A sesquiterpene compound oxyphylleudne F isolated from Alpinia oxyphylla Miq. and its applications

Abstract

The present invention relates to a sesquiterpenoid compound oxyphylleudne F separated from Alpinia oxyphylla seeds and its application, and a compound with a novel structure is found from Alpinia oxyphylla seeds, and the application of the new compound in the development of kidney protection drugs is realized. The technical solution is to separate and identify a new sesquiterpenoid compound oxyphylleudne F from the n-butanol part of Alpinia oxyphylla seeds, and find through experimental research that the compound can inhibit and attenuate the TGF-β-catenin induced by TGF-β-catenin in a dose-dependent manner. β 1 induces the expression of fibronectin, type I collagen and α The expression of SMA has anti-renal fibrosis activity and can be effectively used to prepare renal protective drugs. The preparation method has good reproducibility and the obtained compound has high purity, which is conducive to further pharmacological and clinical research on it, and has expanded the medicinal value of Alpinia oxyphylla, with huge economic and social benefits.

Description

1. Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a sesquiterpene compound oxyphylleudne F (Compound IV) isolated from Alpinia oxyphylla Miq. and its applications. 2. Background Art

[0002] Chronic kidney disease (CKD) is one of the major global public health problems, with a global prevalence of 9.1% (about 697.5 million cases) and causing 1.2 million deaths annually. According to the statistical data from 1997 to 2017, the prevalence has increased by 29.3%, while the incidence of dialysis and kidney transplantation has also increased by 32.8% and 21.6% respectively. These results indicate the urgent need to improve CKD and the importance of finding targeted therapies. The development of renal fibrosis is one of the important characteristics of the biological and pathological changes in CKD patients. Certain fibrosis molecules, such as fibronectin, transforming growth factor-β1 (TGF-β1), and collagens I, III, and IV, are important indicators of the progression of renal fibrosis. Fibroblasts can also proliferate and express α-smooth muscle actin (α-SMA), which has been identified as a renal fibrosis marker. In addition, during the process of progressive tubulointerstitial nephropathy, damaged or apoptotic cells attract the infiltration of inflammatory cells such as macrophages and granulocytes to induce an inflammatory response, leading to the progression of renal injury. Therefore, anti-fibrosis has recently been considered a new strategy for the treatment of CKD.

[0003] Traditional Chinese medicine often has the characteristics of multiple effects and bidirectional regulation in the treatment of chronic kidney disease, as well as advantages such as fewer adverse reactions and richer sources. It has been attracting more and more attention. Searching for and developing traditional Chinese medicine and its active ingredients with renal protective effects has gradually become one of the research hotspots in modern medicine. Alpinia oxyphylla Miq. is the dried mature fruit and seeds of the plant Alpinia oxyphylla Miq. of the Zingiberaceae family. It is a natural plant that is both a medicine and a food announced by the Ministry of Health in 1998. It is mainly produced in the mountainous areas of Hainan Island in China and is one of the four major southern Chinese medicines. Alpinia oxyphylla Miq. is warm in nature and pungent in taste, and belongs to the heart, spleen, and kidney meridians. It has the effects of warming and tonifying, warming the spleen to stop diarrhea, fixing saliva and arresting drooling, warming the kidney to consolidate essence and reduce urination. The main chemical components in Alpinia oxyphylla Miq. include terpenoids, diphenylheptanes, flavonoids, sterols, and phenolic acids. However, there has been no patent or literature report on how to extract the sesquiterpene compound oxyphylleudne F from Alpinia oxyphylla Miq. and realize its application in the preparation of anti-renal fibrosis drugs. 3. Summary of the Invention

[0004] In view of the above situation, to solve the deficiencies of the prior art, the object of the present invention is to provide a sesquiterpene compound oxyphylleudne F isolated from Alpinia oxyphylla Miq. and its application, to discover a compound with a novel structure from Alpinia oxyphylla Miq., and to realize the application of the new compound in the development of kidney protection drugs.

