Phillygenin glucuronic acid derivative as well as preparation method and application thereof

Abstract

The present invention provides a novel phillygenin glucuronic acid derivative shown as a general formula (I). ##STR00001##
wherein, R.sub.1=H, R.sub.2=C.sub.nH.sub.2n+1, R.sub.3=C.sub.nH.sub.2n+1 or R.sub.1=C.sub.nH.sub.2n+1, R.sub.2=C.sub.nH.sub.2n+1,R.sub.3=H or R.sub.1-R.sub.2=CH.sub.2, R.sub.3=C.sub.nH.sub.2n+1; n=1-30. The present invention further relates to a preparation method of the compound, a pharmaceutical composition taking the compound as an active ingredient, as well as application of the compound in the present invention in antiviral diseases.

Claims

1. A preparation method of a phillygenin glucuronic acid derivative, the method sequentially comprises the following steps: 1) mixing forsythia leaves and extraction solvent water and heating, decocting and extracting for 2-3 times, and collecting and merging the extracting solution, and thereby obtaining a forsythia water extract; 2) separating the forsythia water extract by adopting a macroporous resin column, and collecting and merging the eluent, and thereby obtaining a forsythia resin column eluent; 3) performing silica-gel column chromatography on the forsythia resin column eluent, collecting the eluent at stages, respectively drying the eluent, thereby obtaining the phillygenin glucuronic acid derivative, and the phillygenin glucuronic acid derivative comprises a general molecular formula shown as a formula (I): ##STR00009## wherein, R.sub.1=H, R.sub.2=C.sub.nH.sub.2n+1, R.sub.3=C.sub.nH.sub.2n+1 or R.sub.1=C.sub.nH.sub.2n+1, R.sub.2=C.sub.nH.sub.2n+1, R.sub.3=H or R.sub.1-R.sub.2=CH.sub.2, R.sub.3=C.sub.nH.sub.2n+1; n=1.

2. The preparation method of claim 1, wherein in each decocting process in the step 1), a weight ratio of the forsythia leaves to the extraction solvent water is 1:6-10.

3. The preparation method of claim 1, wherein the method further comprises the following steps: concentrating the forsythia water extract in the step 1), preparing a forsythia concentrated solution, and performing macroporous resin column separation treatment.

4. The preparation method of claim 3, wherein a ratio of the volume of the forsythia concentrated solution prepared by concentration treatment to the weight of the forsythia leaves is a range from 1:1 to 5:1.

5. The preparation method of claim 1, wherein in the step 3), C18 reversed-phase silica-gel serves as packing in the silica-gel column chromatography process; specifications of a chromatographic column comprise an internal diameter of 10-100 mm and a length of 10-300 mm; a particle size of the packing is 5-10 m; and a mobile phase is eluted in an isocratic elution manner or a gradient elution manner.

6. The preparation method of claim 5, wherein the mobile phase in the silica-gel column chromatography process is a mixed solution of methanol and water, wherein a volume ratio of the methanol to the water is 8:2-10:1.

7. A method of treating a human patient in need of treatment for influenza virus infection, wherein the method comprises administering to the patient a phillygenin glucuronic acid derivative, and the phillygenin glucuronic acid derivative comprises a general molecular formula shown as a formula (I): ##STR00010## wherein, R.sub.1=C.sub.nH.sub.2n+1, R.sub.2=C.sub.nH.sub.2n+1, R.sub.3H or R.sub.1-R.sub.2=CH.sub.2, R.sub.3=C.sub.nH.sub.2n+1;n=1-30.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The present invention is further described below through embodiments. However, the embodiments are only for illustrating the present invention, but should not be construed as limiting the scope of the present invention.

