Application of flavonoids in fructus aurantii

Abstract

This invention discloses the application of a flavonoid compound from Citrus aurantium, specifically the application of a flavonoid compound from Citrus aurantium or its physiologically acceptable salt in the preparation of anti-inflammatory and / or therapeutic drugs for diseases related to lactate dehydrogenase release. The flavonoid compound from Citrus aurantium has the structural formula shown in Formula I, wherein R1 is selected from methoxy, hydrogen, ethoxy, or hydroxy; R2 is selected from methoxy, hydrogen, ethoxy, or hydroxy; R3 is selected from methoxy, hydrogen, ethoxy, or hydroxy; R4 is selected from methoxy, hydrogen, ethoxy, or hydroxy; R5 is selected from methoxy, hydrogen, ethoxy, or hydroxy; R6 is selected from methoxy, hydrogen, ethoxy, or hydroxy; and the C2-C3 bond is a double bond or a saturated bond. This invention clarifies that among the flavonoid compounds from Citrus aurantium, isopyroside, apigenin, norihesperidin, hesperidin, and isohesperidin can significantly inhibit LPS-induced LDH release in HL-1 cells and can serve as lead compounds for antiseptic myocardial injury drugs.

Description

Technical Field

[0001] This invention relates to the application of a flavonoid compound from Citrus aurantium, belonging to the pharmaceutical field. Background Technology

[0002] Sepsis cardiomyopathy (SCM) is a common complication of sepsis, leading to reversible left ventricular systolic dysfunction, decreased cardiac output, shock, left ventricular dilation with or without right heart failure, and increasing the mortality rate of sepsis patients to 70%–90%, making it one of the leading causes of death in sepsis patients. In recent years, guided by traditional Chinese medicine theory and with continuous updates to modern drug research methods, traditional Chinese medicine has achieved significant results in the treatment of sepsis and its related complications through mechanisms such as clearing heat and detoxifying, strengthening the body's resistance, and purging the bowels.

[0003] Citrus (Citrus) belongs to the Citrus subfamily of the Rutaceae family. Common Chinese medicinal herbs within this genus include dried tangerine peel, immature bitter orange (Citrus aurantium), and tangerine peel (Citrus reticulata peel), which are frequently used in clinical practice for regulating Qi. According to traditional Chinese medicine theory, the dried, immature fruit of Citrus aurantium L. and its cultivated varieties, known as immature bitter orange (Citrus aurantium peel), is a key herb for resolving phlegm and regulating Qi, and can be used to treat myocardial damage caused by sepsis. Domestic and international research indicates that the main components of immature bitter orange peel include flavonoids, alkaloids, coumarins, and volatile oils. Flavonoids are the primary component, and previous studies have confirmed their pharmacological activities, including antitumor, anti-inflammatory, and antioxidant effects. However, it remains unclear which specific herbal monomer within the flavonoids, the main therapeutic component of immature bitter orange peel, plays a key role. In addition to myocardial damage, patients with septic cardiomyopathy often suffer from other organ dysfunctions on the basis of sepsis. In order to reduce the secondary damage to various target organs caused by drugs with ineffective or weak active ingredients, it is necessary to isolate and screen commonly used Chinese medicines in treatment, and to find the key flavonoid monomers in Citrus aurantium that have a therapeutic effect on septic myocardial damage. Providing scientific evidence for the prevention and treatment of septic myocardial damage by Citrus aurantium and its main flavonoid components has significant clinical and translational value. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide the use of flavonoids from Citrus aurantium or physiologically acceptable salts thereof in the preparation of anti-inflammatory and / or therapeutic drugs for diseases related to lactate dehydrogenase release.

