Phellinus igniarius extract with anti-influenza virus activity

Abstract

The present invention belongs to the field of medicinal chemistry and natural product separation technology, and provides a phellinus linteus extract with anti-influenza virus activity. The extract is 9-oxo-10E, 12E-octadecadienoic acid, or porphyrone. The phellinus linteus is a fungus of the species Porites crassifolia ( Unfinished hairy ) fruiting bodies or mycelia. The monomeric compounds isolated from the extract of Phellinus igniarius contain components with high inhibitory activity against the H1N1 influenza virus. Their inhibition rate against the H1N1 influenza virus exhibits an S-shaped curve, with significant inhibition increasing with increasing concentration. The present invention provides two potential antiviral compounds with potential for further development.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal chemistry and natural product separation, and particularly relates to a phellinus igniarius extract. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be regarded as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] The traditional Chinese medicine Phellinus igniarius, also known as mulberry ear, was first recorded in the Shennong Bencao Jing (Shen Nong's Herbal Classic). It is known for its beneficial effects on the five internal organs, softening hard masses, detoxifying, stopping bleeding, activating blood circulation, and soothing the stomach and stopping diarrhea. Currently, Phellinus igniarius is primarily derived from the fruiting bodies of the fungus Phellinus igniarius, the poplar Phellinus igniarius, and similar species. Studies have shown that the ethanol extract of the fermentation broth of Phellinus igniarius and hispolon and hispidin isolated and identified from its fruiting bodies exhibit significant anti-influenza A (H1N1) and H3N2 viruses, as well as influenza B virus. Five polyphenolic compounds, hispidin, hypolomine B, inoscavin A, davallilactone, and phelligridin D, isolated from the ethanol extract of Phellinus igniarius fruiting bodies, all exhibit non-competitive inhibition of H1N1, H5N1, and H3N2 neuraminidase activity. In addition, Li Qingjie's research has shown that polyphenols such as phellibaumin A and phelligridin D from the fungus also exhibit significant anti-inflammatory activity, demonstrating their potential antiviral properties. However, the underlying mechanism of their anti-H1N1 influenza virus activity remains unclear.

[0004] Previous studies have shown that the antiviral activity of Phellinus igniarius is primarily based on polyphenolic compounds with a styryl α-pyrone backbone, such as hispidin and its derivatives. These compounds exhibit potential antiviral mechanisms. However, their low concentration in Phellinus igniarius, the difficulty in preparing them, and the instability of their components make it difficult to meet industrial demands, presenting significant challenges for their practical application and industrial development. To further identify the antiviral active components in Phellinus igniarius, researchers conducted a study to isolate the active components from Phellinus igniarius extracts. Summary of the Invention

[0005] In response to the problems in the prior art, the present invention provides a phellinus igniarius extract with good biological activity and simple structure.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] A use of a phellinus linteus extract in the preparation of an anti-influenza virus drug, wherein the extract is 9-oxo-10E,12E-octadecadienoic acid: ;

[0008] Or, porphyrone: .

[0009] The preparation method of the above-mentioned Phellinus igniarius extract comprises the following steps:

[0010] (1) Extract Phellinus igniarius with 75% v / v ethanol solution to obtain a crude extract;

[0011] (2) The aqueous dispersion of the crude extract was adsorbed on ADS-17 macroporous resin and then gradient eluted with water, 10% v / v ethanol solution, 30% v / v ethanol solution, 50% v / v ethanol, 70% v / v ethanol, and 95% v / v ethanol to obtain six corresponding parts Fr.1 to Fr.6;

[0012] (3) The methanol solution of the Fr.3 part was subjected to silica gel column chromatography and gradient elution with dichloromethane-methanol at a volume ratio of 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, and 1:1. The fractions were collected and concentrated under reduced pressure. After TLC identification and merging, a total of 22 fractions Fr.3-1 to Fr.3-22 were obtained. The methanol solution of Fr.3-18 was separated and purified by preparative liquid chromatography to obtain purpurone.

