A sesquiterpenoid compound oxyphylleudne I isolated from Alpinia oxyphylla seeds and its application

Abstract

The present invention relates to a sesquiterpenoid compound oxyphylleudne I 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 I 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 invention relates to the technical field of medicinal chemistry, in particular to a sesquiterpenoid compound oxyphylleudne I (compound III) separated from Alpinia oxyphylla seeds and application thereof. 2. Background Technology

[0002] Chronic kidney disease (CKD) is one of the major public health problems in the world, with a global prevalence of 9.1% (about 697.5 million cases) and 1.2 million deaths each year. According to statistics from 1997 to 2017, the prevalence increased by 29.3%, while the incidence of dialysis and kidney transplantation 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 biological and pathological changes in CKD patients. Certain fibrotic molecules, such as fibronectin, transforming growth factor (TGF-β1), and collagen I, III, IV (collagen I, III, 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 progressive tubulointerstitial nephropathy, damaged or apoptotic cells attract infiltration of inflammatory cells such as macrophages and granulocytes to induce inflammatory responses, leading to the progression of renal damage. Therefore, anti-fibrosis has recently been considered as a new technology for the treatment of CKD.

[0003] In the treatment of chronic kidney disease, traditional Chinese medicine often has the characteristics of multiple effects and bidirectional regulation, as well as advantages such as fewer adverse reactions and rich sources. It has attracted more and more attention. Finding and developing traditional Chinese medicine with kidney protection effect and its effective ingredients has gradually developed into one of the research hotspots of modern medicine. Alpinia oxyphylla Miq. is the dried mature fruit and seeds of the ginger plant Alpinia oxyphylla Miq. It is a natural plant with medicinal and edible properties announced by the Ministry of Health in 1998. It is mainly produced in the mountainous areas of Hainan Island, my country, and is one of the four major southern medicines in my country. Alpinia oxyphylla Miq. is warm in nature and pungent in taste. It belongs to the heart, spleen, and kidney meridians. It has the effects of warming, tonifying, consolidating, warming the spleen and stopping diarrhea, consolidating saliva, warming the kidney and consolidating essence and reducing urine. Alpinia oxyphylla Miq. mainly contains chemical components such as terpenes, diphenylheptanes, flavonoids, sterols and phenolic acids. However, there is no patent or literature report on how to extract oxyphylleudneI, a sesquiterpenoid compound, from Alpinia oxyphylla Miq. and realize its application in the preparation of anti-renal fibrosis drugs. III. Summary of the invention

[0004] In view of the above situation, in order to solve the defects of the prior art, the purpose of the present invention is to provide a sesquiterpenoid compound oxyphylleudne I isolated from Alpinia oxyphylla kernel and its application, to discover novel structural compounds from Alpinia oxyphylla kernel, and to realize the application of new compounds in the development of renal protective drugs.

[0005] The technical solution provided by the present invention is to separate and identify a new sesquiterpenoid compound oxyphylleudne I from the n-butanol part of Alpinia oxyphyllae seeds, and the structural formula is as follows:

[0006]

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

[0008] 1) drying and crushing the Alpinia oxyphylla kernel, extracting with 6-10 times the amount of 70% ethanol under reflux for 3 times, each time for 2 hours, combining the extracts and concentrating under reduced pressure to obtain an extract, suspending the extract with 2-4 times the amount of water, extracting with ethyl acetate and n-butanol in sequence until the extract is colorless (more than 10 times), and concentrating to obtain an ethyl acetate fraction, an n-butanol fraction, and a water fraction;

[0009] 2) The n-butanol fraction in step 1) was loaded onto an MCI Gel CHP-20 column (Mitsubishi Chemical Corporation, Japan), and gradient eluted with an ethanol-water system (0:100 to 95:5) to obtain a water fraction, a 10% ethanol fraction, a 30% ethanol fraction, a 50% ethanol fraction, a 70% ethanol fraction, and a 95% ethanol fraction in sequence;

[0010] 3) The 50% ethanol fraction of MCI in step 2) was subjected to gradient elution using a Sephadex LH-20 (Parmacia Biotech) column with methanol-water (0:100 to 95:5) as the eluent to obtain Fr.D.1 to Fr.D.7 in sequence;

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

[0012] 5) Fr.D.1.3 in step 4) was subjected to ODS (TOSOH, Japan) column chromatography and gradient eluted with methanol-water (0:100 to 95:5) to obtain Fr.D.1.3.1 to Fr.D.1.3.5;

[0013] 6) The component Fr.D.1.3.2 of step 5) was purified by semi-preparative HPLC to obtain compound oxyphylleudneI.