[0005] Technical solution: This invention provides the use of Citrus aurantium flavonoids or their physiologically acceptable salts in the preparation of anti-inflammatory and / or therapeutic drugs for diseases related to lactate dehydrogenase release, wherein the structural formula of the Citrus aurantium flavonoids is shown in Formula I:

[0006]

[0007] Wherein, R1 is selected from methoxy, hydrogen, ethoxy, or hydroxy; R2 is selected from methoxy, hydrogen, ethoxy, or hydroxy; R3 is selected from methoxy, hydrogen, ethoxy, or hydroxy; R4 is selected from methoxy, hydrogen, ethoxy, or hydroxy; R5 is selected from methoxy, hydrogen, ethoxy, or hydroxy; R6 is selected from methoxy, hydrogen, ethoxy, or hydroxy; and the C2-C3 bond is a double bond or a saturated bond.

[0008] Furthermore, the flavonoid compounds of the bitter orange peel are selected from isopyroxin, apigenin, norihesperidin, hesperidin, or isohesperidin.

[0009] Furthermore, the diseases associated with lactate dehydrogenase release include sepsis-related myocardial injury.

[0010] Furthermore, the drug also includes other pharmaceutically acceptable excipients.

[0011] Furthermore, the concentration of flavonoids from Citrus aurantium or their physiologically acceptable salts is 10–50 μM / L.

[0012] Furthermore, the concentration of flavonoids from Citrus aurantium or their physiologically acceptable salts is 10–25 μM / L.

[0013] Furthermore, the flavonoids from the bitter orange peel are obtained by the following extraction method:

[0014] (1) The bitter orange peel was crushed, extracted with an organic solvent, and the solvent was recovered to obtain the extract.

[0015] (2) The extract was separated into four components by silica gel column chromatography, and the mobile phase used was dichloromethane-methanol 100:1-:1;

[0016] (3) After separating components 1 to 3 by silica gel column chromatography, purifying by gel column chromatography, or recrystallizing, the flavonoids of Citrus aurantium are obtained.

[0017] The mobile phase used for separation on silica gel column chromatography was petroleum ether-ethyl acetate 10:1-1:5.

[0018] Further, in step (3), component 1 obtained in step (2) is separated into three subcomponents by silica gel column chromatography. The second subcomponent is purified by gel column chromatography to obtain isopropionol. The mobile phase used for purification is dichloromethane-methanol.

[0019] Further, in step (3), the component 2 obtained in step (2) is separated into 6 sub-components by silica gel column chromatography. The second sub-component is dissolved in petroleum ether-ethyl acetate and recrystallized to obtain apigenin.

[0020] Furthermore, the fourth subfraction was separated again by silica gel column chromatography to obtain three more subfractions. The second subfraction was then purified by gel column chromatography to obtain nodosumetin.

[0021] Further, in step (3), after separating and purifying component 3 obtained in step (2) by silica gel column chromatography, the component obtained by eluting with the mobile phase of petroleum ether-ethyl acetate 3:1 is extracted to obtain hesperidin.

[0022] Further, in step (3), after separating and purifying component 3 obtained in step (2) by silica gel column chromatography, isoadenosine is obtained by eluting the mobile phase petroleum ether-ethyl acetate 2:1.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The present invention clarifies that among the flavonoids derived from Citrus aurantium, isopyroxin, apigenin, norihesperidin, hesperidin and isohesperidin can significantly inhibit LPS-induced LDH release in HL-1 cells, indicating that the above compounds play a protective role against septic cardiomyocyte injury and can be used as lead compounds for antiseptic cardiomyocyte injury drugs. Attached Figure Description

[0024] Figure 1 The results show that different concentrations of flavonoids from Citrus aurantium inhibit the release of lactate dehydrogenase. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0026] Example 1: Preparation of 95% ethanol extract of Citrus aurantium

[0027] Take 1.5 kg of dried Citrus aurantium (purchased from the herbal medicine shop of the Affiliated Hospital of Yangzhou University), cut it into pieces, and grind it to 5-20 mesh. Extract it by reflux with 25 L of 95% ethanol for 1.5 h, repeat the process 3 times, combine the extracts and concentrate under reduced pressure until there is no ethanol odor, thus obtaining 167 g of 95% ethanol extract.