(2) Embodiment 1

(3) 1. Decocting Treatment 1-1) grinding forsythia leaves and enabling the forsythia leaves to pass through a 20-mesh sieve so as to obtain forsythia leaf powder, adding 10 kg of water into 1 kg of the forsythia leaves, uniformly mixing and heating, and performing the first decocting treatment, wherein a weight ratio of the added water to the forsythia leaves is 10:1; and heating and boiling, decocting and extracting for 2 hours, filtering so as to obtain a first extracting solution, and a first drug residue; 1-2) adding 8 kg of water into the first drug residue, heating and boiling, and performing the second decocting treatment, wherein a weight ratio of the added water to the forsythia leaves is 8:1; and decocting and extracting for an hour, filtering so as to obtain a second extracting solution, and a drug residue (discarding); 1-3) merging the first extracting solution and the second extracting solution so as to prepare a forsythia water extract; 1-4) performing vacuum concentration treatment on the forsythia water extract in a vacuum rotary evaporator, and recovering a solvent, thereby obtaining a forsythia concentrated solution (2 L) for later use, wherein a ratio of the weight of the forsythia leaves to the volume of the forsythia concentrated solution is 1:2;

(4) 2. Macroporous Resin Column Chromatography 2-1) loading the forsythia concentrated solution to a macroporous resin column, and performing macroporous resin column separation treatment, wherein the macroporous resin is selected from an AB-8 type macroporous resin; a column volume of the macroporous resin in the macroporous resin column is 1 L (the chromatographic column has a diameter of 60 mm and a height of 500 mm, and a height of the filled resin is 354 mm); a ratio of the volume of the resin in the resin column to the weight (dry weight) of the forsythia leaves is 1:1 (e.g. if the dry weight of the forsythia leaves is 1 kg, the volume of the macroporous resin is 1 L; and if dry weight of medicinal materials is1 g, the volume of the macroporous resin is 1 ml); washing with water in an amount of 4 times the volume of the column (that is, 4 L) and removing the eluent after a concentrated supernatant completely flows into the resin column; eluting with an ethanol solution at a mass concentration of 3% in an amount of 8 times the volume of the column (that is, 8 L), and removing the eluent; and eluting with an absolute ethyl alcohol in an amount of 8 times the volume of the column (that is, 8 L), and collecting the eluent, thereby obtaining a forsythia-macroporous resin eluent; 2-2) performing vacuum concentration treatment on the forsythia-macroporous resin eluent in a rotary evaporator, recovering a solvent, drying the concentrated residue, thereby obtaining 62 g of crude forsythia product;

(5) 3. Performing Silica-Gel Column Chromatography 3-1) weighing 0.5 g of the crude forsythia product, adding a proper amount of water (2.5 ml, and stirring and dissolving for later use; 3-2) performing chromatography treatment on the crude forsythia product by adopting a high-performance liquid chromatograph (a Shimadzu SCL-10AVP semi-preparative high-performance liquid chromatograph, an LC-8A pump and an SPD-20A monitor), separating and purifying the crude forsythia product, injecting (loading) the dissolved crude forsythia product into the semi-preparative high-performance liquid chromatograph, and performing gradient elute by taking a methanol-water solution as an eluent, wherein: a size of a chromatographic column in the high-performance liquid chromatograph is 22.2250 mm, C18 reversed-phase silica-gel serves as packing, the particle size is 10 m, the loading quantity is 500 mg, the methanol-water solution serves as a mobile phase, conditions of the gradient elute are that: gradient elute: 0-25min, the methanol concentration is 30%-50%; gradient elute: 25-50 min, the methanol concentration is 50%-50%; the flow velocity is 4 ml/min; the column temperature is 20 C.; and an UV detection wavelength is 273 nm; and respectively collecting fractions at retention time of 25.5-27.5 min, 30.5-32.5 min and 35.5-37.5 min; 3-3) respectively performing vacuum concentration on the three collected fractions, performing vacuum drying, thereby obtaining a compound I (70.5 mg), a compound II (53.2 mg) and a compound III (46.6 mg) respectively.

(6) The content of the compounds I, II and III is respectively detected by adopting HPLC (High Performance Liquid Chromatography), and detection conditions of the HPLC comprise: instrument: Water 515 pump; 2487 detector; chromatographic column: Kromasil RP-C18; mobile phase: acetonitrile: 0.1% phosphoric acid solution (13:87); detection wavelength: 230 nm; and flow velocity: 1.0 ml/min.