[0014] The invention discloses an application of the sesquiterpenoid compound oxyphylleudne I in preparing an active drug for resisting renal fibrosis.

[0015] The invention relates to a novel sesquiterpenoid compound oxyphylleudne I (compound III) extracted and separated from Alpinia oxyphyllae Oxyphyllae Radix. Experimental studies have shown that the compound can inhibit and reduce the expression of fibronectin, type I collagen and alpha-SMA in rat renal tubular duct epithelial cells (NRK 52E cells) induced by TGF-β1 in a dose-dependent manner, has anti-renal fibrosis activity, can be effectively used for preparing kidney protection drugs, and the preparation method thereof has good reproducibility and the obtained compound has high purity, which is conducive to further pharmacological and clinical research on the compound, expands the medicinal value of Alpinia oxyphyllae Radix, and has huge economic and social benefits. IV. Description of the drawings

[0016] Figure 1 is the HMBC of the compound of the present invention 1 H- 1 H COSY Related spectra.

[0017] Figure 2 The experimental and calculated NMR images of the compounds of the present invention are shown in FIG.

[0018] Figure 3 It is the experimental ICD diagram of the compound of the present invention.

[0019] Figure 4 The graph shows the anti-renal fibrosis activity of the compounds of the present invention on NRK-52E cells induced by TGF-β1.

[0020] Figure 5 is the HR-ESI-MS spectrum of the compound of the present invention.

[0021] Figure 6 It is the UV spectrum of the compound of the present invention.

[0022] Figure 7 is the IR spectrum of the compound of the present invention.

[0023] Figure 8 The compounds of the present invention 1 H-NMR spectrum.

[0024] Fig. 9 The compounds of the present invention 13 C-NMR spectrum.

[0025] Fig.10 It is the COSY spectrum of the compound of the present invention.

[0026] Fig.11 It is the HSQC spectrum of the compound of the present invention.

[0027] Fig.12 It is the HMBC spectrum of the compound of the present invention. V. Specific implementation methods

[0028] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0029] Example 1

[0030] In the specific implementation of the present invention, the preparation method of the compound oxyphylleudne I may include the following steps:

[0031] 1) 40.00 kg of Alpinia oxyphylla kernel was dried and crushed, and refluxed and extracted with 8 times the amount of 70% ethanol for 2 h (3 times), and the extracts were combined and concentrated under reduced pressure to obtain 8.30 kg of extract, and the extract was suspended with 3 times the amount of water and extracted with 30 L of ethyl acetate and n-butanol in sequence until the extract was colorless (more than 10 times), and then concentrated to obtain ethyl acetate fraction (1.45 kg), n-butanol fraction (628.33 g) and water fraction (6.60 kg);

[0032] 2) The n-butanol fraction in step 1) was subjected to an MCI Gel CHP-20 column (Mitsubishi Chemical Corporation, Japan) and eluted with an ethanol-water system (0:100 to 95:5) in a gradient manner to obtain a water 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);

[0033] 3) The 50% ethanol fraction of MCI in step 2) was subjected to gradient elution using a Sephadex LH-20 (Parmacia Biotech) column with methanol-water (0:100 to 95:5) as the eluent to obtain Fr.D.1 to Fr.D.7 in sequence;

[0034] 4) Fr.D.1 in step 3) was subjected to MCI (Mitsubishi Chemical Corporation, Japan) column chromatography, and gradient eluted with methanol-water (0:100-95:5) as eluent to obtain four components, namely Fr.D.1.1-Fr.D.1.4;

[0035] 5) Fr.D.1.3 in step 4) was subjected to ODS (TOSOH, Japan) column chromatography and gradient eluted with methanol-water (0:100 to 95:5) to obtain Fr.D.1.3.1 to Fr.D.1.3.5;

[0036] 6) The component Fr.D.1.3.2 of step 5) was purified by semi-preparative HPLC to obtain compound oxyphylleudne I (33.64 mg; MeOH / H2O, 28:72, flow rate: 3 mL / min; t R =28.5min).

