Flavonoid compound, preparation method and application thereof

Abstract

The embodiment of the present application discloses a flavonoid compound and a preparation method and application thereof.The flavonoid compound has the following structural formula:8,2'-diisopentenyl quercetin-3-methyl ether and 8-isopentenyl kaempferol separated from an extract of Radix Scutellariae have good inhibitory effect on methicillin-resistant Staphylococcus, and provide a better scientific basis for clinical treatment of methicillin-resistant Staphylococcus infection, and development and utilization of active ingredients 8,2'-diisopentenyl quercetin-3-methyl ether and 8-isopentenyl kaempferol of Radix Scutellariae.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a flavonoid compound, its preparation method, and its application. Background Technology

[0002] MRSA is one of the most common causes of multidrug-resistant infections, with significant morbidity and mortality, and has become a major pathogen in hospital-acquired and community-acquired infections. As MRSA gradually develops resistance to all types of antibiotics, it becomes increasingly difficult to control. Therefore, seeking low-toxicity and effective treatments is of great significance. The search for new drugs to combat MRSA is crucial. Mongolian medicine, with its broad efficacy and few side effects, is one of the four major ethnic medicines in my country. In the treatment of infections, Mongolian medicine can exert antibacterial effects not only by directly inhibiting bacterial growth or killing bacteria, but also by enhancing the body's immunity. However, due to the extremely complex chemical composition and numerous pharmacological mechanisms of Mongolian medicine, the understanding of the interrelationships between its multiple targets is insufficient, resulting in relatively slow research progress. Therefore, the screening and identification of its active ingredients is of paramount importance.

[0003] *Thladiantha dubia* fruit, the dried, ripe fruit of the Cucurbitaceae plant *Thladiantha dubia*, is a commonly used medicine in Mongolian medicine. Known in Mongolian as "Aolemose," it has the effects of promoting blood circulation, removing blood stasis, and regulating menstruation. It is mainly used for vaginal diseases, blood stasis in the uterus, blood clots, amenorrhea, blood vessel diseases, skin diseases, stillbirth, and retained placenta. Furthermore, studies by scholars both domestically and internationally have found that *Thladiantha dubia* fruit possesses antioxidant, analgesic, and anti-inflammatory effects. Research by Kang Yan et al. has found that the main effective components for the anti-inflammatory and analgesic effects of *Thladiantha dubia* fruit are total organic acids. In recent years, studies by Zhang Xing et al. have found that the volatile oil of *Thladiantha dubia* fruit has significant antibacterial effects against various bacteria, including *Staphylococcus aureus*, *Escherichia coli*, *Enterococcus faecalis*, *Acinetobacter baumannii*, *Salmonella paratyphi A*, and *Salmonella paratyphi B*.

[0004] Currently, most studies on the antibacterial components of *Citrus aurantium* involve volatile oils, and there are no reports on the antibacterial activity of flavonoids from *Citrus aurantium*. Summary of the Invention

[0005] Therefore, this invention provides a flavonoid compound, its preparation method, and its application. This paper studies the anti-MRSA activity and active components of *Cephalotaxus fortunei*. Using the activity tracking method, substances with anti-MRSA activity were extracted and isolated from *Cephalotaxus fortunei*, in order to lay the foundation for the development of novel anti-MRSA drugs.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of the present invention, the present invention provides a flavonoid compound having the following structural formula:

[0008]

[0009] According to a second aspect of the present invention, the present invention provides a method for preparing the flavonoid compound as described above, the method comprising: using ethanol as a solvent, ultrasonically extracting pulverized *Gnaphalium affine*, filtering, concentrating, eluting the crude extract using a silica gel column with gradient elution, and then separating it using a high performance liquid chromatography column.

[0010] Furthermore, the concentration of ethanol is 95%, the red peony is crushed into coarse powder of 24 mesh, the material-to-liquid ratio is 1:5, the ultrasonic extraction power is 500w, the time is 30min, and the extraction is performed 3 times.

[0011] Furthermore, the silica gel column is eluted with a gradient of petroleum ether and ethyl acetate at volume ratios of 50:1, 20:1, 15:1, 10:1, 5:1, and 3:1.

[0012] The high-performance liquid chromatography column uses an acetonitrile-trifluoroacetic acid water system with a volume ratio of 60%:40% as the mobile phase, wherein the trifluoroacetic acid water system is composed of water and trifluoroacetic acid with a volume ratio of 1000:0.1.

[0013] According to a third aspect of the present invention, the present invention provides the use of the flavonoids described above in the preparation of drugs against methicillin-resistant Staphylococcus aureus.

