Acetylated flavonoid glycoside compound extracted from fructus alpiniae oxyphyllae as well as preparation method and application of acetylated flavonoid glycoside compound

Abstract

The invention relates to an acetylated flavonoid glycoside compound extracted from fructus alpiniae oxyphyllae as well as a preparation method and application of the acetylated flavonoid glycoside compound, which can effectively solve the problem of application of the acetylated flavonoid glycoside compound Oxyphyllvide G extracted from the fructus alpiniae oxyphyllae in preparation of kidney protection medicines. A new acetylated flavonoid glycoside compound Oxyphyllvide G is separated and identified from an n-butyl alcohol part of fructus alpiniae oxyphyllae, the molecular formula of the acetylated flavonoid glycoside compound Oxyphyllvide G is C23H20O14, and the acetylated flavonoid glycoside compound Oxyphyllvide G with a rare structure is extracted and separated from the fructus alpiniae oxyphyllae through an extraction and separation technology of natural active ingredients. Experimental research finds that the compound can inhibit and weaken the expression of fibronectin, I-type collagen and alpha-SMA in NRK 52E cells induced by TGF-beta1 in a dose-dependent manner, has a kidney protection effect, can be effectively used for preparing kidney protection drugs, develops the medicinal value of fructus alpiniae oxyphyllae, and has huge economic and social benefits.

Description

1. Technical Field

[0001] The invention relates to the field of medicine, in particular to an acetylated flavonoid glycoside compound extracted from Alpinia oxyphylla seeds and a preparation method 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, 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 marker of renal fibrosis. In addition, during progressive tubulointerstitial nephropathy, damaged or apoptotic cells attract inflammatory cells such as macrophages and granulocytes to infiltrate to induce an inflammatory response, leading to the progression of renal damage. Therefore, anti-fibrosis has recently been considered as a new strategy 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 abundant sources. It is increasingly valued by people. Finding and developing traditional Chinese medicine with kidney-protective effects and its active ingredients has gradually become one of the research hotspots in modern medicine.

[0004] Alpinia oxyphylla Miq. is the dried mature fruit and seed of the ginger plant Alpinia oxyphylla Miq., which is a natural plant with medicinal and edible properties announced by the Ministry of Health in 1998. It is mainly produced in the mountainous area of ​​Hainan Island in 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 enters the heart, spleen and kidney meridians and has the effects of warming, strengthening and consolidating, warming the spleen and stopping diarrhea, consolidating saliva and 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. Flavonoids, as the main biologically active components of Alpinia oxyphylla Miq., have multiple pharmacological effects, among which galangin has a potential therapeutic effect on rats with overactive bladder. However, there has been no public report on the acetylated flavonoid glycoside compound Oxyphyllvonide G extracted from Alpinia oxyphylla Miq. III. Summary of the invention

[0005] 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 an acetylated flavonoid glycoside compound extracted from Alpinia oxyphylla kernel and its preparation method and application, which can effectively solve the problem of the acetylated flavonoid glycoside compound Oxyphyllvonide G extracted from Alpinia oxyphylla kernel and realize its application in the preparation of therapeutic kidney protection drugs.

[0006] The technical solution of the present invention is to separate and identify a new acetylated flavonoid glycoside compound Oxyphyllvonide G from the n-butanol part of Alpinia oxyphylla kernel, and its molecular formula is C 23 H 20 O 14 , the structural formula is as follows:

[0007]

[0008] The preparation method thereof comprises the following steps:

[0009] 1) Dry and crush the Alpinia oxyphylla kernel, extract with 8 times the amount of 70% ethanol under reflux for 3 times, each time for 2 hours, combine the extracts and concentrate under reduced pressure to obtain an extract;

[0010] 2) suspending the extract obtained in step 1) with 3 times the amount of water, extracting with 30L of 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;

[0011] 3) The n-butanol fraction obtained in step 2) was loaded onto an MCI Gel CHP-20 column and eluted with an ethanol-water system (0:100 to 95:5) in a gradient manner 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;