[0037] The high-resolution mass spectrum of the present invention is measured by TripleTOF 6600 high-performance liquid chromatography-high-resolution mass spectrometry (ABSCIEX); the compound is purified by a Syplex LC-52 semi-preparative liquid chromatograph (Syplex Beijing Technology Co., Ltd.); the specific rotation is measured by an Anton Paar MCP 5100 polarimeter (Anton Paar, Austria); the NMR spectrum is measured by a Bruker AM-500 MHz nuclear magnetic resonance spectrum using a superconducting nuclear magnetic resonance instrument (TMS as an internal standard, Bruker, Germany); the ultraviolet spectrum is measured by a Thermo EVO300 ultraviolet spectrophotometer, and the infrared spectrum is measured by a Thermo Nicolet IS10 infrared spectrometer (Thermo Scientific, USA).

[0038] 1. Structural identification of compounds

[0039]

[0040] Compound III, light yellow oil, (c 0.1, MeOH); IR (MeOH) v max 3415,1708,1659,1461,1425,1365,1203,1034cm -1 ; UV(MeOH)λ max (logε):200.0(2.51),236.0(2.97)nm; HR-ESI-MS[M+Na]+m / z 291.1573(calcd for C 15 H 24 O4Na 291.1567) suggests that its molecular formula is C 15 H 24 O4.

[0041] 1 There is a carbon-carbon double bond signal δ in H NMR (CD3OD, 500MHz) H 6.06 (1H, s, H-6), six methylene signals [δ H3.59 (1H, d, J = 11.2 Hz, H-12a), 3.49 (1H, d, J = 11.2 Hz, H-12b), 2.45 (2H, dd, J = 9.0, 4.4 Hz, H-8), 2.45 (2H, dd, J = 9.0, 4.4 Hz, H-3), 1.96 (1H, dt, J = 13.4, 5.8 Hz, H-9a), 1.78 (1H, dt, J = 13.4, 5.2 Hz, H-9b), 1.53 (1H, m, H-1a), 1.49 (1H, m, H-2a), 1.45 (1H, m, H-1b), 1.38 (1H, m, H-2b)] and three methyl signals [δ H 2.10 (3H, s, H-14), 1.29 (3H, s, H-13), 1.05 (3H, s, H-15)]. 13 A total of 15 carbon signals were observed in the CNMR (CD3OD, 125 MHz) spectrum combined with the HSQC spectrum, including two carbonyl signals [δ C 211.7 (C-4), 207.2 (C-5)], a carbon-carbon double bond signal [δ C 123.9(C-6),170.1(C-7)], two quaternary carbons [δ C 44.7 (C-10), 76.1 (C-11)], six methylene signals [δ C 36.9 (C-1), 19.4 (C-2), 44.5 (C-3), 24.1 (C-8), 34.6 (C-9), 69.4 (C-12)] and three methyl signals [δ C 23.7 (C-13), 29.8 (C-14), 22.2 (C-15)]. In the HMBC spectrum, H2-3 (δ H 2.45) / H3-14(δ H 2.10) and C-4(δ C 211.7) has related signals; H-2a (δ H 1.49) / H3-15(δ H 1.05) and C-1(δ C 36.9) has related signals; H-6 (δ H 6.06) / H2-8(δ H 2.45) / H-12a(δ H 3.59) / H3-13(δ H 1.29) and C-11(δ C 76.1) There is a related signal; combined 1 H- 1A series of related signals of H2-1 / H2-2 / H2-3 and H2-8 / H2-9 in the HCOSY spectrum indicate that compound III is a eucalyptane-type sesquiterpene with ring opening at C-4 and C-5. The two chiral centers C-10 and C-11 of compound III are far apart in space, and there are no related signals in the NOESY spectrum. Therefore, the calculated NMR was used to determine its relative configuration as (10R, 11S) and (10S, 11R). First, we calculated the ECD spectra of the two relative configurations (10R, 11S) and (10S, 11R) at the B3LYP / 6-31G (d, p) level using the TD-DFT method. After comparison, we found that the measured ECD spectrum was closer to the trend of (10R, 11S), and determined its absolute configuration to be (10R, 11S). Since the chiral center C-11 is located on the flexible side chain outside the ring, in order to ensure the rigor of the absolute configuration, the vicinal diol structural fragments at C-11 and C-12 can be further determined by using the transition metal Mo2(OAc)4 reagent to determine the ICD spectrum. Compound III was dissolved in DMSO solution, and after adding Mo2(OAc)4 reagent for full chelation, a Mo derivative with cotton effect was generated, and the ICD spectrum was determined. Because compound III has an absorption signal at 300-400nm in the ECD spectrum, we chose the difference spectrum between the ICD spectrum of the Mo derivative of compound III and the ECD spectrum of compound III as the analysis object. The cotton effect at 300nm in the difference spectrum is negative. The absolute configuration of C-11 is determined to be S by the Snatzke rule, so the absolute configuration of compound III is determined to be (10R, 11S). Therefore, the structure of the compound was determined to be (10R,11S)-7-(11,12-dihydroxy-11-methylethyl)-1-methyl-1-(4-oxopentyl)-3-cyclohexen-2-one and was named oxyphylleudneI.

