Steroid compound Aglatestate A as well as preparation method and application thereof

Abstract

The invention relates to a steroid compound Aglatestatin A as well as a preparation method and application thereof, and belongs to the field of medicines. The compound is separated and extracted from branches and leaves of Pachyrhizus erosus and is simple to prepare and separate. The compound disclosed by the invention has a relatively good effect of inhibiting the release of inflammatory factors of RAW264.7 cells induced by LPS, the compound has a good effect of inhibiting the release of inflammatory factors NO of the RAW264.7 cells induced by the LPS at a non-toxic concentration, and the anti-inflammatory effect IC50 of the compound is 3.41 + / -0.05 and is close to that of a positive drug PDTC (NF-kappa B inhibitor / antioxidant), so that the compound Aglatestatin A has a good prospect of developing anti-inflammatory drugs and has a good application prospect. And a solid foundation is laid for development of a technology for preparing anti-inflammatory drugs.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry, and specifically relates to a steroid compound Aglatestate A and a preparation method and application thereof. Background Art

[0002] Inflammation is a basic pathological process that occurs when biological tissues are stimulated by certain stimuli such as trauma, infection and other damaging factors, and is mainly a defensive response. Under normal circumstances, inflammation is beneficial and is an automatic defensive response of the human body. But sometimes, inflammation is also harmful. For example, autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus are inflammatory immune diseases with abnormal inflammatory immune responses. Clinical therapeutic anti-inflammatory drugs can be mainly divided into two categories, non-steroidal anti-inflammatory drugs and steroidal anti-inflammatory drugs. Both drugs have good effects in treating inflammation, but they also have many side effects. Therefore, the research and development of new anti-inflammatory drugs with low toxicity or minimal (or no) side effects is of great clinical significance.

[0003] Natural products are an important source of drug discovery. my country is rich in plant resources, and medicinal plants, as my country's treasures, have long played an important role. Finding anti-inflammatory therapeutic agents from medicinal plants has become a research hotspot, and many active ingredients for treating inflammation have been found. The horse kidney fruit is a tree of the genus Aglaia in the Meliaceae family. It is mainly distributed in Southeast Asian countries and southern my country. It has the effects of treating dizziness, fever, cough, skin inflammation, as well as promoting blood circulation, removing blood stasis, reducing swelling and relieving pain, and promoting qi and relieving depression. Modern pharmacology shows that the plant extract has multiple activities such as anti-tumor, insecticidal, antibacterial and antiviral. The chemical composition of the horse kidney fruit was studied and it was found that its main chemical components include cyclopentanebenzofuran, limonin, diamide, steroids, flavonoids and other structural types, showing a diversity of structural types. Summary of the invention

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

[0005] The technical solutions adopted to achieve the above purpose are as follows:

[0006] 1. A steroidal structural compound Aglatestate A, whose structural formula is formula (1):

[0007]

[0008] The compound is separated and extracted from the branches and leaves of the fruit of the genus Psoralea corylifolia.

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

[0010] The steroidal structural compound Aglatestate A is used in the preparation of anti-inflammatory drugs. The compound Aglatestate A shows 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 comprises the steroidal structural compound Aglatestate A and optionally contains a pharmaceutically acceptable carrier.

[0013] The pharmaceutical composition comprises a steroidal structural compound Aglatestate A and a pharmaceutically acceptable carrier. The pharmaceutical composition contains the steroidal structural compound Aglatestate A in a mass fraction of 0.1-99%, and the rest is a pharmaceutical carrier or excipient.

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

[0015] The preparation method of the steroidal structural compound Aglatestate A comprises the following steps:

[0016] A. 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, recover the alcohol and concentrate it to obtain 5 parts of extract;

[0017] B. The crude extract was subjected to silica gel column chromatography, and a gradient elution was performed using an eluent having a volume ratio of petroleum ether to acetone of 9:1 to 1:1, with 100 portions for each gradient. Different elution fractions were detected using silica gel thin layer chromatography technology, and fractions with the same thin layer chromatography results were combined. According to the thin layer chromatography performance, seven fractions were obtained: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7;

[0018] C. Fr.6 in step B was subjected to MCI column chromatography, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, and the fractions were combined according to their thin layer chromatography performance to be divided into 4 sub-fractions: Fr.6a, Fr.6b, Fr.6c, and Fr.6d; Fr.6b was subjected to reverse column chromatography, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, to be divided into 9 sub-fractions, namely 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 with a volume ratio of dichloromethane to acetone of 9:1 to 1:1 as the eluent to obtain compound 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the H NMR spectrum of compound Aglatestate A

[0020] Figure 2 This is the carbon NMR spectrum of compound Aglatestate A DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0022] Example 1: Preparation of Compound Aglatestate A

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

[0024] (1) 50 parts of the branches and leaves of the spatholobus spatholobus were taken, crushed, and extracted with 200 parts of 95% industrial methanol at room temperature for 4 times, each time for 5 days, and the alcohol was recovered and concentrated to obtain 5 parts of an extract;

[0025] (2) The crude extract was subjected to silica gel column chromatography, using a gradient elution with a volume ratio of petroleum ether to acetone of 9:1 to 1:1, with 100 parts per gradient. Different elution fractions were detected using silica gel thin layer chromatography technology, and fractions with the same thin layer chromatography results were combined to obtain seven fractions according to the thin layer chromatography 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, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, and was combined according to the thin layer chromatography performance to be divided into 4 sub-fractions: Fr.6a, Fr.6b, Fr.6c, and Fr.6d; Fr.6b was subjected to reverse column chromatography, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, and was divided into 9 sub-fractions, namely 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 with a volume ratio of dichloromethane to acetone of 9:1 to 1:1 as the eluent, to obtain compound Aglatestate A.