[0012] 4) The 50% ethanol fraction obtained in step 3) was subjected to gradient elution using a Sephadex LH-20 column (Parmacia Biotech) with methanol-water as the eluent to obtain Fr.D.1 to Fr.D.7 in sequence;

[0013] 5) The Fr.D.5 obtained in step 4) was subjected to silica gel column chromatography (200-300 mesh, Qingdao Ocean Chemical Plant) with dichloromethane-methanol as the eluent for gradient elution to obtain components Fr.D.5.1-Fr.D.5.4, wherein component Fr.D.5.4 was purified by semi-preparative high performance liquid chromatography (YMC-Pack ODS-A column; 250mm×20mm, 5μm) with methanol:water=45:55 as the mobile phase to obtain compound Oxyphyllvonide G.

[0014] The invention discloses an application of the acetylated flavonoid glycoside compound Oxyphyllvonide G extracted from Alpinia oxyphylla seeds in the preparation of kidney protection drugs.

[0015] The present invention extracts and separates a structurally rare acetylated flavonoid glycoside compound Oxyphyllvonide G from Alpinia oxyphylla kernel by using the extraction and separation technology of natural active ingredients, and through experimental research it is found that the compound can 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, has a kidney-protecting effect, can be effectively used for preparing kidney-protecting drugs, expands the medicinal value of Alpinia oxyphylla kernel, and has huge economic and social benefits. IV. Description of the drawings

[0016] Figure 1 HR-ESI-MS spectrum of the compound of the present invention

[0017] Figure 2 UV spectrum of the compound of the present invention

[0018] Figure 3 IR spectrum of the compound of the present invention

[0019] Figure 4 The compounds of the present invention 1 H-NMR spectrum.

[0020] Figure 5 The compounds of the present invention 13 C-NMR spectrum.

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

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

[0023] Figure 8 is the COSY spectrum of the compound of the present invention.

[0024] Fig. 9 The effect of different concentrations of the compound of the present invention on the proliferation of NRK 52E cells (A). V. Specific implementation methods

[0025] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples.

[0026] Example 1

[0027] The present invention, when implemented, comprises the following steps:

[0028] 1) 40.00 kg of Alpinia oxyphylla kernels were 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;

[0029] 2) The extract obtained in step 1) was suspended with 3 times the amount of water, extracted with 30L of ethyl acetate and n-butanol in sequence until the extract was colorless (more than 10 times), and concentrated to obtain 1.45kg of ethyl acetate fraction, 628.33g of n-butanol fraction and 6.60kg of water fraction;

[0030] 3) The n-butanol fraction obtained in step 2) was loaded onto an MCI Gel CHP-20 column (Mitsubishi Chemical Corporation, Japan) and eluted with an ethanol-water system at a ratio of 0:100 to 95:5 to obtain 371.10 g of a water fraction, 8.43 g of a 10% ethanol fraction, 33.36 g of a 30% ethanol fraction, 49.82 g of a 50% ethanol fraction, 20.73 g of a 70% ethanol fraction and 105.06 g of a 95% ethanol fraction;

[0031] 4) The 50% ethanol fraction obtained in step 3) was subjected to gradient elution using a Sephadex LH-20 column (Parmacia Biotech) with methanol-water as the eluent to obtain Fr.D.1 to Fr.D.7 in sequence;

[0032] 5) Fr.D.5 obtained in step 4) was subjected to silica gel column chromatography (200-300 mesh, Qingdao Ocean Chemical Plant) with dichloromethane-methanol as the eluent for gradient elution to obtain components Fr.D.5.1-Fr.D.5.4, wherein component Fr.D.5.4 was purified by semi-preparative high performance liquid chromatography (YMC-Pack ODS-A column; 250mm×20mm, 5μm) with methanol: water = 45:55 as the mobile phase to obtain compound Oxyphyllvonide G (t R =18.8min; 24.6mg).