[0028] Example 2: Preparation of flavonoid monomers from Citrus aurantium (1)

[0030] The 95% ethanol extract obtained in Example 1 was mixed with silica gel (100-200 mesh) (sample to silica gel ratio of 1:1.5) and then subjected to silica gel column chromatography. Elution was performed using dichloromethane-methanol (100:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, 0:1), with three retention volumes (2 L each) for each gradient. The resulting fractions were concentrated under reduced pressure and combined by TLC to obtain four fractions: Fr.1 (100:1-30:1), Fr.2 (20:1-10:1), Fr.3 (8:1-5:1), and Fr.4 (3:1-0:1). (2)

[0032] (2.1) Preparation of isonospermin (compound 1)

[0033] Fraction Fr.1 was separated into three subfractions by silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and elution gradients of (10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5): Fr.1.1 (10:1-5:1), Fr.1.2 (3:1-2:1), and Fr.1.3 (1:1-1:5). Fr.1.2 was purified by repeated purification using Sephadex LH-20 gel column chromatography with dichloromethane-methanol (1:1) as the mobile phase to obtain 2.3 mg of isonophylloidin (compound 1).

[0034] The structure of the compound was confirmed by nuclear magnetic resonance: 1 H-NMR (CDCl3, 400MHz): δ: 7.43 (2H, d, J = 8.7Hz, H-2′, H-6′), 6.98 (2H, d, J = 8.8Hz, H-3′, H-5′), 5.91 (1H, d, J = 2.1Hz, H-6), 5.91 (1H, d, J = 2. 1Hz,H-8),5.41(1H,dd,J=3.0,13.1Hz,H-2),3.13(1H,dd,J=13.1,17.2Hz,H-3α),2.74(1H,dd,J=3.0,17.0Hz,H-3β),3.83(3H,s,4′-OCH3); 13 C-NMR (100MHz, CDCl3) δ: 197.1 (C-4), 168.3 (C-5), 165.5 (C-9), 164.8 (C-7), 161.4 (C-4′), 132.4 (C-1′), 128. 9(C-2′,6′),115.0(C-3′,-5′),103.3(C-10),97.0(C-6),96.1(C-8),80.2(C-2),55.7(4′-OCH3),44.0(C-3).

[0035] (2.2) Preparation of apigenin (compound 2)

[0036] The Fr.2 fraction obtained in (1) was separated into six subfractions by silica gel column chromatography using petroleum ether-ethyl acetate as the mobile phase and eluting at gradients of (10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5): Fr.2.1 (10:1), Fr.2.2 (5:1), Fr.2.3 (3:1), Fr.2.4 (2:1), Fr.2.5 (1:1), and Fr.2.6 (1:3-1:5). Fr.2.2 was dissolved in petroleum ether:ethyl acetate at a ratio of 1:3, and apigenin (compound 2) was obtained by recrystallization.

[0037] The structure of the compound was confirmed by nuclear magnetic resonance: 1 H-NMR (CDCl3, 400MHz): δ: 12.96 (1H, s, 5-OH), 7.91 (2H, d, J = 8.6 Hz, H-2', 6'), 6.92 (2H, d, J =8.6Hz,H-3',5'),6.77(1H,s,H-3),6.47(1H,d,J=2.0Hz,H-8),6.19(1H,d,J=2.0Hz,H-6); 13 C-NMR (100MHz, CDCl3) δ: 181.7 (C-4), 164.1 (C-2), 163.7 (C-7), 161.4 (C-4''), 161.1 (C-9), 157.3 (C-5 ),128.4(C-2',6'),121.1(C-1'),115.9(C-3',5'),103.7(C-10),102.8(C-3),98.8(C-6),93.9(C-8).