[0013] (4) The methanol solution of the Fr.6 fraction was subjected to silica gel column chromatography and eluted with n-hexane-ethyl acetate at a volume ratio of 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, and 1:1, ethyl acetate-methanol at a volume ratio of 1:1, and methanol-water at a volume ratio of 1:1. The fractions were collected and concentrated under reduced pressure. After TLC analysis and merging, a total of 17 fractions, Fr.6-1 to Fr.6-17, were obtained.

[0014] The methanol solution of Fr.6-5 was eluted using a Sephadex LH-20 column with methanol as the eluent. The obtained fractions were combined into four fractions Fr.6-5-1 to Fr.6-5-4 after TLC identification; Fr.6-5-2 was then separated and purified by preparative liquid chromatography to obtain the compound 9-oxo-10E,12E-octadecadienoic acid.

[0015] In step (3), the mobile phase of the preparative liquid chromatography was methanol-water with a volume ratio of 32:68, a flow rate of 8 mL / min, and the retention time of purpurogenol (t R ) is 35 min.

[0016] In step (4), the mobile phase of the preparative liquid chromatography was methanol-water with a volume ratio of 90:10, the flow rate was 8 mL / min, and the retention time (t R ) is 52.5 min.

[0017] The mulberry linterinary is a thick hair fibrous fungus ( Inontus hispidus ) fruiting bodies or mycelium.

[0018] The influenza virus is H1N1 virus.

[0019] The present invention has the following advantages:

[0020] The present invention isolates monomeric compounds from a Phellinus igniarius extract, containing components with high inhibitory activity against the H1N1 influenza virus, including the compounds purpurogenol and 9-oxo-10E,12E-octadecadienoic acid. These compounds exhibit an S-shaped inhibitory effect on the H1N1 influenza virus, with increasing concentration leading to a significant inhibitory effect. This invention provides two potential antiviral compounds with potential for further development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is compound 1 1 H NMR spectrum (600 MHz, Methanol- d 4);

[0022] Figure 2 is compound 1 13 C-NMR spectrum (151 MHz, Methanol- d 4);

[0023] Figure 3 is compound 2 1 H-NMR spectrum (600 MHz, Methanol- d 4);

[0024] Figure 4 is compound 2 13 C-NMR spectrum (151 MHz, Methanol- d 4);

[0025] Figure 5 is compound 3 1 H-NMR spectrum (600 MHz, Methanol- d 4);

[0026] Figure 6 is compound 3 13 C-NMR spectrum (151 MHz, Methanol-d 4). DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0028] Example 1 Isolation and Identification of Active Ingredients from Phellinus igniarius

[0029] 1. Preparation of Phellinus linteus Extract

[0030] 5 kg of the fruiting bodies of the tricholoma were crushed into fine powder, soaked in 10 times the amount of 75 vol% ethanol for 24 h, and subjected to ultrasonic-assisted extraction at a power of 100 W for 40 min at room temperature for three times. The filtrates were combined and concentrated using a rotary evaporator under reduced pressure to recover the solvent to obtain a crude extract, which was then evaporated to dryness in a water bath to obtain a crude extract.

[0031] The 75% ethanol crude extract of P. crassi was dispersed in water and then adsorbed on pretreated ADS-17 macroporous adsorption resin. The crude extract was then gradient eluted with H2O, 10% v / v ethanol, 30% v / v ethanol, 50% v / v ethanol, 70% v / v ethanol and 95% v / v ethanol for 5 column volumes. The effluent was evaporated to dryness under reduced pressure to obtain six effluent fractions, Fr.1 to Fr.6, including H2O, 10% ethanol, 30% ethanol, 50% ethanol, 70% ethanol and 95% ethanol.