[0033] The present invention provides a new acetylated flavonoid glycoside compound Oxyphyllvonide G, which is extracted and separated from the dried mature fruit and seed of the ginger plant Alpinia oxyphylla, and can reduce the renal fibrosis of NRK 52E cells induced by TGF-β1 and reduce the expression of fibronectin, type I collagen and α-SMA in NRK 52E cells in a dose-dependent manner, and can be used as a new drug component or lead compound with renal protective effect. The relevant experimental data are as follows:

[0034] 1. Structural Identification

[0035] like Figure 1-8As shown, the high-resolution mass spectrum was measured using a TripleTOF 6600 HPLC-HRMS (AB SCIEX); the compound was purified using a Syplex LC-52 semi-preparative liquid chromatograph (Syplex Beijing Technology Co., Ltd.); the specific rotation was measured using an Anton Paar MCP 5100 polarimeter (Anton Paar, Austria); the NMR spectrum was measured using a Bruker AM-500 MHz nuclear magnetic resonance spectrum using a superconducting nuclear magnetic resonance instrument (TMS as internal standard, Bruker, Germany); the UV spectrum was measured using a Thermo EVO300 UV spectrophotometer, and the IR spectrum was measured using a Thermo Nicolet IS10 infrared spectrometer (Thermo Scientific, USA).

[0036] Oxyphyllvonide G, yellow amorphous powder; (c 0.1, MeOH); IR (MeOH) v max 3336,1653,1605,1303,1202,1087cm -1 ; UV(MeOH)λ max (logε):206.0(3.08),255.0(2.82),354.0(2.73)nm; HR-ESI-MS[M+Na] + m / z 543.0742 (calcd for C 23 H 20 O 14 Na 543.0745) suggests that its molecular formula is C 23 H 20 O 14 .

[0037] 1 The H-NMR (500 MHz, CD3OD) spectrum showed a signal of a 1,3,4,5 tetrasubstituted benzene ring [δ H 6.36 (1H, br s, H-8), 6.17 (1H, br s, H-6)], a signal of an ABX benzene ring system [δ H 7.71 (1H, br s, H-2'), 7.60 (1H, dd, J = 8.4, 1.5 Hz, H-6'), 6.85 (1H, d, J = 8.4 Hz, H-5')], one methyl signal δ H 2.04 (3H, s, 4”-COCH3), in addition, a group of hydrogen signals of glucuronic acid were found [δ H5.41(1H,d,J=7.7Hz,H-1”),3.62(1H,m,H-2”),3.69(1H,t,J=9.2Hz,H-3”),5.01(1H,t,J=9.6Hz,H-4”),3.93(1H,d,J=10.0Hz,H-5”)]. 13 There are two benzene ring signals in the C-NMR (125MHz, CD3OD) spectrum [δ C 162.9 (C-5), 99.9 (C-6), 165.9 (C-7), 94.7 (C-8), 159.0 (C-9), 105.6 (C-10), 123.3 (C-1'), 116.0 (C-2'), 145.9 (C-3'), 149.9 (C-4'), 117.3 (C-5'), 122.7 (C-6')], an α, β unsaturated ketone signal [δ C 158.3 (C-2), 135.3 (C-3), 179.1 (C-4)], an acetyl signal [δ C 171.8(4”-COCH3),20.8(4”-COCH3)] and a glucuronic acid signal [δ C 103.9(C-1”),75.5(C-2”),75.3(C-3”),73.0(C-4”),74.4(C-5”),169.8(C-6”)]. 1 H- 1In the H COSY spectrum, a series of correlation signals of H-1” / H-2” / H-3” / H-4” / H-5” proved the connection mode of carbon chains in glucuronic acid. Analysis of its HMBC spectrum, the correlation signal of H-4” and 4”-COCH3 confirmed that the acetyl group was connected to C-4”; the HMBC correlation between H-1” and C-3 confirmed that glucuronic acid was connected to C-3. By analyzing the hydrogen coupling constant of the glucuronic acid terminal group (J=7.8Hz), it was determined to be β configuration. The absolute configuration was determined to be β-D-glucuronic acid by LC-MS analysis after acid hydrolysis and derivatization: Take the compound (about 1 mg), add 2 mol / L HCl (3 mL) was heated and hydrolyzed at 90°C for 2.5 h. The mixture was concentrated and dried under vacuum, and then H2O (1.0 mL) was added, followed by extraction with ethyl acetate (1 mL*3). The ethyl acetate layer was concentrated and dried to obtain aglycone; the aqueous layer was concentrated and dried to obtain glucose. After concentration, the sample of the aqueous layer was added with L-cysteine ​​methyl ester hydrochloride (1 mg) and pyridine (1 mL) and heated in a 60°C water bath for 1.5 h, and then o-toluene isothiocyanate (20 μL) was added and heated in a 60°C water bath for 1.5 h. Another D-glucuronic acid standard was added with L-cysteine ​​methyl ester hydrochloride (1 mg) and pyridine (1 mL) and heated in a 60°C water bath for 1.5 h, and then o-toluene isothiocyanate (20 μL) was added and heated in a 60°C water bath for 1.5 h. The obtained glucuronic acid test sample derivative and D-glucuronic acid standard derivative were subjected to LC-MS analysis. LC-MS analysis conditions: TripleTOF6600UPLC / MS (AB SCIEX); Kinetex C18 (2.6μm, 2.1mm×50mm); gradient elution: 0.1% formic acid water (A)-methanol (B), (0-20min) 10%-100% B; column temperature 40℃, flow rate 0.3mL / min; mass spectrometry conditions: positive ion mode. By comparing the glucuronic acid test derivatives (t R =14.360min,m / z257.1096[M+H] + ) and D-glucuronic acid standard derivative (t R =14.347min,m / z 257.1095[M+H] + ) and mass spectrometry data to determine the absolute configuration of the sugar in the compound. Therefore, the compound structure was determined to be quercetin3-O-[4”-O-acetyl]-β-D-glucuronide. It was named Oxyphyllvonide G.