[0038] (2.3) Preparation of nonocitretin (compound 3)

[0039] The subfraction Fr.2.4 obtained in (2.2) was subjected to silica gel column chromatography again, with petroleum ether-ethyl acetate as the mobile phase and eluted in gradients of (10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5) to further obtain three subfractions, namely Fr.2.4.1 (10:1-5:1), Fr.2.4.2 (3:1-2:1), and Fr.2.4.3 (1:1-1:5). Among them, Fr.2.4.2 was purified by Sephadex LH-20 gel column chromatography with methanol as the mobile phase to obtain 1.9 mg of nonotrimonin (compound 3).

[0040] The structure of the compound was confirmed by nuclear magnetic resonance: 1H-NMR (CDCl3, 400MHz): δ: 7.68 (1H, dd, J = 2.0, 8.5Hz, H-6′), 7.56 (1H, d, J = 2.0Hz, H-2′), 7.15 (1H, d, J = 8.9Hz, H-5′), 6.72 (1H, s, H-3 ),4.12(3H,s,5-OCH3),4.09(3H,s,6-OCH3),4.05(3H,s,7-OCH3),3.95(3H,s,8-OCH3),3.91(3H,s,3′-OCH3),3.90(3H,s,4′-OCH3); 13 C-NMR (100MHz, CDCl3) δ: 163.6 (C-2), 107.4 (C-3), 179.6 (C-4), 144.6 (C-5), 132 .1(C-6),151.8(C-7),145.5(C-8),145.5(C-9),112.8(C-10),124.8(C-1′),109 .6(C-2′),150.9(C-3′),153.9(C-4′),110.3(C-5′),121.2(C-6′),56.0(3′-OCH 3), 56.0(4′-OCH3), 62.5(5-OCH3), 62.0(6-OCH3), 61.9(7-OCH3), 61.7(8-OCH3).

[0041] (2.4) Preparation of hesperidin (compound 4)

[0042] The Fr.3 fraction obtained in (1) was subjected to silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and eluted in gradients of (10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5). The fraction eluted with mobile phase 3:1 yielded a yellow powdery compound, which was hesperidin (compound 4) 1.3 mg.

[0043] The structure of the compound was confirmed by nuclear magnetic resonance: 1 H-NMR (CDCl3, 400MHz): δ: 7.99-7.91 (2H, d, J = 8.4Hz, H-2′ / H-6′), 7.12-7.06 (2H, m, J = 8.4Hz, H-3′ / H-5′), 6.6 9(1H,s,H-3),4.12(3H,s,5-OCH3),4.04(3H,s,7-OCH3),3.94(3H,s,6-OCH3),3.91(6H,s,4′-OCH3 / 3′-OCH3); 13C-NMR(100MHz, CDCl3)δ:178.2(C-4),163.8(C-2),162.4(C-4′),151.9( C-9),147.9(C-5),147.8(C-7),145.6(C-6),138.3(C-8),129.1(C-2′ / C- 6′),124.5(C-1′),115.7(C-3′ / C-5′),114.0(C-10),106.7(C-3),62.6( 5-OCH3), 62.6(7-OCH3), 62.1(6-OCH3), 62.1(4′-OCH3), 56.0(3′-OCH3).

[0044] (2.5) Preparation of isohesperidin (compound 5)

[0045] The Fr.3 fraction obtained in (1) was subjected to silica gel column chromatography with petroleum ether-ethyl acetate as the mobile phase and eluted in gradients of (10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5). The fraction obtained by elution with mobile phase 2:1 was eluted with isoauropinolone (compound 5) 1.5 mg.

[0046] The structure of the compound was confirmed by nuclear magnetic resonance: 1 H-NMR (CDCl3, 400MHz): δ: 7.67 (1H, dd, J = 8.6, 2.1Hz, H-6′), 7.56 (1H, d, J = 2.1Hz, H-2′), 7.14 (1H, d, J = 8.6Hz, H-5′), 6.70 (1H, s, H -3),6.67(1H,s,H-6),4.05(3H,s,7-OCH3),3.96(3H,s,4′-OCH3),3.96(3H,s,3′-OCH3),3.96(3H,s,8-OCH3),3.96(3H,s,5-OCH3); 13 C-NMR(100MHz, CDCl3)δ:160.2(C-2),105.6(C-3),175.6(C-4),151.7(C-5) ,97.2(C-6),157.3(C-7),140.4(C-8),153.8(C-9),111.6(C-10),123.0(C-1 ′),109.1(C-2′),148.9(C-3′),151.5(C-4′),111.8(C-5′),119.6(C-6′),55 .6(5-OCH3),61.9(7-OCH3),55.8(8-OCH3),60.1(3′-OCH3),56.5(4′-OCH3).