[0014] Furthermore, the drug against methicillin-resistant Staphylococcus aureus is a preparation made by adding pharmaceutically acceptable excipients to the flavonoids or their salts as active ingredients.

[0015] The embodiments of the present invention have the following advantages:

[0016] This study investigated the antibacterial active ingredients of the Mongolian medicine *Chibaozi*. First, four compounds were isolated from the *Chibaozi* extract, and their structures were determined using modern spectroscopic techniques combined with physical and chemical methods. Second, the minimum inhibitory concentrations (MICs) of the monomeric compounds and antibacterial drugs were determined according to the CLSI-recommended microbroth dilution method. The results showed that 8,2′-diisopentenylquercetin-3-methyl ether and 8-isopentenylkaempferol exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), with MICs of 32 μg / ml and 16 μg / ml, respectively. The above results fully confirm that the active ingredients in the ethanol extract of *Staphylococcus aureus* that inhibit methicillin-resistant Staphylococcus aureus are 8,2′-diisopentenylquercetin-3-methyl ether and 8-isopentenylkaempferol. This provides a better scientific basis for the clinical treatment of methicillin-resistant Staphylococcus aureus infection and the development and utilization of the active ingredients 8,2′-diisopentenylquercetin-3-methyl ether and 8-isopentenylkaempferol from *Staphylococcus aureus*. Detailed Implementation

[0017] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1

[0019] Preparation and identification of flavonoids

[0020] 1. Materials and Methods

[0021] 1.1 Experimental materials: Red peony root, Inner Mongolia Yikang Chinese Medicine Pieces Co., Ltd.; MRSA (clinical isolate preserved by the Institute of Microbiology, Chinese Academy of Sciences).

[0022] 1.2 Reagents and Instruments: MH medium; PBS (Beijing Hongyue Innovation Technology Co., Ltd.); methanol (chromatographic grade) (Tianjin Siyou Chemical Reagent Co., Ltd.); acetonitrile (chromatographic grade) (Fisher Scientific, ChemAlert); n-hexane (analytical grade) (Beijing Chemical Reagent Co., Ltd.); ethyl acetate, dichloromethane, acetone, methanol, ethanol, dimethyl sulfoxide (analytical grade, Beijing Chemical Reagent Co., Ltd.). Bruker Avance 500MHz NMR spectrometer; Agilent 6520LC / MS high-resolution mass spectrometer; Bruker Esquire 3000plus low-resolution mass spectrometer; Agilent 1200 high-performance liquid chromatograph; RP-C18 150mm × 4.6mm column (Agilent and YMC); EL800 microplate reader (BIO-TEK, USA); CO2 incubator (Thermo Fisher Scientific, USA); Waters 1525 high-performance liquid chromatograph (Waters, USA); 2996 PDA detector; Empower chromatography workstation; Agela Venusil XBP-C18 (Agela Corporation); ACB-4A1 clean bench (Singapore Yisi Gao Technology Co., Ltd.); AL240 electronic analytical balance (Mettler AG, Switzerland & Toledo Instruments AG); KQ3200DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); RE-52AA rotary evaporator (Shanghai Yarong Biochemical Instrument Factory). Column chromatography silica gel, GF254 thin-layer chromatography silica gel, HG / T2354-92 thin-layer chromatography silica gel, Sephadex LH-20 (Fluka Corporation, USA); column chromatography silica gel (Qingdao Haiyang Chemical Co., Ltd.); 10% H2SO4-ethanol solution (self-made) as colorimetric reagent.

[0023] 1.3 Experimental Methods:

[0024] 1.3.1 Preparation of Red Peony Extract: The medicinal material was pulverized and passed through a 24-mesh sieve. The resulting coarse powder was extracted with 95% ethanol as solvent, with a material-to-liquid ratio of 1:5, ultrasonic extraction time (500w, 30min), and extraction times (3 times). The filtrates were combined, concentrated under vacuum, and freeze-dried.

[0025] 1.3.2 Separation of *Citrus aurantium* extract: The crude extract was separated by silica gel column chromatography with gradient elution of petroleum ether-ethyl acetate (volume ratio: 50:1, 20:1, 15:1, 10:1, 5:1, 3:1) to obtain 42 fractions (Fr.1-42). Four compounds were prepared by high performance liquid chromatography (60% acetonitrile-trifluoroacetic acid water, wherein the trifluoroacetic acid water is composed of water and trifluoroacetic acid in a volume ratio of 1000:0.1).

[0026] 2 Results

[0027] 2.1 Compound Isolation and Structural Identification: Four compounds were isolated from the extract of *Citrus aurantium*. The structures of the four compounds were determined using modern spectroscopic techniques combined with physical and chemical methods. They were identified as 8,2′-diisopentenylquercetin-3-methyl ether (compound 1), deoxypodophyllotoxin (compound 2), yatein (compound 3), and 8-isopentenylkaempferol (compound 4).