(7) According to the HPLC detection, the purity of the compound I is 99.6%, the purity of the compound II is 98.1%, and the purity of the compound III is 98.3%.

(8) The compound I is a white solid with a melting point of 111 C. and is soluble in water and ethanol. When expanded on a TLC board (a chromatographic solution of chloroform/methanol 3:1, Rf of 0.25) and sprayed with a 10% H.sub.2SO.sub.4-ethanol reagent, the compound I is purplish red.

(9) ESI-MS: m/z 533.1658[M-H].sup., molecular weight: 534.

(10) Hydrogen nuclear magnetic resonance (400 MHz, d.sub.6-DMSO): (ppm):12.0(1H, s, COOH), 7.119-7.099(1H, d, J=8.0 Hz, ArH), 6.530-6.943(2H, d, J=4.0 Hz, ArH), 6.872(3H, s, ArH), 5.39(2H, s, J=4.8 Hz), 5.23(1H, d, J=4.8 Hz), 5.1(1H, d, J=4.8 Hz), 4.800(1H, d, J=4.8 Hz), 4.374-4.388(1H, d, J=9.6 Hz), 4.105-4.085(1H, d, J=8.0 Hz), 4.005-3.982(1H, d, J=9.2 Hz), 3.75(8H, d, J=8.4 Hz), 3.422 (1H, t, J=8.7 Hz), 3.08(1H, t, J=8.1 Hz), 2.85(1H, d, J=7.2 Hz);

(11) Carbon nuclear magnetic resonance (100 MHz, d.sub.6-DMSO): (ppm):172.75(C-17), 149.51(C-9), 148.95(C-34), 148.09(C-33), 145.74(C-8), 136.26(C-11), 131.67(C-30), 118.55(C-12), 118.05(C-31), 115.72(C-13), 112.03(C-32), 111.07(C-10), 109.92(C-35), 100.21(C-2), 87.11(C-26), 81.74(C-22), 76.26(C-6), 75.70(C-3), 73.41(C-5), 71.91(C-4), 70.81(C-28), 69.46(C-24), 56.15(C-21), 55.99(C-38), 54.47(C-29), 49.79(C-25).

(12) According to test data of ESI-MS, .sup.1H-NMR and .sup.13C-NMR, the compound I is determined to have an English name of 33-Hydroxy phillygenin-8-O--D-glucuronide. A structural formula of the compound I is as follows:

(13) ##STR00006##

(14) The compound II is a white solid with a melting point of 113 C. and is soluble in water and ethanol.

(15) When expanded on the TLC board (a chromatographic solution of chloroform/methanol 3:1, Rf of 0.32) and sprayed with the 10% H.sub.2SO.sub.4-ethanol reagent, the compound II is purplish red.

(16) ESI-MS m/z 533.1641 [M-H].sup., molecular weight: 534.

(17) Hydrogen nuclear magnetic resonance (400 MHz, d.sub.6-DMSO): (ppm):12.0(1H, s, COOH), 7.119-7.099(1H, d, J=8.0 Hz, ArH), 6.530-6.943(2H, d, J=4.0 Hz, ArH), 6.872(3H, s, ArH), 5.39(2H, s, J=4.8 Hz), 5.23(1H, d, J=4.8 Hz), 5.1(1H, d, J=4.8 Hz), 4.800(1H, d, J=4.8 Hz), 4.374-4.388(1H, d, J=9.6 Hz), 4.105-4.085(1H, d, J=8.0 Hz), 4.005-3.982(1H, d, J=9.2 Hz), 3.75(8H, d, J=8.4 Hz), 3.422(1H, t, J=8.7 Hz), 3.08(1H, t, J=8.1 Hz), 2.85(1H, d, J=7.2 Hz);

(18) Carbon nuclear magnetic resonance (100 MHz, d.sub.6-DMSO): (ppm): 173.72(C-17), 149.51(C-33), 148.95(C-34), 148.09(C-9), 144.74(C-8), 136.26(C-11), 131.67(C-30), 121.45(C-12), 119.72(C-31), 118.05(C-13), 115.07(C-10), 113.03(C-32), 109.92(C-35), 100.21(C-2), 87.11(C-26), 81.74(C-22), 76.26(C-6), 75.70(C-3), 73.41(C-5), 71.91(C-4), 70.81(C-28), 69.46(C-24), 56.15(C-21), 55.99(C-38), 54.47(C-29), 50.16(C-25).

