[0002] Inflammation is a fundamental pathological process, primarily a defensive response, that occurs in biological tissues in response to stimuli such as trauma or infection. Normally, inflammation is beneficial, serving as the body's automatic defense mechanism. However, it can also be harmful, as seen in autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, which are characterized by abnormal inflammatory immune responses. Clinically, therapeutic anti-inflammatory drugs can be broadly classified into two categories: nonsteroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SSAIDs). Both types are effective in treating inflammation but also have numerous side effects. Therefore, the research and development of novel anti-inflammatory drugs with low toxicity or minimal (or no) side effects is of significant clinical importance.

[0003] Natural products are an important source of drug discovery. my country is rich in plant resources, and medicinal plants, as a national treasure, have long played a vital role. The search for anti-inflammatory agents from medicinal plants has become a research hotspot, and several active ingredients with anti-inflammatory properties have already been discovered. *Aglaia odorata*, a tree belonging to the genus *Aglaia* of the family Meliaceae, is mainly distributed in Southeast Asian countries and southern my country. It has therapeutic effects on dizziness, fever, cough, and skin inflammation, as well as promoting blood circulation, reducing swelling and pain, and relieving depression. Modern pharmacology shows that extracts from this plant possess various activities, including anti-tumor, insecticidal, antibacterial, and antiviral activity. Chemical composition studies of *Aglaia odorata* have revealed that its main chemical components include cyclopentanebenzofuran, limonene, diamides, steroids, flavonoids, and other structural types, exhibiting structural diversity. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a steroidal compound with anti-inflammatory activity.

[0005] The technical solution adopted to achieve the above objectives is as follows:

[0006] 1. A steroidal compound, Aglatestate A, has the structural formula (1):

[0007]

[0008] The compound was isolated and extracted from the branches and leaves of the horse kidney fruit.

[0009] The application of the steroidal compound Aglatestate A in the preparation of anti-inflammatory drugs.

[0010] The application of the steroidal compound Aglatestate A in the preparation of anti-inflammatory drugs, wherein the compound Aglatestate A exhibits anti-inflammatory activity in a dose-dependent manner.

[0011] The compound AglatestateA has an anti-inflammatory effect on LPS-induced RAW264.7 cells.

[0012] A pharmaceutical composition comprising the steroidal compound AglatestateA, optionally including a pharmaceutically acceptable carrier.

[0013] The pharmaceutical composition comprises a steroidal structural compound, AglatestateA, and a pharmaceutically acceptable carrier. The pharmaceutical composition contains 0.1-99% by mass of the steroidal structural compound, AglatestateA, with the remainder being a pharmaceutical carrier or excipient.

[0014] The dosage form of the drug is any one of tablets, capsules, or granules.

[0015] The method for preparing the steroidal compound Aglatestate A includes the following steps:

[0016] A. Take 50 parts of the branches and leaves of the horse kidney fruit, crush them, and extract them 4 times at room temperature using 200 parts of 95% industrial methanol for 5 days each time. The alcohol is recovered and concentrated to obtain 5 parts of extract.

[0017] B. The crude extract was subjected to silica gel column chromatography, using a gradient elution of petroleum ether to acetone in a volume ratio of 9:1 to 1:1, with 100 fractions per gradient. Silica gel thin-layer chromatography was used to detect the different eluted fractions. Fractions with the same thin-layer chromatographic results were combined, and seven fractions were obtained based on their thin-layer chromatographic performance: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7.

[0018] C. Fr.6 from step B was subjected to MCI column chromatography, using a gradient elution of methanol to water in a volume ratio of 1:1 to 9:1. The fractions were combined according to their thin-layer chromatographic properties and separated into four subfractions: Fr.6a, Fr.6b, Fr.6c, and Fr.6d. Fr.6b was subjected to reverse-phase column chromatography, using a gradient elution of methanol to water in a volume ratio of 1:1 to 9:1, and separated into nine subfractions: Fr.6b.1, Fr.6b.2, Fr.6b.3, Fr.6b.4, Fr.6b.5, Fr.6b.6, Fr.6b.7, Fr.6b.8, and Fr.6b.9. Fr.6b.5 was subjected to silica gel column chromatography, using a gradient elution of dichloromethane to acetone in a volume ratio of 9:1 to 1:1 to obtain compound 1. Attached Figure Description

[0019] Figure 1 The proton NMR spectrum of compound Aglatestate A

[0020] Figure 2 The carbon NMR spectrum of compound Aglatestate A Detailed Implementation

[0021] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0022] Example 1: Preparation of compound Aglatestate A

[0023] A method for preparing a steroidal compound includes the following steps:

[0024] (1) Take 50 parts of the branches and leaves of the horse kidney fruit, crush them, use 200 parts of 95% industrial methanol, extract them 4 times at room temperature, each time for 5 days, and recover the alcohol and concentrate to obtain 5 parts of extract.