[0032] 2. Osmundacetone

[0033] The 30% ethanol fraction of the macroporous adsorption resin (Fr.3, 9.51 g) was isolated and purified. After dissolution with an appropriate amount of methanol, the sample was mixed with 100-200 mesh silica gel in a 1:1 v / v ratio. Primary separation was performed using silica gel column chromatography (194.7 g, 100-200 mesh) using a gradient elution system of dichloromethane, dichloromethane-methanol (100:1, 50:1, 20:1, 10:1, 5:1, and 1:1, v / v), with 5 column volumes per elution ratio. Fractions were collected and concentrated under reduced pressure. After TLC analysis and combination, a total of 22 fractions, Fr.3-1 to Fr.3-22, were obtained.

[0034] Fr.3-18 (184.8 mg) was dissolved in methanol and separated and purified by preparative liquid chromatography (methanol-water, 32:68 v / v) at a flow rate of 8 mL / min to obtain compound 1 (t R = 35 min, 6.5 mg).

[0035] Compound 1 is a yellow powder; 1H NMR (600 MHz, Methanol- d 4) Spectrum, d 7.09 and 6.80 can be observed as ortho-hydroxyl groups on the benzene ring, which are ortho-substituted. 13 C NMR (151 MHz, Methanol- d 4) In the score d C 146.9 (C-3'), 150.0 (C-4') are hydroxyl signals. The specific NMR information is as follows: 1 H NMR (600 MHz, Methanol- d 4) d : 7.53 (1H, d, J = 16.2 Hz, H-4), 7.09 (1H, d, J = 2.1 Hz, H-2'), 7.00 (1H, dd, J = 8.0, 2.0 Hz, H-6'), 6.80 (1H, d, J = 8.2 Hz, H-5'), 6.56 (1H, d, J = 16.2 Hz,H-3), 2.34 (3H, s, H-1); 13 C NMR (151 MHz, Methanol- d 4) d : 27.0 (C-1), 201.6 (C-2), 123.5 (C-3), 146.9 (C-4), 127.7 (C-1'), 115.3 (C-2'), 146.9 (C-3'), 150.0(C-4'), 116.6 (C-5'), 124.7 (C-6'). 1 H-NMR and 13 The C-NMR data were basically consistent with the results of the literature, and it was speculated that compound 1 was porphyrone.

[0036] 3. phelligridin D

[0037] The 70% ethanol fraction of the macroporous adsorption resin (Fr.5, 12.8 g) was isolated and purified. After dissolution with an appropriate amount of methanol, the sample was mixed with 100-200 mesh silica gel at a 1:1 v / v ratio. Primary separation was performed using silica gel column chromatography (silica gel: 128.99 g, 100-200 mesh) using a gradient elution system consisting of dichloromethane, dichloromethane-methanol (100:1, 50:1, 20:1, 10:1, 5:1, and 1:1, v / v), ethyl acetate-methanol (1:1, v / v), and methanol-water (1:1, v / v), with 5 column volumes per elution ratio. Fractions were collected, concentrated under reduced pressure, and combined to obtain 21 fractions, Fr.5-1 to Fr.5-21, identified by TLC.

[0038] Fr.5-18 (344.8 mg) was dissolved in methanol, filtered through a 0.22 μm organic filter membrane, and separated and purified by preparative liquid chromatography (methanol-0.1% formic acid water, 60:40 v / v) at a flow rate of 8 mL / min to obtain compound 2 (t R = 50 min, 1.0 mg).