[0005] The technical solution solved by the present invention is to isolate and identify a new sesquiterpene compound oxyphylleudne F from the n-butanol fraction of Alpinia oxyphylla Miq., and its structural formula is as follows:

[0006]

[0007] The preparation method of the novel compound of the present invention includes the following steps:

[0008] 1) Dry and crush Alpinia oxyphylla Miq., reflux extract with 6 - 10 times the amount of 70% ethanol for 3 times, 2 hours each time. After combining the extracts, concentrate under reduced pressure to obtain an extract. Suspend the extract with 2 - 4 times the amount of water and then extract successively with ethyl acetate and n-butanol until the extract is colorless (more than 10 times), and then concentrate to obtain the ethyl acetate fraction, n-butanol fraction and water fraction;

[0009] 2) Load the n-butanol fraction obtained in step 1) onto an MCI Gel CHP-20 chromatographic column (Mitsubishi Chemical Corporation, Japan), and perform gradient elution with an ethanol-water system (0:100 - 95:5) to successively obtain a water fraction, 10% ethanol fraction, 30% ethanol fraction, 50% ethanol fraction, 70% ethanol fraction and 95% ethanol fraction;

[0010] 3) Use a Sephadex LH-20 (Parmacia Biotech) chromatographic column for the MCI 50% ethanol fraction obtained in step 2), and perform gradient elution with methanol-water (0:100 - 95:5) as the eluent to successively obtain Fr.D.1 - Fr.D.7;

[0011] 4) Load Fr.D.1 obtained in step 3) onto an MCI (Mitsubishi Chemical Corporation, Japan) column chromatography, and perform gradient elution with methanol-water (0:100 - 95:5) as the eluent to obtain four components, namely Fr.D.1.1 - Fr.D.1.4;

[0012] 5) Purify Fr.D1.4 obtained in step 4) by semi-preparative HPLC to obtain compound Ⅳ oxyphylleudne F.

[0013] The application of the sesquiterpene compound oxyphylleudne F of the present invention in the preparation of anti-kidney fibrosis active drugs.

[0014] The present invention relates to a novel sesquiterpenoid oxyphylleudne F (Compound IV) isolated from the medicinal material Alpiniae Oxyphyllae Fructus. Through experimental research, it is found that this compound can inhibit and dose-dependently attenuate the expression of fibronectin, type I collagen, and α-SMA in rat renal tubular epithelial cells (NRK 52E cells) induced by TGF-β1, has anti-renal fibrosis activity, can be effectively used for the preparation of renal protective drugs, and its preparation method has good reproducibility and the obtained compound has high purity, which is conducive to further pharmacological and clinical research, expands the medicinal value of Alpiniae Oxyphyllae Fructus, and has great economic and social benefits. IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 HMBC of the compound of the present invention 1 H- 1 H COSY Related spectrogram.

[0016] Figure 2 ECD experiment and calculation diagram of Compound IV of the present invention.

[0017] Figure 3 Anti-renal fibrosis activity diagram of the compound of the present invention against TGF-β1-induced NRK-52E cells.

[0018] Figure 4 HR-ESI-MS spectrogram of the compound of the present invention.

[0019] Figure 5 UV spectrogram of the compound of the present invention.

[0020] Figure 6 IR spectrogram of the compound of the present invention.

[0021] Figure 7 is of the compound of the present invention 1 H-NMR spectrogram.

[0022] Figure 8 is of the compound of the present invention 13 C-NMR spectrogram.

[0023] Figure 9 COSY spectrogram of the compound of the present invention.

[0024] Figure 10 HSQC spectrogram of the compound of the present invention.

[0025] Figure 11 HMBC spectrogram of the compound of the present invention.

[0026] Figure 12 NOE spectrogram of the compound of the present invention. V. Specific Embodiments

[0027] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Example 1

[0029] When the present invention is specifically implemented, the preparation method of compound oxyphylleudne F may include the following steps:

[0030] 1) After drying and pulverizing 40.00 kg of Alpinia oxyphylla, it was refluxed and extracted with 8 times the amount of 70% ethanol for 2 h (3 times). After combining the extraction solutions, they were concentrated under reduced pressure to obtain 8.30 kg of extract. The extract was suspended in 3 times the amount of water and then extracted successively with 30 L of ethyl acetate and n-butanol until the extraction solution was colorless (more than 10 times), and then concentrated to obtain an ethyl acetate fraction (1.45 kg), an n-butanol fraction (628.33 g), and an aqueous fraction (6.60 kg);

[0031] 2) The n-butanol fraction obtained in step 1) was loaded onto an MCI Gel CHP-20 chromatographic column (Mitsubishi Chemical Corporation, Japan) and eluted with an ethanol-water system (0:100 to 95:5) in a gradient manner. Subsequently, an aqueous fraction (371.10 g), a 10% ethanol fraction (8.43 g), a 30% ethanol fraction (33.36 g), a 50% ethanol fraction (49.82 g), a 70% ethanol fraction (20.73 g), and a 95% ethanol fraction (105.06 g) were obtained in sequence;