[0025] (2) The crude extract was subjected to silica gel column chromatography with a gradient elution of petroleum ether to acetone in a volume ratio of 9:1 to 1:1. 100 fractions of each gradient were eluted. Silica gel thin-layer chromatography was used to detect the different eluted fractions. Fractions with the same thin-layer chromatographic results were combined to obtain seven fractions based on their thin-layer chromatographic performance: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7.

[0026] (3) Fr.6 was subjected to MCI column chromatography with a gradient elution of methanol to water in a volume ratio of 1:1 to 9:1. The fractions were combined according to their thin-layer chromatographic properties and were divided into four subfractions: Fr.6a, Fr.6b, Fr.6c, and Fr.6d. Fr.6b was subjected to reverse column chromatography with a gradient elution of methanol to water in a volume ratio of 1:1 to 9:1 and was divided into nine subfractions: Fr.6b.1, Fr.6b.2, Fr.6b.3, Fr.6b.4, Fr.6b.5, Fr.6b.6, Fr.6b.7, Fr.6b.8, and Fr.6b.9. Fr.6b.5 was subjected to silica gel column chromatography with a gradient elution of dichloromethane to acetone in a volume ratio of 9:1 to 1:1 to obtain compound AglatestateA.

[0027] Example 2: Structural identification of compound Aglatestate A

[0028] The compounds were analyzed using a combination of high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy (¹H NMR, ¹³C NMR, 2D-NMR), ultraviolet spectroscopy, infrared spectroscopy, and optical rotation data to determine the structural formula of compound 1 (Aglatestate A), as shown below:

[0029]

[0030] Compound Aglatestate A: white amorphous crystals with the molecular formula C21H30O4. HR-ESI-MS m / z 369.2037 ([M+Na]+, calcd for C21H30O4Na: 369.2036), [α]25D -161.0 (c 0.43, MeOH); IR (KBr) vmax 3402, 2950, ​​2929, 2855, 1708, 1642, 1469, 1434, 1411, 1379, 1259, 1195, 1144, 1064, 1049, 999 cm-1; NMR data are shown in Table 1.

[0031] Table 1: NMR data of compound Aglatestate A, 1H (600MHz) and 13C (150MHz) NMR data in CD3Cl.

[0032]

[0033] Example 3: Verification of anti-inflammatory activity

[0034] To further verify the beneficial effects of the compound described in this invention, the inhibitory activity of the compound AglatestateA on NO release in RAW264.7 cells was studied. The specific experiments are as follows:

[0035] I. Experimental Methods

[0036] Cell culture: Mouse mononuclear macrophage RAW264.7 cells were cultured in high glucose DMEM medium containing 10% fetal bovine serum, with 100 mg·L⁻¹ penicillin and 100 mg·L⁻¹ streptomycin added. The incubation conditions were 37°C and 5% CO₂. The cells were passaged every other day.

[0037] Effect of the compound on RAW 264.7 cell viability: Logarithmically growing RAW 264.7 cells were seeded at 1.5 × 10⁴ cells / well in 96-well plates and incubated at 37°C in a 5% CO₂ incubator for 24 h. The old culture medium was discarded, and 100 μL of DMEM solution containing the compound or PDTC (positive control drug) was added, with three replicates per sample. After culturing for another 24 h, 10 μL of ALMTT solution was added to each well, and the plates were incubated at 37°C in a 5% CO₂ incubator for 4 h. After culturing, the cells were centrifuged at 3000 rpm for 15 min, the supernatant was discarded, and 160 μL of DMSO was added to each well to dissolve formazan, followed by gentle shaking. The absorbance of each well was measured at 490 nm using a microplate reader, and cell viability was calculated based on the measured OD values.