[0042] Table 1 Compound III 1 H-NMR and 13 C-NMR data (in CD3OD)

[0043]

[0044] II. Study on the renal protective activity of the novel sesquiterpenoid compound III of the present invention

[0045] 2.1 Cell lines

[0046] NRK 52E: normal rat renal tubular epithelial cells

[0047] 2.2 Experimental instruments, consumables and reagents

[0048] Table 2 Instrument names and manufacturers

[0049]

[0050]

[0051] Table 3 Reagent names and manufacturers list

[0052]

[0053] 2.3 Experimental methods

[0054] (1) Cell culture and treatment: NRK 52E cells were cultured in DMEM medium containing 10% fetal bovine serum. After trypsin digestion, the cells were seeded into 12-well plates (2×10 4 cells / mL) until they grow to 70%-80%, TGF-β1 (10ng / mL) is added to induce cell fibrosis, and the compound is added at the same time. After 48h, the cells are collected for Western Blot. The compounds used in the cell experiment are dissolved in dimethyl sulfoxide.

[0055] (20) Cell viability test: NRK 52E cells (2×10 4 cells / mL) were inoculated into 96-well cell culture plates. After overnight culture, cells were treated with different compounds or DMSO for 48 h. The supernatant was removed and 100 μL of culture medium (V 空培养基 / V Cell Count Kit-8 =10 / 1) into each well. After 1 hour, the absorbance of each well was recorded at 450 nm using an ELISA reader.

[0056] (3) Western blot: After the cells were treated with compounds and TGF-β1, the total protein of the cell line was extracted using 100 μL 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 PVDF membranes. They were blocked with 5% skim milk for 1 hour, then coated with the designated antibodies overnight at 4°C, and then incubated with secondary antibodies for 2 hours at room temperature. The bands were visualized and measured using an ECL kit and developer. ImageJ software was used to perform grayscale analysis of the immunoblotting results.

[0057] (4) Data analysis: All experimental data obtained in the present invention were independently repeated three times. The data are presented as the mean with standard error of the mean (SEM). Statistical analysis was performed using Graphpad Prism 8 (Graphpad Software, San Diego, CA, USA) and Excel (Microsoft). * P≤0.05, ** P≤0.01, *** P≤0.001, and **** When P ≤ 0.0001, the difference was considered significant.

[0058] 2.4 Experimental Results

[0059] After adding different concentrations of compounds to the cells, 10 ng / mL TGF-β1 was added for 48 hours. The effects of different concentrations of compounds on cell proliferation were detected by CCK-8 method (A), and the protein levels of fibronectin, type I collagen and α-SMA were detected by immunoblotting, and the GAPDH value was used as a control (B). The (C) fibronectin, (D) type I collagen and (E) α-SMA proteins were quantified by bar graphs. The data represent the mean ± SEM values ​​of three experiments. GW788388 (GW) was used as a positive control. Figure 4 As shown, compared with the positive drug GW788388, the oxyphylleudne I compound of the present invention can significantly reduce the expression of Fibronectin and Collagen I, indicating that it can reduce the renal fibrosis of NRK 52E cells induced by TGF-β1 and has renal protective activity, proving that the compound of the present invention can reduce the renal fibrosis of NRK-52e cells induced by TGF-1.

[0060] 2.5 Conclusion

[0061] The anti-renal fibrosis activity study showed that the compounds in Alpinia oxyphylla could inhibit and attenuate the expression of fibronectin, type I collagen and α-SMA in NRK 52E cells induced by TGF-β1 in a dose-dependent manner.