[0038] Table 1 Compounds Oxyphyllvonide G 1 H-NMR and 13 C-NMR data (in CD3OD)

[0039]

[0040] II. Study on the anti-renal fibrosis activity of the compound Oxyphyllvonide G of the present invention

[0041] 2.1 Cell lines

[0042] NRK 52E: normal rat renal tubular epithelial cells

[0043] 2.2 Experimental instruments, consumables and reagents

[0044] Table 2 Instrument names and manufacturers

[0045]

[0046]

[0047] Table 3 Reagent names and manufacturers list

[0048]

[0049]

[0050] 2.3 Experimental methods

[0051] (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.

[0052] (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.

[0053] (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 10% SDS-PAGE and transferred to PVDF membranes. The membranes 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.

[0054] (4) Data analysis: All experimental data obtained in this study were independently repeated three times. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis, including Student's t test and one-way analysis of variance, was performed using Graphpad Prism 6 (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.

[0055] 2.4 Experimental Results

[0056] like Fig. 9 As shown, the compounds of the present invention can reduce the renal fibrosis of NRK 52E cells induced by TGF-β1. After adding different concentrations of compounds to the cells, 10 ng / mL of 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 GAPDH value was used as control (B). The levels of (C) α-SMA, (D) type I collagen and (E) fibronectin were quantified by bar graphs. The data represent the mean ± SEM values ​​of three experiments. **** P<0.0001 compared with TGF-β1alone. #### P<0.0001 compared with DMSO alone. GW788388 (GW) was used as a positive control.

[0057] 2.5 Conclusion

[0058] 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.

[0059] The present invention has identified a structurally rare acetylated flavonoid glycoside compound Oxyphyllvonide G extracted and separated from Alpinia oxyphylla kernel, and through experimental research, it has been found that the compound has kidney-protecting effects and can be effectively used to prepare kidney-protecting drugs, thus expanding the medicinal value of Alpinia oxyphylla kernel, having huge development prospects, and having good economic and social benefits.