[0039] Compound 2 is a yellow powder; 1 H NMR (600 MHz, Methanol- d 4) spectrum, 7.42 (1H, d, J =15.8 Hz, H-2') and 6.71 (1H, d, J = 15.9 Hz, H-1') signal indicates that the compound contains unsaturated double bonds. 7.08 (1H, d, J = 2.1 Hz, H-4') and 7.00 (1H, dd, J = 8.2, 2.1 Hz, H-8') and 6.80 (1H,d, J = 8.2 Hz, H-7') signal indicates the presence of substituents on the aromatic ring and a remote coupling effect. In addition, 8.43 (1H, s, H-7) and 7.61 (1H, s, H-10) correspond to two hydrogen signals on the aromatic ring. d H 7.42 and 6.71 can be inferred that the compound contains a trans double bond. d H 8.43 and 7.61 are two hydrogen signals on the aromatic ring. The specific NMR information is as follows: 1 H NMR (600 MHz, Methanol- d 4) d : 8.43 (1H, s, H-7), 7.61 (1H, s, H-10), 7.42 (1H, d, J= 15.8 Hz, H-2'), 7.08 (1H, d, J = 2.1 Hz, H-4'), 7.00 (1H,dd, J = 8.2, 2.1 Hz, H-8'), 6.80 (1H, d, J = 8.2 Hz, H-7'), 6.71 (1H, d, J = 15.9Hz, H-1'), 6.54 (1H, s, H-4); 13 C NMR (151 MHz, Methanol- d 4) d : 162.1 (C-1),160.6 (C-3), 99.9 (C-4), 161.1 (C-6), 115.5 (C-7), 148.4 (C-8), 155.2 (C-9),111.9 (C-10), 116.4 (C-1'), 138.1 (C-2'), 128.8 (C-3'), 114.9 (C-4'), 146.9(C-5'), 149.1 (C-6'), 116.6 (C-7'), 122.4 (C-8'), 162.8 (C-4a), 113.3 (C-6a),129.1 (C-10a), 100.6 (C-10b). 1 H-NMR and 13 The C-NMR data were basically consistent with those reported in the literature, and it was speculated that compound 2 was phelligridin D.

[0040] 4. 9-oxo-(10E, 12E)-octadecadienoic acid

[0041] The 95% ethanol fraction of the macroporous resin (Fr.6, 4.79 g) was isolated and purified. After dissolution with an appropriate amount of methanol, the sample was mixed with 100-200 mesh silica gel (1:1 v / v). Separation was performed using silica gel column chromatography (100.27 g, 100-200 mesh) using a gradient elution system consisting of n-hexane, n-hexane-ethyl acetate (100:1, 50:1, 20:1, 10:1, 5:1, and 1:1, v / v), ethyl acetate-methanol (1:1, v / v), and methanol-water (1:1, v / v), with 5 column volumes per elution ratio. Fractions were collected, concentrated under reduced pressure, and combined to yield 17 fractions, Fr.6-1 to Fr.6-17, identified by TLC.

[0042] Fr.6-5 (41.6 mg) was dissolved in methanol and filtered through a 0.22 μm organic filter membrane. It was purified by Sephadex LH-20 with methanol as the eluent. A total of 21 test tubes of fractions were obtained. After TLC identification, they were initially combined into 4 fractions. Fr.6-5-2 was separated and purified by preparative liquid chromatography (methanol-water, 90:10 v / v) at a flow rate of 8 mL / min to obtain compound 3 (t R = 52.5 min, 1.0 mg).