[0062] The invention has identified a new sesquiterpenoid compound oxyphylleudne I extracted and separated from the seeds of Alpinia oxyphylla, and through experimental research, it is found that the compound has an anti-renal fibrosis effect and can be used to prepare kidney protection drugs, thus expanding the medicinal value of the seeds of Alpinia oxyphylla, having great development prospects and good economic and social benefits.

[0063] It should be pointed out that the above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make changes or modify the technical contents disclosed above into equivalent embodiments with equivalent changes without departing from the scope of the technical solution of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A sesquiterpenoid compound oxyphylleudne I isolated from Alpinia oxyphylla kernel, characterized in that: The structural formula is as follows:

2. The method for preparing oxyphylleudne I, a sesquiterpenoid compound isolated from Alpinia oxyphyllae seeds according to claim 1, is characterized in that: The following steps are involved: 1) drying and crushing the Alpinia oxyphylla kernel, extracting with 6-10 times the amount of 70% ethanol under reflux for 3 times, each time for 2 hours, combining the extracts and concentrating under reduced pressure to obtain an extract, suspending the extract with 2-4 times the amount of water, and extracting with ethyl acetate and n-butanol for more than 10 times in sequence until the extract is colorless, and concentrating to obtain an ethyl acetate fraction, an n-butanol fraction, and a water fraction; 2) placing the n-butanol fraction in step 1) on an MCI Gel CHP-20 column and eluting with an ethanol-water system at a ratio of 0:100 to 95:5 to obtain a water fraction, a 10% ethanol fraction, a 30% ethanol fraction, a 50% ethanol fraction, a 70% ethanol fraction and a 95% ethanol fraction in sequence; 3) The 50% ethanol fraction of MCI in step 2) was subjected to gradient elution using a Sephadex LH-20 column with methanol-water in a ratio of 0:100 to 95:5 as the eluent to obtain Fr.D.1 to Fr.D.7 in sequence; 4) subjecting Fr.D.1 in step 3) to MCI column chromatography, and gradient eluting with methanol-water in a ratio of 0:100 to 95:5 as the eluent to obtain four components, namely Fr.D.1.1 to Fr.D.1.4; 5) subjecting Fr.D.1.3 in step 4) to ODS column chromatography and gradient eluting with methanol-water at a ratio of 0:100 to 95:5 to obtain Fr.D.1.3.1 to Fr.D.1.3.5; 6) The component Fr.D.1.3.2 of step 5) was purified by semi-preparative HPLC to obtain compound oxyphylleudne I.

3. The method for preparing oxyphylleudne I, a sesquiterpenoid compound isolated from Alpinia oxyphyllae seeds according to claim 1, is characterized in that: The following steps are involved: 1) 40.00 kg of Alpinia oxyphylla kernel was dried and crushed, and refluxed and extracted with 8 times the amount of 70% ethanol for 3 times, each time for 2 hours. The extracts were combined and concentrated under reduced pressure to obtain 8.30 kg of extract. The extract was suspended with 3 times the amount of water and then extracted with 30 L of ethyl acetate and n-butanol in sequence until the extract was colorless, and then concentrated to obtain ethyl acetate fraction, n-butanol fraction and water fraction; 2) placing the n-butanol fraction in step 1) on an MCI Gel CHP-20 column, and eluting with an ethanol-water system at a gradient ratio of 0:100 to 95:5 to obtain a water fraction, a 10% ethanol fraction, a 30% ethanol fraction, a 50% ethanol fraction, a 70% ethanol fraction, and a 95% ethanol fraction in sequence; 3) The 50% ethanol fraction of MCI in step 2) was subjected to gradient elution using a Sephadex LH-20 column with methanol-water in a ratio of 0:100 to 95:5 as the eluent to obtain Fr.D.1 to Fr.D.7 in sequence; 4) subjecting Fr.D.1 in step 3) to MCI column chromatography, and gradient eluting with methanol-water in a ratio of 0:100 to 95:5 as the eluent to obtain four components, namely Fr.D.1.1 to Fr.D.1.4; 5) subjecting Fr.D.1.3 in step 4) to ODS column chromatography and gradient eluting with methanol-water at a ratio of 0:100 to 95:5 to obtain Fr.D.1.3.1 to Fr.D.1.3.5; 6) The component Fr.D.1.3.2 of step 5) was purified by semi-preparative HPLC to obtain compound oxyphylleudne I.

4. Use of the sesquiterpenoid compound oxyphylleudne I according to claim 1 in the preparation of an active drug against renal fibrosis.

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