(19) According to test data of ESI-MS, and .sup.1H-NMR, and .sup.C-NMR, the compound II is determined to have an English name of 9-Hydroxy phillygenin-8-O--D-glucuronide.

(20) A structural formula of the compound II is as follows:

(21) ##STR00007##

(22) The compound III is a white solid with a melting point of 119 C. and is soluble in water and ethanol. When expanded on the TLC board (a chromatographic solution of chloroform/methanol 3:1, Rf of 0.36) and sprayed with the 10% H.sub.2SO.sub.4-ethanol reagent, the compound II is purplish red.

(23) ESI-MS m/z 531.4933[M-H].sup., molecular weight: 532.

(24) Hydrogen nuclear magnetic resonance (400 MHz, d.sub.6-DMSO): (ppm):12.0(1H, s, COOH), 7.119-7.099(1H, d, J=8.0 Hz, ArH), 6.530-6.943(2H, d, J=4.0 Hz, ArH), 6.872(3H, s, ArH), 6.12(2H, s), 5.39(2H, s, J=4.8 Hz), 5.23(1H, d, J=4.8 Hz), 5.1(1H, d, J=4.8 Hz), 4.800(1H, d, J=4.8 Hz), 4.374-4.388(1H, d, J=9.6 Hz), 4.105-4.085(1H, d, J=8.0 Hz), 4.005-3.982(1H, d, J=9.2 Hz), 3.75(8H, d, J=8.4 Hz), 3.422(1H, t, J=8.7 Hz), 3.08(1H, t, J=8.1 Hz), 2.85(1H, d, J=7.2 Hz);

(25) Carbon nuclear magnetic resonance (100 MHz, d.sub.6-DMSO): (ppm):169.75(C-17), 149.51(C-9), 148.95(C-34), 148.09(C-33), 145.74(C-8), 136.26(C-11), 131.67(C-30), 118.55(C-12), 118.05(C-13), 115.72(C-31), 112.03(C-32), 111.07(C-10), 109.92(C-35), 101.21(C-2), 100.29(C-38), 87.11(C-26), 81.74(C-22), 76.26(C-6), 75.70(C-3), 73.41(C-16), 71.91(C-4), 70.81(C-28), 69.46(C-24), 56.15(C-21), 54.47(C-29), 49.79(C-25).

(26) According to test data of ESI-MS, 1H-NMR and 13C-NMR, the compound III is determined to have an English name of 33,34-Methylenedioxy phillygenin-8-O--D-glucuronide.

(27) A structural formula of the compound III is as follows:

(28) ##STR00008##

(29) Embodiment 2

(30) 1. Decocting Treatment 1-1) grinding forsythia leaves and enabling the forsythia leaves to pass through a 20-mesh sieve so as to obtain forsythia leaf powder, adding 9 kg of water into 1 kg of the forsythia leaves, uniformly mixing and heating, and performing the first decocting treatment, wherein a weight ratio of the added water to the forsythia leaves is 9:1; and heating and boiling, decocting and extracting for 2.5 hours, filtering so as to obtain a first extracting solution, and a first drug residue; 1-2) adding 8 kg of water into the first drug residue, heating and boiling, and performing the second decocting treatment, wherein a weight ratio of the added water to the forsythia leaves is 8:1; and decocting and extracting for an hour, filtering so as to obtain a second extracting solution, and a drug residue (discarding); 1-3) merging the first extracting solution and the second extracting solution so as to prepare a forsythia water extract; 1-4) performing vacuum concentration treatment on the forsythia water extract in a vacuum rotary evaporator, and recovering a solvent, thereby obtaining a forsythia concentrated solution (2.5 L) for later use, wherein a ratio of the weight of the forsythia leaves to the volume of the forsythia concentrated solution is 1:2.5;

