An acetylated flavonoid glycoside compound extracted from fructus evodiae and a preparation method and application thereof

Abstract

The application relates to an acetylated flavonoid glycoside compound extracted from Fructus Schisandrae and a preparation method and application thereof, and can effectively solve the problem of the application of the acetylated flavonoid glycoside compound Oxyphyllvonide G extracted from Fructus Schisandrae in preparing a kidney protection medicine, and the technical solution is that a new acetylated flavonoid glycoside compound Oxyphyllvonide G is separated and identified from a n-butanol part of Fructus Schisandrae, the molecular formula of the compound is C 23 H 20 O 14 The application extracts and separates a rare acetylated flavonoid glycoside compound Oxyphyllvonide G from Fructus Schisandrae through a natural active ingredient extraction and separation technology, and it is found through test research that the compound can inhibit and weaken the expression of fibronectin, type I collagen and alpha-SMA in NRK 52E cells induced by TGF-beta 1 in a dose-dependent manner, has a kidney protection effect, can be effectively used for preparing a kidney protection medicine, and develops the medicinal value of Fructus Schisandrae, and has huge economic and social benefits.

Description

I. Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to an acetylated flavonoid glycoside compound extracted from Alpinia oxyphylla, its preparation method, and its application. II. Background Technology

[0002] Chronic kidney disease (CKD) is one of the major public health problems worldwide, with a global prevalence of 9.1% (approximately 697.5 million cases) and causing 1.2 million deaths annually. 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 highlight the urgent need to improve CKD and the importance of finding targeted therapies. The development of renal fibrosis is one of the important biological and pathological changes in CKD patients. Certain fibrotic molecules, such as fibronectin, transforming growth factor-β1 (TGF-β1), and collagen I, III, and IV, are important indicators of renal fibrosis progression. Fibroblasts can also proliferate and express α-smooth muscle actin (α-SMA), which has been identified as a marker of renal fibrosis. Furthermore, in progressive tubulointerstitial nephropathy, damaged or apoptotic cells attract inflammatory cells such as macrophages and granulocytes to infiltrate, inducing an inflammatory response and leading to the progression of kidney damage. Therefore, anti-fibrosis has recently been considered a new strategy for treating CKD.

[0003] Traditional Chinese medicine (TCM) is increasingly valued for its versatility, bidirectional regulatory effects, fewer adverse reactions, and abundant sources in the treatment of chronic kidney disease. The search for and development of TCM with renal protective effects and its effective components has gradually become one of the research hotspots in modern medicine.

[0004] Alpinia oxyphylla Miq., the dried, mature fruit and seeds of the ginger family plant, is a natural plant used for both food and medicine, 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 Chinese medicinal herbs. Alpinia oxyphylla is warm in nature and pungent in taste, entering the heart, spleen, and kidney meridians. It has the effects of warming and tonifying, consolidating the spleen and stopping diarrhea, reducing saliva production, and warming the kidneys and consolidating essence to reduce urination. Alpinia oxyphylla mainly contains terpenes, diphenylheptanes, flavonoids, sterols, and phenolic acids. Flavonoids, as the main bioactive components of Alpinia oxyphylla, have various pharmacological effects, including the potential therapeutic effect of galangin on rats with overactive bladder. However, there are currently no publicly reported acetylated flavonoid glycosides, Oxyphyllvonide G, extracted from Alpinia oxyphylla. III. Summary of the Invention

[0005] In view of the above situation and to overcome the shortcomings of the prior art, the purpose of this invention is to provide an acetylated flavonoid glycoside compound extracted from Alpinia oxyphylla, its preparation method and application, which can effectively solve the problem of applying the acetylated flavonoid glycoside compound Oxyphyllvonide G extracted from Alpinia oxyphylla in the preparation of nephroprotective drugs.

[0006] The technical solution provided by this invention is the isolation and identification of a novel acetylated flavonoid glycoside compound, Oxyphyllvonide G, from the n-butanol fraction of Alpinia oxyphylla, with the molecular formula C. 23 H 20 O 14 The structural formula is as follows:

[0007]

[0008] Its preparation method includes the following steps:

[0009] 1) After drying, Alpinia oxyphylla is pulverized and extracted by reflux with 8 times the amount of 70% ethanol 3 times, 2 hours each time. The extracts are combined and concentrated under reduced pressure to obtain an extract.