[0047] The substitution of the flavonoids prepared in this embodiment is shown in Table 1.

[0048] Table 1. Substitution of general formulas for flavonoids

[0049]

[0050] The specific structural formula is as follows:

[0051]

[0052] Example 3: Study on the inhibitory effect of Citrus aurantium flavonoids on LPS-induced LDH release in HL-1 cells. The information on the drugs and reagents used in this example is shown in Table 2, and the information on the experimental instruments is shown in Table 3.

[0053] Table 2 Drugs and Reagents

[0054]

[0055] Table 3 Experimental Instruments

[0056]

[0057] Experimental methods:

[0058] 1. Culture of HL-1 cell line

[0059] (1) Cell resuscitation

[0060] HL-1 cells were removed from liquid nitrogen and placed in a 37°C water bath until completely lysed. The lysed cells were then transferred to DMEM high-glucose medium and centrifuged at 1000 rpm for 5 min at room temperature. The supernatant was discarded, and the cells were resuspended in complete medium (DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% antibiotics). The cell suspension was seeded into culture flasks, mixed well, and incubated in a 37°C, 5% CO2 saturated humidity incubator.

[0061] (2) Cell passage and culture

[0062] Observe the cells. If the cell density is below 50%, replace with complete culture medium, discard the culture medium, wash with PBS, and replace with fresh complete culture medium. When the cell density is above 80%, passage the cells. Discard the culture medium and wash with PBS. Add trypsin to digest the cells for 3-4 minutes. Observe the adherent cells detach. Add an equal amount of complete culture medium to stop the digestion. Transfer to centrifuge tubes and centrifuge at 1000 rpm, 4°C, for 5 minutes. Remove the supernatant, add complete culture medium to make a cell suspension, passage at a ratio of 1:2, mix well, and incubate in a 37°C, 5% CO2 saturated humidity incubator.

[0063] 2. Cell grouping and treatment

[0064] (1) Preparation of Citrus aurantium flavonoid monomer solution

[0065] 5 mg of Chinese herbal monomers (the five kinds of flavonoid monomers of Citrus aurantium prepared in Example 2) were dissolved in DMSO solution to prepare a final concentration of 200 mM. After being dispensed, the solution was stored at -20°C in the dark.

[0066] (2) Different dosage groups of flavonoid monomers from Citrus aurantium

[0067] NC group: HL-1 cells + 200 μl DMEM;

[0068] LPS group: HL-1 cells were incubated with 200 μl DMEM + dimethyl sulfoxide (DMSO) for 1 h, followed by the addition of LPS (20 μg / ml);

[0069] Citrus aurantium flavonoid monomer group: HL-1 cells + 200 μl DMEM + Citrus aurantium flavonoid monomer (concentrations of 10 μM / L, 25 μM / L and 50 μM / L, respectively) were incubated for 1 h and then LPS (20 μg / ml) was added.

[0070] 3. LDH detection

[0071] Cell viability was assessed using an LDH cytotoxicity assay kit. Specifically, after grouping and treatment according to the above protocol, HL-1 cells were cultured for another 24 hours. LDH release reagent was added to one well in the NC group 1 hour beforehand (at 23 hours). One hour later, 120 μl of supernatant from each well was transferred to a clear 96-well plate. The LDH reaction solution was prepared using an LDH cytotoxicity assay kit with a ratio of 1x enzyme solution: 1x xint reagent: 1x lactate = 1:1:1. 60 μl of the LDH reaction solution was added to each well of the clear 96-well plate, and the plates were incubated at 37°C for 15 minutes. The absorbance was measured at 490 nm using a microplate reader. The mean absorbance values ​​of the 6 wells in the NC group were taken, and cell viability was calculated using the following formula: Cell viability % = (Absorbance value per well - Mean absorbance value of the NC group) / (Absorbance value of the release well - Mean absorbance value of the NC group) × 100%. Each experiment was repeated three times.