[0032] 3) The MCI 50% ethanol fraction obtained in step 2) was chromatographed on a Sephadex LH-20 (Parmacia Biotech) column using methanol-water (0:100 to 95:5) as the eluent for gradient elution, and Fr.D.1 to Fr.D.7 were obtained in sequence;

[0033] 4) The Fr.D.1 obtained in step 3) was chromatographed on an MCI (Mitsubishi Chemical Corporation, Japan) column using methanol-water (0:100 to 95:5) as the eluent for gradient elution to obtain four components, namely Fr.D.1.1 to Fr.D.1.4;

[0034] 5) The Fr.D1.4 (1.54 g) obtained in step 4) was purified by semi-preparative HPLC to obtain compound Ⅳ oxyphylleudne F (77.70 mg; MeCN / H2O; 26∶74, flow rate: 3 mL / min; t R = 22.1 minutes).

[0035] This invention was measured by using an ABSCIEX TripleTOF 6600 high-performance liquid chromatography-high-resolution mass spectrometer for high-resolution mass spectrometry; the compound was purified by using a Sepurest LC-52 semi-preparative liquid chromatograph (Sepurest Beijing Science and Technology Co., Ltd.); the specific rotation was measured by using an Anton Paar MCP 5100 polarimeter (Anton Paar, Austria); the NMR spectrum was measured by using a Bruker AM-500 MHz superconducting nuclear magnetic resonance spectrometer with TMS as the internal standard (Bruker, Germany); the ultraviolet spectrum was measured by using a Thermo EVO300 ultraviolet spectrophotometer, and the infrared spectrum was measured by using a Thermo Nicolet IS10 infrared spectrometer (Thermo Scientific, USA).

[0036] I. Structure Identification of Compound IV

[0037]

[0038] Compound IV, a colorless oil, (c 0.05, MeOH); IR (MeOH) ν max 3396, 1659, 1460, 1380, 1202, 1032 cm-1; UV (MeOH) λ max (logε): 241.0 (2.86) nm; HR-ESI-MS [M+Na]+ m / z 233.1141 (calcd for C 12 H 18 O3Na 233.1148) indicated that its molecular formula was C 12 H 18 O3.

[0039] 1 In 1H NMR (CD3OD, 500 MHz), there was a carbon-carbon double bond at δ H 6.01 (1H, s, H-6), six methylene signals [δ H 3.69 (1H, d, J = 11.2 Hz, H-14a), 3.43 (1H, d, J = 11.2 Hz, H-14b), 2.64 (1H, ddd, J = 17.7, 15.1, 5.2 Hz, H-9a), 2.32 (1H, m, H-9b), 2.10 (1H, m, H-2a), 1.89 (1H, m, H-8a), 1.74 (1H, m, H-8b), 1.74 (2H, m, H-3), 1.66 (1H, dd, J = 13.1, 4.2 Hz, H-1a), 1.55 (1H, dq, J = 17.2, 3.6 Hz, H-2b), 1.36 (1H, m, H-1b)] and a methyl signal at δH 1.45 (3H, s, H-15). Analyze its 13 \(^{13}\)C NMR (CD₃OD, 125 MHz) spectrum and a total of 12 carbon signals were observed in combination with the HSQC spectrum, including a carbonyl signal at δ C 203.6 (C-7), a carbon-carbon double bond signal [δ C 170.0 (C-5), 125.1 (C-6)], two quaternary carbons [δ C 73.8 (C-4), 36.9 (C-10)], six methylene signals [δ C 41.9 (C-1), 17.7 (C-2), 35.8 (C-3), 40.9 (C-8), 34.8 (C-9), 68.3 (C-14)] and a methyl signal at δ C 24.9 (C-15). Based on the above NMR data, it was speculated to be a eudesmane-type sesquiterpene. According to the HMBC correlations of H-1a (δ H 1.65) with C-2 (δ C 17.7) / C-5 (δ C 170.0) / C-10 (δ C 36.9); H-6 (δ H 6.29) with C-4 (δ C 73.8) / C-5 (δ C 170.0) / C-7 (δ C 203.6) / C-8 (δ C 40.9), and combined with a series of 1 H- 1 H COSY correlations of H₂-1 / H₂-2 / H-3 and H₂-8 / H₂-9, it was proved that there are two six-membered rings in the structure. The HMBC correlations of H-14a (δ H 3.67) with C-3 (δ C 35.8) / C-4 (δ C 73.8) / C-5 (δ C 170.0) determined the positions of the hydroxyl and hydroxymethyl groups attached to C-4. The relative configuration of the compound was determined by the presence of a correlation signal between H₂-14 and H₃-15 in its NOESY spectrum. Then, the ECD spectra of the two relative configurations were calculated by the TD-DFT method at the B3LYP / 6-31G(d,p) level. After comparison with the measured ECD spectrum, the absolute configuration was determined to be (4S,10S). Therefore, the structure of the compound was determined to be (4S,5E,10S)-7-oxo-tri-nor-eudesm-5-en-4,14-diol, named oxyphylleudne F.