[0010] 2) The extract obtained in step 1) is suspended in 3 times the amount of water, and extracted successively with 30L of ethyl acetate and n-butanol until the extract is colorless (more than 10 times), and concentrated to obtain the ethyl acetate fraction, n-butanol fraction and 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) gradient to obtain the water fraction, 10% ethanol fraction, 30% ethanol fraction, 50% ethanol fraction, 70% ethanol fraction and 95% ethanol fraction in sequence.

[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 sequentially;

[0013] 5) The Fr.D.5 obtained in step 4) was subjected to silica gel column chromatography (200-300 mesh, Qingdao Marine Chemical Plant) with dichloromethane-methanol as the eluent gradient to obtain fractions Fr.D.5.1 to Fr.D.5.4. Among them, fraction 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 present invention relates to the application of Oxyphyllvonide G, an acetylated flavonoid glycoside compound extracted from Alpinia oxyphylla, in the preparation of a nephroprotective drug.

[0015] This invention utilizes natural active ingredient extraction and separation technology to extract and isolate a structurally rare acetylated flavonoid glycoside compound, Oxyphyllvonide G, from Alpinia oxyphylla. Experimental studies have shown that this compound can inhibit and, in a dose-dependent manner, reduce the expression of fibronectin, type I collagen, and α-SMA induced by TGF-β1 in NRK 52E cells, exhibiting a renal protective effect. It can be effectively used to prepare renal protective drugs, expanding the medicinal value of Alpinia oxyphylla and bringing significant economic and social benefits. IV. Description of the attached drawings

[0016] Figure 1 The HR-ESI-MS spectrum of the compound of this invention.

[0017] Figure 2 The UV spectrum of the compound of this invention.

[0018] Figure 3 The IR spectrum of the compound of this invention.

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

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

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

[0022] Figure 7 This is the HMBC spectrum of the compound of this invention.

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

[0024] Figure 9 The effect of different concentrations of the compound of the present invention on the proliferation of NRK 52E cells (A). V. Detailed Implementation Methods

[0025] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples.

[0026] Example 1

[0027] In its specific implementation, this invention includes the following steps:

[0028] 1) After drying 40.00 kg of Alpinia oxyphylla, it was pulverized and extracted three times with 8 times the amount of 70% ethanol, 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) is suspended in 3 times the amount of water, and extracted successively with 30L of ethyl acetate and n-butanol until the extract is colorless (more than 10 times). The extract is then 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 in a gradient ratio of 0:100 to 95:5, yielding 371.10 g of water fraction, 8.43 g of 10% ethanol fraction, 33.36 g of 30% ethanol fraction, 49.82 g of 50% ethanol fraction, 20.73 g of 70% ethanol fraction, and 105.06 g of 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 sequentially;

[0032] 5) The Fr.D.5 obtained in step 4) was subjected to silica gel column chromatography (200-300 mesh, Qingdao Marine Chemical Plant) with a gradient elution of dichloromethane-methanol as the eluent to obtain fractions Fr.D.5.1 to Fr.D.5.4. Among them, fraction 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 =18.8 min; 24.6 mg).

[0033] This invention provides a novel acetylated flavonoid glycoside compound, Oxyphyllvonide G, extracted and isolated from the dried mature fruit and seeds of Alpinia oxyphylla (a plant in the ginger family). It can alleviate TGF-β1-induced renal fibrosis in NRK 52E cells and attenuate the expression of fibronectin, type I collagen, and α-SMA in NRK 52E cells in a dose-dependent manner. It can serve as a novel drug component or lead compound with renal protective effects. Relevant experimental data are as follows:

[0034] I. Structural Assessment

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

[0036] Oxyphyllvonide G, a 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) indicates its molecular formula is C 23 H 20 O 14 .

