Flavone glycoside with antiviral activity, and preparation method and application thereof

Abstract

The application provides a flavone glycoside with antiviral activity and a preparation method and application thereof. The structural formula of the flavone compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside of the application is shown as formula (I), and the compound is isolated from dried or fresh products of fruits, stems or leaves of Averrhoa carambola. In vitro pharmacological experiments prove that the compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside has good anti-H1N1 virus ability, and the EC 50 value thereof is 8.916 muM, indicating that the compound has good antiviral activity. The flavone compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside is expected to be used as a lead compound to develop a new antiviral drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of natural medicine, and particularly relates to a new alkaloid compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside, a preparation method thereof, and application thereof in preparing antiviral drugs. BACKGROUND

[0002] The H1N1 virus belongs to the Orthomyxoviridae, Influenzavirus A, and its genetic material is RNA. The virus particle is spherical, with a diameter of 80nm-120nm, and has a capsule, which poses a serious threat to human health. There is no specific drug for human infection with H1N1 influenza at present, and the commonly used drugs include amantadine and viral neuraminidase inhibitors (oseltamivir, zanamivir), etc. Although the above drugs have anti-H1N1 virus efficacy, they all have certain toxic side effects, and therefore it is urgent to find safe and low-toxicity antiviral drugs. Natural products have always been an important source of drug development, and it is of great significance to develop new antiviral drugs by searching for active ingredients with inhibitory effects on H1N1 virus from nature.

[0003] Averrhoa carambola L. is a plant of the family Oxalidaceae and the genus Averrhoa, and Averrhoa carambola L. is rich in nutrients and also contains rich active substances such as polysaccharides, flavonoids, phenolic compounds, volatile oil, and anthocyanins. At present, there is no report on the research of phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside in Averrhoa carambola L. which has antiviral activity. SUMMARY

[0004] The purpose of the present application is to provide a new alkaloid component phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside, a preparation method thereof, and application thereof in preparing antiviral drugs.

[0005] The new alkaloid component phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside or an ester derivative thereof according to the present application has a structure represented by formula (I).

[0006]

[0007] The new alkaloid component phlorin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside is first isolated from the dried or fresh products of various tissue parts of Averrhoa carambola L., and the specific parts can be pericarp, pulp.

[0008] The new alkaloid component phlorin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside has good anti-H1N1 virus activity, and the half inhibitory concentration is 8.916 μM, as confirmed by in-vitro pharmacological experiments. The new flavonoid glycoside component 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside, or a pharmaceutically acceptable salt thereof, or an ester derivative thereof can be used for preparing an antiviral drug.

[0009] The new flavonoid glycoside component phlorin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside, or an ester derivative thereof, can be combined with a formulation or pharmaceutically acceptable excipient or carrier to prepare a drug or pharmaceutical composition having an anti-H1N1 virus effect. The drug or pharmaceutical composition can adopt a wettable powder, tablet, granule, capsule, oral liquid, dripping pill, injection, aerosol and other dosage forms; and can also adopt a controlled-release or sustained-release dosage form or nano-preparation known in the modern pharmaceutical industry.

[0010] Therefore, the present application also provides an antiviral drug, which comprises the new alkaloid component phlorin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside, or an ester derivative thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0011] The antiviral drug also includes preparation or pharmaceutically acceptable excipient or carrier, such as wettable powder, tablet, granule, capsule, oral liquid, dripping pill, injection, aerosol and other dosage forms; the controlled release or sustained release dosage form or nano preparation known in modern pharmaceutical industry can also be used.

[0012] The antiviral drug is an anti-influenza drug.

[0013] The application also provides a preparation method of the new flavonoid glycoside component phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside, which is isolated from Averrhoa carambola L. plants, including dried or fresh fruits or leaves of Averrhoa carambola L. Preferably, the new flavonoid glycoside component phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside is isolated from dried or fresh fruits or leaves of Averrhoa carambola L.

[0014] The preparation method of the new flavonoid glycoside component phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside of the application has the following specific steps:

[0015] Preparation of the extract: Averrhoa carambola L. is cut and then extracted with an ethanol aqueous solution, the extract is collected, and the extract is obtained after removal of the organic solvent under reduced pressure;

[0016] Separation and purification: the extract is subjected to normal phase silica gel column chromatography, eluted with chloroform / methanol from a volume ratio of 100:0 to 60:40, and the component F10 eluted with a volume ratio of 80:20 of chloroform / methanol is collected; the component F10 is subjected to ODS-A medium pressure column chromatography, eluted with methanol / water from a volume ratio of 10:90 to 70:30, and the component F10-14 eluted with a volume ratio of 30:70 of methanol / water is collected; the component F10-14 is subjected to dextran gel chromatography for purification with methanol as the eluent, and phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside is obtained.