[0040] Table 1 of Compound Ⅳ 1 H-NMR and 13 C-NMR data (in CD3OD)

[0041]

[0042]

[0043] II. Research on the Renoprotective Activity of the Novel Sesquiterpenoid Compound Ⅳ of the Present Invention 2.1 Cell Line

[0044] NRK 52E: Normal Rat Renal Tubular Epithelial Cells

[0045] 2.2 Experimental Instruments, Consumables and Reagents

[0046] Table 2 List of Instrument Names and Manufacturers

[0047]

[0048]

[0049] Table 3 List of Reagent Names and Manufacturers

[0050]

[0051] 2.3 Experimental Methods

[0052] (1) Cell Culture and Treatment: NRK 52E cells were cultured in DMEM medium containing 10% fetal bovine serum. Through trypsin digestion, the cells were seeded into a 12-well plate (2×10 4 cells / mL) and waited until they grew to 70%-80%. Then, TGF-β1 (10 ng / mL) was added to induce cell fibrosis, and the compound was added simultaneously. After 48 h, the cells were harvested for Western Blot. The compound used in the cell experiment was dissolved in dimethyl sulfoxide.

[0053] (2) Cell Viability Test: NRK 52E cells (2×10 4 cells / mL) were seeded into a 96-well cell culture plate with 10% fetal bovine serum DMEM medium. After overnight culture, the cells were treated with different compounds or DMSO for 48 h. The supernatant was removed, and 100 μL of medium (V 空培养基 / V Cell Count Kit-8 = 10 / 1) was added to each well. After 1 h, the absorbance of each well was recorded at 450 nm using a microplate reader.

[0054] (3) Western blot: After the cells were treated with the compound and TGF-β1, the total protein of the cell line was extracted using 100 μL of RIPA (strong) lysis buffer (1 mM PMSF + protease inhibitor + nuclease), and the protein samples were quantified using the BCA assay. Equal amounts of protein extracts were separated by 8% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 hour, then incubated overnight with the specified antibody at 4°C, and then incubated with the secondary antibody for 2 hours at room temperature. The bands were visualized and measured using an ECL kit and a developer. The immunoblot results were analyzed by gray scale using ImageJ software.

[0055] (4) Data analysis: All experimental data obtained in this invention were independently repeated three times. The data were provided in the form of the mean of the standard error of the mean (SEM). Statistical analysis was performed using Graphpad Prism 8 (Graphpad Software, San Diego, CA, USA) and Excel (Microsoft). When * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001, the difference was considered significant.

[0056] 2.4 Experimental results

[0057] After the cells were added with different concentrations of the compound and then 10 ng / mL of TGF-β1 was added and allowed to act for 48 h, the CCK-8 method was used to detect the effect of different concentrations of the compound on cell proliferation (A), and the protein levels of fibronectin, collagen type I, and α-SMA were detected by immunoblotting, with the GAPDH value used as a control (B). The fibronectin, collagen type I, and α-SMA proteins were quantified by histograms (C), (D), and (E). The data represent the mean ± SEM values of three experiments. GW788388 (GW) was used as a positive control. As Figure 3 shown, compared with the positive drug GW788388, the oxyphylleudne F compound of this invention could significantly reduce the expression of Fibronectin and Collagen I, indicating that it could alleviate renal fibrosis in NRK 52E cells induced by TGF-β1 and had renal protective activity, proving that the compound of this invention could alleviate renal fibrosis in NRK-52e cells induced by TGF-1.

[0058] 2.5 Conclusion

[0059] The anti-renal fibrosis activity study shows that the compounds in Alpiniae Oxyphyllae Fructus can inhibit and dose-dependently attenuate the expression of fibronectin, type I collagen, and α-SMA in NRK 52E cells induced by TGF-β1.