[0038] NO content assay in cells: After counting RAW264.7 cells, the cell concentration was adjusted to 2 × 10⁵ cells / mL, and 2 mL was seeded into each well of a 6-well plate. After cell adhesion, different concentrations of steroids (0, 25, 50, 100 μmol·L⁻¹), LPS (0.5 μg·L⁻¹), and PDTC (1 μmol·L⁻¹) were administered. The mixture was incubated at 37°C in the dark for 10 minutes. Then, 50 μL of GriessReagent II was added to each well, and the mixture was shaken to mix thoroughly. After incubation at 37°C in the dark for 10 minutes, the OD value of each group was measured at 540 nm. The inhibitory effect of compound LPS on the release of the inflammatory factor NO from RAW264.7 cells was calculated for each well using the following formula: NO content = [1 - (drug administration well A - control well A) / control well A)] × 100%. The IC50 of the compound was also calculated. 50 value.

[0039] The cytotoxicity (Table 2) and anti-inflammatory activity (Table 3) of the compounds are shown below:

[0040] Table 2. Cytotoxicity of compound Aglatestate A

[0041]

[0042] Note: bPDTC positive control pyrrolidine dithiocarbamate ammonium

[0043] Table 3. Anti-inflammatory activity of compound 1 (Aglatestate A)

[0044]

[0045]

[0046] Note: bPDTC positive control pyrrolidine dithiocarbamate ammonium

[0047] Experimental results showed that, at non-toxic concentrations, the compound effectively inhibited the release of the inflammatory cytokine NO induced by LPS in RAW264.7 cells, and its anti-inflammatory effect had an IC50 value of [missing value]. 50 =3.41±0.05, which is close to that of the positive drug PDTC (NF-κB inhibitor / antioxidant), therefore, compound AglatestateA has good prospects for the development of anti-inflammatory drugs.

[0048] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A steroid compound Aglatestate A, having the formula (1) : 。 2. Use of the steroid compound Aglatestate A as described in claim 1 in the preparation of an anti-inflammatory drug.

3. Use of the steroid structuring compound Aglatestate A according to claim 2 for the preparation of an anti-inflammatory medicament, characterized in that, The compound Aglatestate A shows anti-inflammatory activity in a dose-dependent manner.

4. Use of the steroid structuring compound Aglatestate A according to claim 2 for the preparation of an anti-inflammatory medicament, characterized in that, The compound Aglatestate A has anti-inflammatory effect on LPS-induced RAW264.7 cells.

5. A pharmaceutical composition, characterized by, A pharmaceutical composition comprising the steroid compound Aglatestate A as described in claim 1, optionally comprising a pharmaceutically acceptable carrier.

6. The pharmaceutical composition of claim 5, comprising the steroid compound Aglatestate A and a pharmaceutically acceptable carrier, which contains 0.1-99% of the steroid compound Aglatestate A by mass fraction, and the rest is a pharmaceutical carrier or excipient.

7. The pharmaceutical composition of claim 5, wherein, The dosage form of the drug is any one of tablet, capsule, granule.

8. A process for the preparation of the steroid structuring compound Aglatestate A according to claim 1, characterized in that: Comprising the following steps: A. Take 50 parts of horse kidney fruit branch and leaf, crush, and extract 4 times at room temperature with 200 parts of 95% industrial methanol, each time for 5 days, and recover the alcohol concentrate to get 5 parts of extract; B. The extract of the crude extract is subjected to silica gel column chromatography, and eluted with eluent of petroleum ether and acetone in a volume ratio of 9:1~1:1 gradient, 100 parts for each gradient, and detected by silica gel thin layer chromatography technology. Fractions with the same thin layer chromatography results are combined, and seven fractions are obtained according to the thin layer chromatography: Fr. 1, Fr. 2, Fr. 3, Fr. 4, Fr. 5, Fr. 6, Fr. 7; C. Fr. 6 in step B is subjected to MCI column chromatography, eluted with eluent of methanol and water in a volume ratio of 1:1~9:1 gradient, and combined according to the thin layer chromatography, and divided into 4 sub-fractions: Fr. 6a, Fr. 6b, Fr. 6c, Fr. 6d; Fr. 6b is subjected to reverse column chromatography, eluted with eluent of methanol and water in a volume ratio of 1:1~9:1 gradient, and divided into Fr. 6b.1, Fr. 6b.2, Fr. 6b.3, Fr. 6b.4, Fr. 6b.5, Fr. 6b.6, Fr. 6b.7, Fr. 6b.8 and Fr. 6b.9 sub-fractions; Fr. 6b.5 is subjected to silica gel column chromatography, eluted with eluent of dichloromethane and acetone in a volume ratio of 9:1~1:1 gradient to obtain the compound of formula (1).

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