[0027] Example 2: Structural Identification of Compound Aglatestate A

[0028] The compound was comprehensively analyzed by high-resolution mass spectrometry, nuclear magnetic resonance spectra (1HNMR, 13C NMR, 2D-NMR), ultraviolet spectra, infrared spectra, optical rotation and other data to determine the structural formula of compound 1 (Aglatestate A), as shown below:

[0029]

[0030] Compound Aglatestate A: white amorphous crystals, 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 (600 MHz) and 13C (150 MHz) NMR Data of 1 in CD3Cl.

[0032]

[0033] Example 3: Anti-inflammatory activity verification

[0034] In order to further verify the beneficial effects of the compounds of the present invention, the NO release inhibitory activity of the compound Aglatestate A in RAW264.7 cells was studied. The specific experiment is as follows:

[0035] 1. Experimental Methods

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

[0037] Effect of compounds on RAW 264.7 cell viability: Take RAW 264.7 cells in the logarithmic growth phase and inoculate 96-well plates at 1.5×104 / well, and incubate in a 37°C, 5% CO. incubator for 24 hours. Discard the old culture medium, add 100 μL of DMEM solution containing the compound or PDTC (positive drug), and replicate each sample in 3 wells. After 24 hours of continuous culture, add 10 μL of MTT solution to each well and incubate in a 37°C, 5% CO2 incubator for 4 hours. After the culture is completed, centrifuge at 3000 r / min for 15 minutes and discard the supernatant. Add 160 μL of DMSO to each well to dissolve formazan and shake slowly. Detect the absorbance of each well at a wavelength of 490 nm on an ELISA reader, and calculate the cell survival rate based on the measured OD value.

[0038] Determination of cell NO content: After counting RAW264.7 cells, adjust the cell concentration to 2×105 / mL and plate them in a 6-well plate, 2mL per well. After the cells adhere to the wall, give different concentrations of steroids (0, 25, 50, 100μmol·L-1), LPS (0.5μg·L-1), PDTC (1μmol·L-1), and react at 37℃ in the dark for 10 minutes. Then add 50μL of Griess ReagentⅡ to each well, shake and mix evenly, react at 37℃ in the dark for 10 minutes, and detect the OD value of each group at a wavelength of 540nm. The inhibitory effect of compound LPS-induced inflammatory factor NO release in RAW264.7 cells is calculated according to the following formula for each well: NO content = [1-(drug well A-control well A) / control well A)]×100%. The compound simultaneously calculates its IC 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 AglatestateA

[0041]

[0042] Note: bPDTC positive control ammonium pyrrolidine dithiocarbamate

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

[0044]

[0045]

[0046] Note: bPDTC positive control ammonium pyrrolidine dithiocarbamate

[0047] The experimental results showed that at non-toxic concentrations, the compound had a good inhibitory effect on the release of inflammatory factor NO in RAW264.7 cells induced by LPS, and its anti-inflammatory effect IC 50 =3.41±0.05, which is close to the positive drug PDTC (NF-κB inhibitor / antioxidant), so compound AglatestateA has good prospects for the development of anti-inflammatory drugs.

[0048] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A steroidal structural compound Aglatestate A, whose structural formula is formula (1):

2. The steroidal structural compound Aglatestate A according to claim 1, characterized in that: The compound is separated and extracted from the branches and leaves of the fruit of the genus Psoralea corylifolia.

3. Use of the steroidal structural compound Aglatestate A described in claim 1 in the preparation of anti-inflammatory drugs.

4. The use of the steroidal structural compound Aglatestate A according to claim 3 in the preparation of anti-inflammatory drugs, characterized in that: The compound AglatestateA exhibits anti-inflammatory activity in a dose-dependent manner.

5. The use of the steroidal structural compound Aglatestate A according to claim 3 in the preparation of anti-inflammatory drugs, characterized in that: The compound AglatestateA has an anti-inflammatory effect on LPS-induced RAW264.7 cells.

6. A pharmaceutical composition, characterized in that The steroidal structural compound Aglatestate A according to claim 1 optionally comprises a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, comprising a steroidal structural compound Aglatestate A and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition contains 0.1-99% by mass of the steroidal structural compound Aglatestate A, and the rest are pharmaceutical carriers or excipients.

8. A pharmaceutical composition according to claim 6, characterized in that: The dosage form of the medicine is any one of tablets, capsules and granules.

9. A method for preparing the steroidal structure compound Aglatestate A according to claim 1, characterized in that: The following steps are involved: A. 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, recover the alcohol and concentrate it to obtain 5 parts of extract; B. The crude extract was subjected to silica gel column chromatography, and a gradient elution was performed using an eluent having a volume ratio of petroleum ether to acetone of 9:1 to 1:1, with 100 portions for each gradient. Different elution fractions were detected using silica gel thin layer chromatography technology, and fractions with the same thin layer chromatography results were combined. According to the thin layer chromatography performance, seven fractions were obtained: Fr.1, Fr.2, Fr.3, Fr.4, Fr.5, Fr.6, and Fr.7; C. Fr.6 in step B was subjected to MCI column chromatography, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, and the fractions were combined according to their thin layer chromatography performance to be divided into 4 sub-fractions: Fr.6a, Fr.6b, Fr.6c, and Fr.6d; Fr.6b was subjected to reverse column chromatography, using a gradient elution with a volume ratio of methanol to water of 1:1 to 9:1 as the eluent, to be divided into 9 sub-fractions, namely 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 with a volume ratio of dichloromethane to acetone of 9:1 to 1:1 as the eluent to obtain compound 1.

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