[0060] The present invention has identified a new sesquiterpene compound oxyphylleudne F isolated from Alpiniae Oxyphyllae Fructus, and through experimental studies, it has been found that this compound has anti-renal fibrosis effects and can be used to prepare renal protection drugs, thus expanding the medicinal value of Alpiniae Oxyphyllae Fructus, having great development prospects, and having good economic and social benefits.

[0061] It should be noted that the above are only the preferred embodiments of the present invention, and do not impose any formal restrictions on the present invention. Any person skilled in the relevant art, without departing from the scope of the technical solution of the present invention, can make changes or modifications to equivalent embodiments with equivalent changes by using the technical content disclosed above, and all fall within the protection scope of the present invention.

Claims

1. A sesquiterpene compound oxyphylleudne F isolated from Alpinia oxyphylla Miq., characterized in that, The structural formula is as follows:

2. The preparation method of sesquiterpene compound oxyphylleudne F isolated from Alpinia oxyphylla Miq. according to claim 1, characterized in that, It includes the following steps: 1) Dry and crush the Alpinia oxyphylla Miq., reflux extract it 3 times with 6 - 10 times the volume of 70% ethanol for 2 hours each time. After combining the extraction solutions, concentrate them under reduced pressure to obtain an extract. Suspend the extract with 2 - 4 times the volume of water, and then extract it with ethyl acetate and n-butanol successively for more than 10 times until the extraction solution is colorless. Then concentrate to obtain the ethyl acetate fraction, n-butanol fraction, and water fraction; 2) Load the n-butanol fraction obtained in step 1) onto an MCI Gel CHP-20 chromatographic column, and elute it with an ethanol-water system in a gradient from 0:100 to 95:

5. Successively obtain the water fraction, 10% ethanol fraction, 30% ethanol fraction, 50% ethanol fraction, 70% ethanol fraction, and 95% ethanol fraction; 3) Use an Sephadex LH-20 chromatographic column for the MCI 50% ethanol fraction obtained in step 2), and perform gradient elution with methanol-water in a ratio from 0:100 to 95:5 as the eluent, successively obtaining Fr.D.1 - Fr.D.7; 4) Perform column chromatography on Fr.D.1 obtained in step 3) using an MCI column, and perform gradient elution with methanol-water in a ratio from 0:100 to 95:5 as the eluent to obtain four components, namely Fr.D.1.1 - Fr.D.1.4; 5) Purify Fr.D1.4 obtained in step 4) by semi-preparative HPLC to obtain the compound oxyphylleudne F.

3. The preparation method of the sesquiterpene compound oxyphylleudne F isolated from Alpinia oxyphylla Miq. according to claim 1, characterized in that, It includes the following steps: 1) Dry and crush 40.00 kg of Alpinia oxyphylla Miq., reflux extract it 3 times with 8 times the volume of 70% ethanol for 2 hours each time. After combining the extraction solutions, concentrate them under reduced pressure to obtain 8.30 kg of an extract. Suspend the extract with 3 times the volume of water, and then extract it with 30 L of ethyl acetate and n-butanol successively until the extraction solution is colorless. Then concentrate to obtain the ethyl acetate fraction, n-butanol fraction, and water fraction; 2) Load the n-butanol fraction obtained in step 1) onto an MCI Gel CHP-20 chromatographic column, and elute it with an ethanol-water system in a gradient from 0:100 to 95:

5. Successively obtain the water fraction, 10% ethanol fraction, 30% ethanol fraction, 50% ethanol fraction, 70% ethanol fraction, and 95% ethanol fraction; 3) Use an Sephadex LH-20 chromatographic column for the MCI 50% ethanol fraction obtained in step 2), and perform gradient elution with methanol-water in a ratio from 0:100 to 95:5 as the eluent, successively obtaining Fr.D.1 - Fr.D.7; 4) Perform column chromatography on Fr.D.1 obtained in step 3) using an MCI column, and perform gradient elution with methanol-water in a ratio from 0:100 to 95:5 as the eluent to obtain four components, namely Fr.D.1.1 - Fr.D.1.4; 5) Purify Fr.D1.4 obtained in step 4) by semi-preparative HPLC to obtain the compound Ⅳ oxyphylleudne F.

4. Use of the sesquiterpenoid compound oxyphylleudne F described in claim 1 in the preparation of an active drug against renal fibrosis.

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