[0043] Compound 3 is a white powder; 1 H NMR (600 MHz, Methanol- d 4) In the spectrum, 7.23 (1H, dd, J = 15.6, 9.9 Hz, H-11), 6.12 (1H, d, J = 15.6 Hz, H-10) shows a hydrogen signal of an unsaturated double bond, and 6.30 (1H, m, H-13) and 6.27 (1H, t, J = 5.4 Hz, H-12) correspond to another hydrogen signal of an unsaturated double bond. 13 C NMR (151 MHz, Methanol- d 4) Spectrum, d C 177.8 (C-1) is the carbon signal of the carboxylic acid group, and d C 203.9 (C-9) is the characteristic signal of ketone carbonyl carbon, such as d C 35.0 (C-2), 30.4 (C-4), 26.1 (C-5), 30.0 (C-6), 25.3 (C-7), 41.5 (C-8) show the carbon environment of the long chain structure in the compound and the conjugated double bond carbon signal d C 147.3 (C-13), 130.4 (C-12), 143.9 (C-11) and 128.2 (C-10). 1 H NMR and 13 The signals in C NMR are all located in high field, suggesting that the compound is an aliphatic hydrocarbon compound. The specific NMR information is as follows: 1 H NMR (600 MHz, Methanol- d 4) d : 7.23 (1H, dd, J = 15.6, 9.9 Hz, H-11), 6.30(1H, m, H-13), 6.27 (1H, t, J= 5.4 Hz, H-12), 6.12 (1H, d, J = 15.6 Hz, H-10),2.61 (2H, t, J = 7.4 Hz, H-8), 2.27 (2H, t, J = 7.4 Hz, H-2), 2.20 (2H, m, H-14), 1.60 (4H, m, H-5, 7), 1.33 (12H, m, H-3, 4, 6, 15, 16, 17), 0.92 (3H, t, J =7.1 Hz, H-18); 13 C NMR (151 MHz, Methanol- d 4) d The molecular weight of the compound 3 was 177.8 (C-1), 35.0 (C-2), 29.1 (C-3), 30.4 (C-4), 26.1 (C-5), 30.0 (C-6), 25.3 (C-7), 41.5 (C-8), 203.9 (C-9), 128.2 (C-10), 143.9 (C-11), 130.4 (C-12), 147.3 (C-13), 34.1 (C-14), 30.2 (C-15), 32.6 (C-16), 23.6 (C-17), and 14.3 (C-18). After comparison with the published literature, compound 3 was determined to be 9-oxo-10E,12E-octadecadienoic acid.

[0044] Example 2 Activity of the active ingredients in Phellinus igniarius against H1N1 influenza virus

[0045] 1. Extract activity

[0046] Weigh 34.08 mg of the water part Fr.1, 32.22 mg of the 10% ethanol part Fr.2, 25.63 mg of the 30% ethanol part Fr.3, 20.45 mg of the 50% ethanol part Fr.4, 22.03 mg of the 70% ethanol part Fr.5, and 20.23 mg of the 95% ethanol part Fr.6, add DMSO solution to maximize dissolution (250 μg / mL for the water part and 10% ethanol part, 500 μg / mL for the 30%, 50%, 70% and 95% ethanol parts), and take 10 μL of the mother solution and add it to 990 μL of virus isolation serum-free culture medium containing 1.5 μg / mL TPCK trypsin. The highest concentration in the drug group was 1 mg / mL. Nine gradients of two-fold dilution were made using virus isolation serum-free culture medium containing 1.5 μg / mL TPCK trypsin. Four replicate wells were set up in the drug group, and eight replicate wells were set up in the cell control group and solvent zero adjustment group to determine the cytotoxicity of each part on MDCK cells and the inhibitory effect on H1N1 virus at different concentrations.

[0047] Table 1 IC values ​​of different polarity fractions of Phellinus igniarius against H1N1 virus 50 and CC on MDCK cells 50

[0048]

[0049] Note: With water part IC 50 Compared with the value, p <0.001; there was no significant difference between the 10% ethanol site and the 70% ethanol site.

[0050] The results of the anti-H1N1 influenza virus activity and cytotoxicity of the fibronectin fungus on MDCK cells are shown in Table 1: IC values ​​of the water fraction against H1N1 virus 50 The IC value of the 10% ethanol fraction for H1N1 virus was 103.12±25.78 μg / mL. 50 The IC value of the 70% ethanol site for H1N1 virus was 25.21±6.51 μg / mL. 50 Although the 30%, 50%, and 95% ethanol fractions failed to exhibit significant anti-H1N1 viral activity, this does not rule out the possibility that they contain components with potential antiviral activity but did not exhibit inhibitory activity due to their low content.