[0037] 1 The H-NMR (500MHz, CD3OD) spectrum shows 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)], the signal [δ] of an ABX benzene ring system H 7.71 (1H,br s,H-2'), 7.60 (1H,dd,J=8.4,1.5Hz,H-6'), 6.85 (1H,d,J=8.4Hz,H-5')], a methyl signal δ H 2.04 (3H, s, 4”-COCH3), and a set of hydrogen signals for glucuronic acid were also 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 Two benzene ring signals were observed 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”)]. In 1 H- 1In the H COSY spectrum, a series of correlation signals from H-1” / H-2” / H-3” / H-4” / H-5” confirmed the carbon chain linkage in glucuronic acid. Analysis of its HMBC spectrum showed that the correlation signal between H-4” and 4”-COCH3 confirmed the acetyl group at C-4”; the HMBC correlation between H-1” and C-3 confirmed the glucuronic acid group at C-3. Analysis of the terminal hydrogen coupling constant (J = 7.8 Hz) determined it to be a β-configuration. LC-MS analysis after acid hydrolysis and derivatization confirmed its absolute configuration as β-D-glucuronic acid: (Approximately 1 mg of the compound was added to a 2 mol / L...) Hydrolyze the mixture with HCl (3 mL) at 90 °C for 2.5 h. Concentrate and dry the mixture under vacuum, then add H₂O (1.0 mL) and extract with ethyl acetate (1 mL * 3). Concentrate and dry the ethyl acetate layer to obtain the aglycone; concentrate and dry the aqueous layer to obtain glucose. Add L-cysteine ​​methyl ester hydrochloride (1 mg) and pyridine (1 mL) to the concentrated aqueous sample and heat in a 60 °C water bath for 1.5 h, then add o-toluene isothiocyanate (20 μL) and heat in a 60 °C water bath for 1.5 h. Separately, add L-cysteine ​​methyl ester hydrochloride (1 mg) and pyridine (1 mL) to the D-glucuronic acid standard and heat in a 60 °C water bath for 1.5 h, then add o-toluene isothiocyanate (20 μL) and heat in a 60 °C water bath for 1.5 h. Perform liquid chromatography-mass spectrometry (LC-MS) analysis on the obtained glucuronic acid test derivative and D-glucuronic acid standard derivative. LC-MS conditions: TripleTOF 6600 UPLC / MS (AB) SCIEX); Kinetex C18 (2.6 μm, 2.1 mm × 50 mm); gradient elution: 0.1% formic acid water (A) - methanol (B), (0–20 min) 10%–100% B; column temperature 40 °C, flow rate 0.3 mL / min; mass spectrometry conditions: positive ion mode. By comparing the glucuronic acid test sample derivative (t... R =14.360 min, m / z 257.1096 [M+H] + ) and D-glucuronic acid standard derivatives (t R =14.347min,m / z 257.1095[M+H] + The retention time and mass spectrometry data of the compound were used to determine the absolute configuration of the sugar. Therefore, the structure of the compound was determined to be quercetin3-O-[4”-O-acetyl]-β-D-glucuronide, and named Oxyphyllvonide G.

[0038] Table 1. Compound 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 this 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 List of Instrument Names and Manufacturers

[0045]

[0046]

[0047] Table 3. List of Reagent Names and Manufacturers

[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. Cells were seeded into 12-well plates (2 × 10⁻⁶ cells / well) by trypsin digestion. 4 Cells were allowed to grow to 70%-80% of their target size, and then TGF-β1 (10 ng / mL) was added to induce cell fibrosis, along with the added compound. Cells were harvested after 48 hours for Western blotting. The compound used in the cell experiments was dissolved in dimethyl sulfoxide.

[0052] (20) Cell viability test: NRK 52E cells (2 × 10⁶ cells / year) were cultured in DMEM medium containing 10% fetal bovine serum. 4 Cells were seeded in 96-well cell culture plates at a density of 100 cells / mL. After overnight culture, cells were treated with different compounds or DMSO for 48 hours. The supernatant was removed, and 100 μL of culture medium (V) was added. 空培养基 / V Cell Count Kit-8 =10 / 1) into each well. After 1 hour, record the absorbance of each well at 450 nm using a microplate reader.

[0053] (3) Western blot: After treatment with the compound and TGF-β1, total protein was extracted from the cell line using 100 μL of RIPA (strong) lysis buffer (1 mM PMSF + protease inhibitor + nuclease), and protein samples were quantified using the BCA assay. Equal volumes of protein extract were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 1 hour, then incubated overnight at 4°C with the specified antibody, and finally incubated with secondary antibody at room temperature for 2 hours. The bands were visualized and measured using an ECL kit and a spectrophotometer. ImageJ software was used for 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 (SEM). Statistical analysis, including Student's t-test and one-way ANOVA, 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 **** A difference is considered significant when P ≤ 0.0001.