[0017] Preferably, the ethanol aqueous solution is an ethanol aqueous solution with a volume fraction of 95%.

[0018] The application extracts and separates an antiviral active substance from carambola fruits, and the preparation process is controllable, convenient, environment-friendly, and has potential good economic benefits, and the obtained new flavonoid glycoside substance phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside is stable in property and easy to store. The H1N1 virus activity is good, and the new type of antiviral drug is expected to be developed as a lead compound. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is the HR-EI-MS spectrum of the compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside. 1 H NMR spectrum;

[0020] Figure 2 is the C NMR spectrum of the compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside. 13 C NMR spectrum;

[0021] Figure 3 is the HR-EI-MS spectrum of the compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside.

[0022] Figure 4 is the anti-H1N1 virus activity result of the compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside. DETAILED DESCRIPTION

[0023] The following examples are further illustrations of the application and are not intended to limit the application, and simple improvements made to the application according to the essence of the application all belong to the scope of the application claimed.

[0024] Example 1: Preparation of flavonoid compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-beta-D-glucosyl)-beta-D-fucosyl-6'-O-(2-O-beta-D-fucosyl)-alpha-L-arabinofuranoside

[0025] 1.1 Plant source and identification

[0026] The fruit sample of Averrhoa carambola L. for extraction was purchased from Tianpijia Fruit Wholesale Market in Tianhe District, Guangzhou City.

[0027] 1.2 Extraction and isolation

[0028] The Averrhoa carambola L. was homogenized with 95% ethanol aqueous solution and then extracted for 48 hours. The extract was collected and concentrated under reduced pressure to remove the organic solvent to obtain the extract. The extract was subjected to normal phase silica gel column chromatography, eluted with chloroform / methanol from 100:0 to 60:40 by volume ratio, and the fraction F10 eluted with chloroform / methanol at 80:20 by volume ratio was collected. The fraction F10 was subjected to ODS-A medium pressure column chromatography, eluted with methanol / water from 10:90 to 70:30 by volume ratio, and the fraction F10-14 eluted with methanol / water at 30:70 by volume ratio was collected. The fraction F10-14 was further separated and purified by dextran gel chromatography with methanol as the eluent to obtain compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside (44 mg).

[0029] 1.3 Structural identification of the new alkaloid compound

[0030] Figure 1 is the HR-EI-MS spectrum of compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside 1 H NMR spectrum; Figure 2 is the HR-EI-MS spectrum of compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside 13 C NMR spectrum; Figure 3 is the HR-EI-MS spectrum of compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside

[0031] Phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside, colorless oil; ESI-MS m / z 1007.3331 [M+H] + ; 1 H(500MHz)and 13 C(125MHz)NMR data of the compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside are shown in Table 1 below.

[0032] Table 1. NMR data of the compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside (in CD3OD)

[0033]

[0034]

[0035] According to the comprehensive analysis of the above mass spectrum and nuclear magnetic spectrum related data, the structure of the compound is deduced as shown in formula (I), which is named as phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside:

[0036]

[0037] Example 2: Detection of the antiviral activity of the flavonoid compound phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside (AC-1)

[0038] 1. Instruments and materials

[0039] Dimethyl sulfoxide was produced by American Cambridge Company; Trolox reagent, fluorescein, 2,2'-azobis(2-methylpropylamidine) hydrochloride (AAPH), 2',7'-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma Company in the United States; the enzyme marker was Genois microplate reader (Tecan GENios, Switzerland). Madin-Darby canine kidney cells (MDCK) were from American Type Culture Collection (ATCC CCL-34™, Rockville, USA). H1N1 influenza virus was isolated from clinical samples at Guangzhou Women and Children's Medical Center, Guangzhou Medical University. Fetal bovine serum (FBS), trypsin and modified Eagle's medium (DMEM) were purchased from Gibco (California, USA), and cell counting kit-8 (CCK-8 kit) was purchased from Biyun Tian Biotechnology Co., Ltd. (Shanghai, China).