[0028]

[0029] Compound 1: Yellow powder (MeOH), positive for HCl-Mg reaction. HR-ESI-MS m / z: 453.1912 [M+H] + (calc.453.1908). 1 H NMR(CDCl3,500MHz)δ6.37(1H,s,H-6),6.88(1H,d,J=8.3Hz,H-6′),6.86(1H,d,J=8.3Hz,H-5′),3.40(4H,m,H-1″and H-1″′),5.16(2H,m,H-2″and H-2″′), 1.61(3H,d,J=1.4Hz,H-4″), 1.60(3H,d,J=1.4Hz,H-5″), 1.54(3H,s,H-4″′), 1.64(3H,d,J=1.4Hz,H-5″′), 3.63(3H,s,-OCH3). 13 C NMR(MeOD,125MHz)δ161.9(C-1),140.4(C-3),180.4(C-4),160.8(C-5),99.2(C-6),163.1(C- 7),107.9(C-8),156.3(C-9),106.3(C-10),123.3(C-1′),129.5(C-2′),144.7(C-3′),148.3(C -4′),113.1(C-5′),122.5(C-6′),22.4(C-1″),123.4(C-2″),132.4(C-3″),17.7(C-4″),25.7( C-5″), 26.6(C-1″′), 124.2(C-2″′), 132.1(C-3″′), 17.8(C-4″′), 26.0(C-5″′), 60.9(-OCH3).

[0030] Compound 2: White solid. HR-ESI-MS m / z: 399.1446 [M+H] + (calcd.for C 22 H23 O7,399.1444). 1 1H-NMR(500 MHz, CD3OD) δ 6.71 (1H, s, H-6), 6.45 (1H, s, H-3), 6.38 (2H, s, H-2', 6'), 5.90 (2H, d, J = 2.4 Hz, -OCH2O-), 4.57 (1H, d, J = 4.8 Hz, H-7'), 4.44 (1H, dd, J = 8.4, 6.9 Hz, H-9a), 3.96 (1H, dd, J = 9.6, 8.4 Hz, H-9b), 3.71 (6H, s, 3', 5'-OCH3), 3.70 (3H, s, 4'-OCH3), 3.08 (1H, dd, J = 14.3, 4.8 Hz, H-7b), 2.88 (1H, dd, J = 14.3, 4.8 Hz, H-7a), 2.75 (2H, m, H-8, 8'); 13 13C-NMR(125 MHz, CD3OD) δ 177.7 (C-9'), 153.7 (C-3', 5'), 148.5 (C-5), 148.1 (C-4), 138.6 (C-1', 4'), 132.0 (C-2), 130.4 (C-1), 111.1 (C-3, 6), 109.5 (C-2', 6'), 102.5 (-OCH2O-), 73.6 (C-9), 61.1 (4'-OCH3), 56.5 (3', 5'-OCH3), 48.3 (C-8'), 45.0 (C-7'), 34.4 (C-8), 33.6 (C-7).

[0031] Compound 3: Colorless oil. ESI-MS m / z: 401 [M + H] + , C 22 H 24 O7. 1 1H NMR (MeOD, 500 MHz, δ, ppm, J / Hz): 6.81 (1H, d, J = 7.6, H-5), 6.68 (1H, d, J = 1.6, H-2), 6.58 (1H, dd, J = 7.6, 1.6, H-6), 6.51 (2H, s, H-2′, 6′), 5.98 (2H, dd, J = 4.4, 1.2, -OCH2O-), 4.16 (1H, m, H-9a), 3.92 (1H, m, H-9b), 3.64–3.76 (9H, s, 3′, 4′, 5′-OCH3), 2.76 (2H, m, H-7′), 2.44–2.56 (4H, m, H-7, 8, 8'). 13C NMR(MeOD,125MHz,δ,ppm):181.3(C-9′),154.5(C-3′,5′),149.3(C-3),14 7.7(C-4),137.8(C-4′),135.5(C-1′,133.7(C-1),122.8(C-6),110.0(C-2 ),109.1(C-5),107.7(C-2′,6′),102.3(-OCH2O-),72.9(C-9),61.1(4′-OC H3),56.6(3′,5′-OCH3),47.7(C-8′),42.5(C-8),39.1(C-7),36.1(C-7′).