[0072] 4. Statistical processing

[0073] Statistical data were analyzed using Graphpad Prism 8.0 software and expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, and repeated measures ANOVA was used for comparisons within groups. A p-value < 0.05 was considered statistically significant.

[0074] 5. Results

[0075] The LDH release rate of LPS-induced HL-1 cells was measured before and after intervention with different doses of isopyroside, hesperidin, apigenin, norihesperidin, and isohesperidin from Citrus aurantium extract. The results showed that all five flavonoids significantly inhibited LDH release rate (P < 0.05). Figure 1 As shown in the figure, the effects of different doses of apigenin and isanoic acid were not significantly different. The effect of hesperidin at a concentration of 10 μM / L was significantly better than that at a concentration of 50 μM / L. The effects of norihesperidin and isohesperidin were best at concentrations of 10 μM / L and 25 μM / L, respectively.

[0076] Flavonoids isolated from the extract of Citrus aurantium significantly inhibited LPS-induced LDH release in HL-1 cells, which may be the main material basis for the anti-inflammatory effect of Citrus aurantium and are expected to become lead compounds for more targeted and efficient antiseptic myocardial injury drugs.

Claims

1. A method for extracting flavonoids from Citrus aurantium, characterized in that... Includes the following steps: (1) The bitter orange peel was crushed, extracted with an organic solvent, and the solvent was recovered to obtain the extract. (2) The extract was separated into four components by silica gel column chromatography. The mobile phases used were dichloromethane-methanol at ratios of 100:1, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, and 0:

1. Among them, 100:1-30:1 yielded component Fr.1, 20:1-10:1 yielded component Fr.2, 8:1-5:1 yielded component Fr.3, and 3:1-0:1 yielded component Fr.

4. (3) The components Fr.1 to Fr.3 were separated by silica gel column chromatography, purified by gel column chromatography, or recrystallized to obtain the flavonoids of Citrus aurantium; wherein the mobile phase used for silica gel column chromatography was petroleum ether-ethyl acetate 10:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:5; the flavonoids of Citrus aurantium were selected from isopyroxin, apigenin, norihesperidin, hesperidin or isohesperidin; The organic solvent mentioned in step (1) is selected from 95% ethanol; In step (3), the component Fr.1 obtained in step (2) is separated into three subcomponents by silica gel column chromatography. The subcomponents are Fr.1.1 (10:1-5:1), Fr.1.2 (3:1-2:1), and Fr.1.3 (1:1-1:5). The second subcomponent Fr.1.2 is purified by gel column chromatography to obtain isopropionol. The mobile phase is dichloromethane-methanol. In step (3), the component Fr.2 obtained in step (2) was separated by silica gel column chromatography to obtain 6 subcomponents, of which 10:1 yielded component Fr.2.1, 5:1 yielded component Fr.2.2, 3:1 yielded component Fr.2.3, 2:1 yielded component Fr.2.4, 1:1 yielded component Fr.2.5, and 1:3-1:5 yielded component Fr.2.

6. The second subcomponent Fr.2.2 was dissolved in petroleum ether-ethyl acetate and recrystallized to obtain apigenin. The fourth subcomponent Fr.2.4 was separated again by silica gel column chromatography to obtain 3 more subcomponents. The second subcomponent was purified by gel column chromatography to obtain nodosumetin. In step (3), the component Fr.3 obtained in step (2) is separated and purified by silica gel column chromatography. The component obtained by elution with petroleum ether-ethyl acetate 3:1 is hesperidin; the component obtained by elution with petroleum ether-ethyl acetate 2:1 is isohesperidin.

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