[0040] 2. Evaluation of antiviral activity

[0041] The CCK-8 method was used to determine the antiviral effect of AC-1. Cells were seeded in a 96-well plate at a density of 1 x 10 5 / mL and grown for 24 h, then 100 TCID50 of H1N1 influenza virus was added to the cells, and incubated in a 33°C incubator for 2 h, then the virus solution was removed and gradually increasing concentrations of AC-1 (8 μM / mL, 16 μM / mL, 32 μM / mL) were added to the cells and incubated for 48 h, then 10 μL of CCK-8 dye was added to each well and incubated at 37°C for 2 h. The absorbance at 450 nm was measured using a microplate reader. The antiviral effect (%) = {(Abs sample) - (Abs blank)} / {(Abs negative control) - (Abs blank)} x 100%. Then the CCK-8 assay was performed as described above to determine the OD value. Each experiment was repeated 3 times (n = 3).

[0042] 3. Experimental data are shown in Table Figure 4 , and the EC50 of flavonoid phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside against H1N1 virus was 8.916 μM.

[0043] 4. Experimental conclusion:

[0044] This experiment showed that flavonoid phloretin 3'-C-(2-O-trans-p-coumaroyl-3-O-β-D-glucosyl)-β-D-fucosyl-6'-O-(2-O-β-D-fucosyl)-α-L-arabinofuranoside had good anti-H1N1 virus activity, and could be developed for the preparation of antiviral drugs, with wide application potential and good prospects.

[0045] The above merely describes the preferred embodiments of the present application, and it should be pointed out that the above preferred embodiments should not be regarded as a limitation to the present application, and the protection scope of the present application should be defined by the scope of the claims. For those skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. Flavonoid glycosides, specifically phlorizin 3'- C -(2- O - trans - p -Coumaranoyl-3- O -β-d-glucosyl)-β-d-fucosylate-6'- O -(2- O -β-d-fucosylate)-α-l-arabinofuranoside, the structure of which is shown in formula (I): Equation (Ⅰ).

2. A method for preparing the flavonoid glycoside compound phlorizin 3'-C-(2-O-trans-p-coumaryl-3-O-β-D-glucosyl)-β-D-fucosylate-6'-O-(2-O-β-D-fucosylate)-α-L-arabinofuranoside according to claim 1, characterized in that, The phloretin 3'-C-(2-O-trans-p-coumaryl-3-O-β-D-glucosyl)-β-D-fucosylate-6'-O-(2-O-β-D-fucosylate)-α-L-arabinofuranoside was isolated from star fruit; The specific steps are as follows: a. Extract preparation: Star fruit was chopped and extracted with an aqueous ethanol solution. The extract was collected, concentrated under reduced pressure to remove organic solvents, and then the extract paste was obtained. b. Separation and purification: The extract was subjected to normal-phase silica gel column chromatography, eluted with chloroform / methanol at a gradient ratio from 100:0 to 60:40 (v / v), and fraction F10 eluted at a chloroform / methanol volume ratio of 80:20 was collected. Fraction F10 was subjected to ODS-A medium-pressure column chromatography, eluted with methanol / water / alcohol at a gradient ratio from 10:90 to 70:30 (v / v), and fraction F10-14 eluted at a methanol / water volume ratio of 30:70 was collected. Fraction F10-14 was purified by dextran gel chromatography using methanol as the eluent to obtain phlorizin 3'-C-(2-O-trans-p-coumaryl-3-O-β-D-glucosyl)-β-D-fucosylate-6'-O-(2-O-β-D-fucosylate)-α-L-arabinofuranoside.

3. The preparation method according to claim 2, characterized in that, The star fruit mentioned refers to the dried or fresh fruit or leaves of the star fruit.

4. The preparation method according to claim 3, characterized in that, The star fruit mentioned refers to the peel and / or pulp of the star fruit.

5. The preparation method according to claim 2, characterized in that, The ethanol aqueous solution is a 95% (v / v) ethanol aqueous solution.

6. The compound phlorizin 3'- according to claim 1 C -(2- O - trans - p -Coumaranoyl-3- O -β-d-glucosyl)-β-d-fucosylate-6'- O -(2- O The use of β-d-fucosylated (α-l-arabinofuranoside) or their medicinal salts in the preparation of antiviral drugs.

7. The application according to claim 6, characterized in that, The antiviral drug mentioned is an antiviral drug for influenza.

8. An antiviral drug, characterized in that, Contains an effective amount of phloretin 3'- as an active ingredient as described in claim 1 C -(2- O - trans - p -Coumaranoyl-3- O -β-d-glucosyl)-β-d-fucosylate-6'- O -(2- O -β-d-fucosylation)-α-l-arabinofuranoside or its medicinal salt.

9. The antiviral drug according to claim 8, characterized in that, It also includes excipients or carriers permitted in the formulation or drug.

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