[0055] 2.4 Experimental Results

[0056] like Figure 9 As shown, the compounds of this invention can alleviate TGF-β1-induced renal fibrosis in NRK 52E cells. Cells were treated with different concentrations of the compounds followed by 10 ng / mL TGF-β1 for 48 h. The effect of different concentrations of the compounds on cell proliferation was detected using the CCK-8 assay (A). Protein levels of fibronectin, type I collagen, and α-SMA were detected using Western blotting, with GAPDH values ​​as a control (B). Bar graphs were used to quantify (C) α-SMA levels, (D) type I collagen, and (E) fibronectin levels. Data represent the mean ± SEM values ​​from 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] Studies on the anti-renal fibrosis activity of compounds in Alpinia oxyphylla have shown that compounds can inhibit and attenuate the expression of fibronectin, type I collagen and α-SMA in TGF-β1-induced NRK 52E cells in a dose-dependent manner.

[0059] This invention identifies and isolates a structurally rare acetylated flavonoid glycoside compound, Oxyphyllvonide G, from Alpinia oxyphylla. Through experimental research, it has been found that this compound has a nephroprotective effect and can be effectively used to prepare nephroprotective drugs. This invention expands the medicinal value of Alpinia oxyphylla, has great development prospects, and has good economic and social benefits.

[0060] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any modifications or alterations made by those skilled in the art without departing from the scope of the present invention to create equivalent embodiments based on the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. A method for preparing Oxyphyllvonide G, an acetylated flavonoid glycoside compound extracted from Alpinia oxyphylla, characterized in that, Includes the following steps: 1) After drying, the Alpinia oxyphylla seeds are pulverized and extracted by reflux with 8 times the amount of 70% ethanol 3 times, 2 hours each time. The extracts are combined and concentrated under reduced pressure to obtain an extract. 2) The extract obtained in step 1) is suspended in 3 times the amount of water, and extracted successively with 30 L of ethyl acetate and n-butanol until the extract is colorless. The extract is then concentrated to obtain the ethyl acetate fraction, the n-butanol fraction and the water fraction. 3) The n-butanol fraction obtained in step 2) was loaded onto an MCI Gel CHP-20 column and eluted with an ethanol-water gradient system to obtain the water fraction, 10% ethanol fraction, 30% ethanol fraction, 50% ethanol fraction, 70% ethanol fraction and 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~Fr.D.7 sequentially; 5) The Fr.D.5 obtained in step 4) was subjected to silica gel column chromatography with dichloromethane-methanol as the eluent gradient to obtain fractions Fr.D.5.1~Fr.D.5.

4. Among them, fraction 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; The acetylated flavonoid glycoside compound Oxyphyllvonide G has the molecular formula C. 23 H 20 O 14 The structural formula is as follows: 。 2. The method for preparing the acetylated flavonoid glycoside compound Oxyphyllvonide G extracted from Alpinia oxyphylla according to claim 1, characterized in that, Includes the following steps: 1) 40.00 kg of Alpinia oxyphylla was dried and pulverized, and extracted three times with 8 times the amount of 70% ethanol for 2 hours each time. 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 in 3 times the amount of water, and extracted successively with 30 L of ethyl acetate and n-butanol until the extract was colorless. The extract was then concentrated to obtain 1.45 kg of ethyl acetate fraction, 628.33 g of n-butanol fraction and 6.60 kg of water fraction. 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 in a gradient ratio of 0:100 to 95:5, yielding 371.10 g of water fraction, 8.43 g of 10% ethanol fraction, 33.36 g of 30% ethanol fraction, 49.82 g of 50% ethanol fraction, 20.73 g of 70% ethanol fraction, and 105.06 g of 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~Fr.D.7 sequentially; 5) The Fr.D.5 obtained in step 4) was subjected to silica gel column chromatography with dichloromethane-methanol gradient elution to obtain fractions Fr.D.5.1 to Fr.D.5.

4. Among them, fraction Fr.D.5.4 was purified by semi-preparative high performance liquid chromatography under the following chromatographic conditions: YMC-PackODS-A column; 250 mm × 20 mm, 5 μm, with methanol:water = 45:55 as the mobile phase to obtain compound Oxyphyllvonide G.

3. The use of the acetylated flavonoid compound Oxyphyllvonide G prepared by the preparation method according to claim 1 or 2 in the preparation of nephroprotective drugs.

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