[0051] 2. Compound Activity

[0052] Each monomer compound was accurately weighed and dissolved in a small amount of DMSO. The mixture was serially diluted 2-fold into 8 gradient concentrations using virus isolation serum-free culture medium containing 1.5 μg / mL TPCK trypsin. The initial concentration range was set between 100 and 800 μmol / L according to the solubility and available amount of the monomer compound. The highest concentration of the positive drug baloxavir group was 100 nmol / mL. The cytotoxicity of the test sample on MDCK cells and the inhibitory effect on H1N1 virus at different concentrations were determined, and the selectivity index (SI) value (SI=CC) was calculated. 50 / IC 50 ).

[0053] Table 2 IC values ​​of different compounds against H1N1 virus 50 and CC on MDCK cells 50

[0054]

[0055] Note: With positive drug baloxavir IC 50 Compared with the value, p <0.001.

[0056] The results are shown in Table 2. Compound 2 has been reported to have the activity of inhibiting H1N1. Compared with compound 2, compounds 1 and 3 not only showed lower IC 50 The results indicate that compounds 1 and 3 have strong inhibitory effects on H1N1 virus activity and are potential antiviral compounds with further development potential.

[0057] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. An application of a Phellinus igniarius extract in the preparation of an anti-influenza virus drug, characterized in that: The extract is 9-oxo-10E,12E-octadecadienoic acid: ; or, Porphyrone: .

2. The use according to claim 1, characterized in that The preparation method of the Phellinus igniarius extract comprises the following steps: (1) Extract Phellinus igniarius with 75% v / v ethanol solution to obtain a crude extract; (2) The aqueous dispersion of the crude extract was adsorbed on ADS-17 macroporous resin and then gradient eluted with water, 10% v / v ethanol solution, 30% v / v ethanol solution, 50% v / v ethanol, 70% v / v ethanol, and 95% v / v ethanol to obtain six corresponding parts Fr.1 to Fr.6; (3) The methanol solution of the Fr.3 part was subjected to silica gel column chromatography and gradient elution with dichloromethane-methanol at a volume ratio of 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, and 1:

1. The fractions were collected and concentrated under reduced pressure. After TLC identification and merging, a total of 22 fractions Fr.3-1 to Fr.3-22 were obtained. The methanol solution of Fr.3-18 was separated and purified by preparative liquid chromatography to obtain purpurone. (4) The methanol solution of the Fr.6 fraction was subjected to silica gel column chromatography and eluted with n-hexane-ethyl acetate at a volume ratio of 100:0, 100:1, 50:1, 20:1, 10:1, 5:1, and 1:1, ethyl acetate-methanol at a volume ratio of 1:1, and methanol-water at a volume ratio of 1:

1. The fractions were collected and concentrated under reduced pressure. After TLC analysis and merging, a total of 17 fractions, Fr.6-1 to Fr.6-17, were obtained. The methanol solution of Fr.6-5 was eluted using a Sephadex LH-20 column with methanol as the eluent. The obtained fractions were combined into four fractions Fr.6-5-1 to Fr.6-5-4 after TLC identification; Fr.6-5-2 was then separated and purified by preparative liquid chromatography to obtain the compound 9-oxo-10E,12E-octadecadienoic acid.

3. The use according to claim 2, characterized in that In step (3), the mobile phase of the preparative liquid chromatography was methanol-water in a volume ratio of 32:68, with a flow rate of 8 mL / min, and the retention time of purpurogenol was 35 min; In step (4), the mobile phase of the preparative liquid chromatography was methanol-water in a volume ratio of 90:10, with a flow rate of 8 mL / min, and the retention time of 9-oxo-10E,12E-octadecadienoic acid was 52.5 min.

4. The use according to claim 1, characterized in that The mulberry linterinary is a thick hair fibrous fungus ( Inontus hispidus ) fruiting bodies or mycelium.

5. The use according to claim 1, characterized in that The influenza virus is H1N1 virus.

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