[0032] Compound 4: Yellow amorphous powder (methanol). ESI-MS m / z: 377.1 [M+Na] + The molecular formula is C 20 H 18 O6. 1 H-NMR(500MHz,CD3OD)δ:6.23(1H,s,H-6),8.10(2H,d,J=9.0Hz,H-2′,6′),6.90(2H,d,J=9.0Hz,H- 3′,5′),3.50(2H,m,H-1″),5.22(1H,t,J=7.6Hz,H-2″),1.80(3H,s,4″-CH3),1.67(3H,s,5″-CH3); 13 C-NMR(125MHz,CD3OD)δ:148.0(C-2),137.0(C-3),177.6(C-4),160.1(C-5),98.7(C-6),162.8(C-7),107.6(C-8),160.1(C-9),104.5(C -10),124.0(C-1′),130.5(C-2′,6′),116.3(C-3′,5′),155.5(C-4′),22.4(C-1″),123.9(C-2″),132.4(C-3″),25.9(C-4″),18.2(C-5″).

[0033] Example 2

[0034] Antibacterial activity test

[0035] 1. Materials and Methods

[0036] 1.1 Sample Preparation

[0037] The compounds and antibacterial drugs were prepared into solutions with a mass concentration of 1.28 mg / mL using DMSO solution, filtered through a 0.22 μm microporous membrane, and compounds 1-4 were serially diluted with liquid culture medium to mass concentrations of 32, 16, 8, 4, 2, and 1 μg / mL. The antibacterial drug vancomycin (Van) was serially diluted with liquid culture medium to mass concentrations of 16, 8, 4, 2, 1, 0.5, and 0.25 μg / mL. The antifungal drug amphotericin B (Amph-B) was serially diluted with liquid culture medium to mass concentrations of 40, 20, 10, 5, 2.5, 1.25, and 0.625 μg / mL.

[0038] 1.2 Preparation of test bacterial suspension

[0039] Taking Staphylococcus aureus as an example, the bacterial suspension stored in glycerol at -80℃ was dissolved at 37℃. In a clean bench, the bacterial suspension was inoculated onto TSA plates using a disposable inoculation loop following a three-zone "Z-shaped" streak pattern for resuscitation. The plates were then incubated at 37℃ for 16 hours. A single colony was then collected using an inoculation loop and placed into a turbidity tube. The turbidity was corrected to 0.5 McFarland standard (concentration approximately 10) with physiological saline. 8 (CFU / mL); then diluted with TSB medium at a ratio of 1:100 to approximately 5×10⁻⁶ CFU / mL. 5 CFU / mL was used as the test bacterial solution.

[0040] Staphylococcus aureus (MRSA) was cultured on TSB medium, EAEC (enterohemorrhagic Escherichia coli) on LB medium, PA (Pseudomonas aeruginosa) on LB medium; Candida albicans on 1640 medium; and Aspergillus fumigatus on PDB medium.

[0041] 1.3 Determination of minimum inhibitory concentration:

[0042] Following the CLSI-recommended microbroth dilution method, five pathogenic bacteria were used to determine the minimum inhibitory concentration (MIC) of monomeric compounds and antimicrobial agents. The MIC was determined based on factors such as whether the broth in the microwells was turbid and whether sediment appeared at the bottom of the wells.

[0043] 2 Results

[0044] 2.1 Antibacterial activity

[0045] The results of antibacterial activity are shown in Table 1: Compounds 1 and 4 showed good antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), and the antibacterial activity of compound 4 was greater than that of compound 1. Compounds 4 had no antibacterial activity against other bacteria, while compounds 2 and 3 had no antibacterial activity against all 5 strains.

[0046] Table 1. Screening results of antibacterial activity of compounds 1–4 (MIC, μg / ml) -1 )

[0047]

[0048]

[0049] Note: Vancomycin is the positive control for Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus; Amphotericin B is the positive control for Candida albicans and Aspergillus fumigatus.

[0050] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing flavonoids, characterized in that, The flavonoids have the following structural formula: ， The method includes: using ethanol as a solvent, ultrasonically extracting the pulverized red peony seeds, filtering, concentrating, and then using a silica gel column for gradient elution and high performance liquid chromatography for separation. The concentration of ethanol is 95%, and the red peony is crushed into coarse powder of 24 mesh, with a material-to-liquid ratio of 1:5; The ultrasonic extraction was performed at a power of 500W for 30 minutes, and the extraction was repeated 3 times. The silica gel column was eluted with a gradient of petroleum ether and ethyl acetate at volume ratios of 50:1, 20:1, 15:1, 10:1, 5:1, and 3:

1. The high-performance liquid chromatography column uses an acetonitrile-trifluoroacetic acid water system with a volume ratio of 60%:40% as the mobile phase, wherein the trifluoroacetic acid water system is composed of water and trifluoroacetic acid with a volume ratio of 1000:0.1.

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