[0060] 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. An acetylated flavonoid glycoside compound extracted from Alpinia oxyphylla kernel, characterized in that: A new acetylated flavonoid glycoside compound, Oxyphyllvonide G, was isolated and identified from the n-butanol fraction of Alpinia oxyphylla. Its molecular formula is C 23 H 20 O 14 , the structural formula is as follows:

2. The method for preparing the acetylated flavonoid glycoside compound extracted from Alpinia oxyphylla kernel according to claim 1, characterized in that: The following steps are involved: 1) Dry and crush the Alpinia oxyphylla seeds, extract them with 8 times the amount of 70% ethanol under reflux for 3 times, each time for 2 hours, combine the extracts and concentrate under reduced pressure to obtain an extract; 2) suspending the extract obtained in step 1) with 3 times the amount of water, extracting with 30L of ethyl acetate and n-butanol in sequence until the extract is colorless, and concentrating to obtain an ethyl acetate fraction, an n-butanol fraction and a water fraction; 3) The n-butanol fraction obtained in step 2) was loaded onto an MCI Gel CHP-20 chromatographic column and eluted with an ethanol-water system gradient 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; 4) The 50% ethanol fraction obtained in step 3) was subjected to gradient elution using a Sephadex LH-20 column with methanol-water as the eluent to obtain Fr.D.1 to Fr.D.7 in sequence; 5) The Fr.D.5 obtained in step 4) was subjected to silica gel column chromatography with dichloromethane-methanol as the eluent for gradient elution to obtain components Fr.D.5.1 to Fr.D.5.4, wherein component Fr.D.5.4 was purified by semi-preparative high performance liquid chromatography with methanol: water = 45:55 as the mobile phase to obtain compound Oxyphyllvonide G.

3. The method for preparing the acetylated flavonoid glycoside compound extracted from Alpinia oxyphylla kernel according to claim 2, characterized in that: The following steps are involved: 1) 40.00 kg of Alpinia oxyphylla kernels were 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; 2) The extract obtained in step 1) was suspended with 3 times the amount of water, extracted with 30L of ethyl acetate and n-butanol in sequence until the extract was colorless, and concentrated to obtain 1.45kg of ethyl acetate fraction, 628.33g of n-butanol fraction and 6.60kg of water fraction; 3) The n-butanol fraction obtained in step 2) was loaded on an MCI Gel CHP-20 column and eluted with an ethanol-water system at a ratio of 0:100 to 95:5 to obtain 371.10 g of a water fraction, 8.43 g of a 10% ethanol fraction, 33.36 g of a 30% ethanol fraction, 49.82 g of a 50% ethanol fraction, 20.73 g of a 70% ethanol fraction, and 105.06 g of a 95% ethanol fraction; 4) The 50% ethanol fraction obtained in step 3) was subjected to gradient elution using a Sephadex LH-20 column with methanol-water as the eluent to obtain Fr.D.1 to Fr.D.7 in sequence; 5) The Fr.D.5 obtained in step 4) was subjected to silica gel column chromatography with dichloromethane-methanol as the eluent for gradient elution to obtain components Fr.D.5.1 to Fr.D.5.4, wherein component Fr.D.5.4 was purified by semi-preparative high performance liquid chromatography under the following chromatographic conditions: YMC-PackODS-A column; 250mm×20mm, 5μm, with methanol:water=45:55 as the mobile phase to obtain compound Oxyphyllvonide G.

4. Use of the acetylated flavonoid glycoside compound Oxyphyllvonide G extracted from Alpinia oxyphylla seeds according to claim 1 in the preparation of kidney protection drugs.

5. Use of the acetylated flavonoid glycoside compound Oxyphyllvonide G prepared by the preparation method according to claim 2 or 3 in the preparation of kidney protection drugs.

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