(31) 2. Macroporous Resin Column Chromatography 2-1) loading the forsythia concentrated solution to a macroporous resin column, and performing macroporous resin column separation treatment, wherein the macroporous resin is selected from an X-5 type macroporous resin; a column volume of the macroporous resin in the macroporous resin column is 1 L (the chromatographic column has a diameter of 60 mm and a height of 500 mm, and a height of the filled resin is 354 mm); a ratio of the volume of the resin in the resin column to the weight (dry weight) of the forsythia leaves is 1:1 (e.g. if the dry weight of the forsythia leaves is 1 kg, the volume of the macroporous resin is 1 L; and if dry weight of medicinal materials is 1 g, the volume of the macroporous resin is 1 ml); washing with deionized water in an amount of 8 times the volume of the column (that is, 8 L) and removing the eluent after a concentrated supernatant completely flows into the resin column; eluting with an ethanol solution at a mass concentration of 3% in an amount of 4 times the volume of the column (that is, 4 L), and removing the eluent; and eluting with an absolute ethyl alcohol in an amount of 8 times the volume of the column (that is, 8 L), and collecting the eluent, thereby obtaining a forsythia-macroporous resin eluent; 2-2) performing vacuum concentration treatment on the forsythia-macroporous resin eluent in a rotary evaporator, recovering a solvent, drying the concentrated residue, thereby obtaining 58 g of crude forsythia product;

(32) 3. Performing silica-gel column chromatography 3-1) weighing 0.8 g of the crude forsythia product, adding a proper amount of water (1.6 ml), and stirring and dissolving for later use; 3-2) performing chromatography treatment on the crude forsythia product by adopting a high-performance liquid chromatograph (a Shimadzu SCL-10AVP semi-preparative high-performance liquid chromatograph, an LC-8A pump and an SPD-20A monitor), separating and purifying the crude forsythia product, injecting (loading) the dissolved crude forsythia product into the semi-preparative high-performance liquid chromatograph, and performing gradient elute by taking a methanol-water solution as an eluent, wherein a size of a chromatographic column in the high-performance liquid chromatograph is 22.2250 mm, C18 reversed-phase silica-gel serves as packing, the particle size is 10 m, the loading quantity is 800 mg, the methanol-water solution serves as a mobile phase, conditions of the gradient elute are that: gradient elute: 0-25 min, the methanol concentration is 30%-50%; gradient elute: 25-50 min, the methanol concentration is 50%-50%; the flow velocity is 4 ml/min; the column temperature is 20 C.; and an UV detection wavelength is 273 nm; and respectively collecting fractions at retention time of 25.5-27.5 min, 30.5-32.5 min and 35.5-37.5 min; 3-3) respectively performing vacuum concentration on the three collected fractions, performing vacuum drying, thereby obtaining a compound A (104.2 mg), a compound B (74.3 mg) and a compound C (58.1 mg) respectively.

(33) The content of the compounds A, B and C is respectively detected by adopting HPLC (High Performance Liquid Chromatography), and detection conditions of the HPLC comprise: instrument: Water 515 pump; 2487 detector; chromatographic column: Kromasil RP-C18; mobile phase: acetonitrile: 0.1% phosphoric acid solution (13:87); detection wavelength: 230 nm; and flow velocity: 1.0 ml/min.

(34) According to the HPLC detection, the purity of the compound A is 99.3%, the purity of the compound B is 98.4%, and the purity of the compound C is 98.5%.

(35) The physicochemical properties, mass spectrums and nuclear magnetic resonance data of the compounds A, B and C prepared in the embodiment 2 are respectively the same as those of the compounds I, II and III prepared in the embodiment 1.

(36) Test Case 1 In-Vitro Antiviral Test

(37) 1.1 Test Material

(38) (1) Drugs

(39) {circle around (1)} Tested Drugs

(40) The phillygenin glucuronide derivatives (that is, the compounds I, II and III) prepared in the embodiment 1 of the present invention.

(41) {circle around (2)} Positive Control Drugs

(42) Ribavirin injection: colorless transparent liquid, produced by Henan Runhong Pharmaceutical Co., Ltd., product batch number: 1206261, SFDA approval number: H19993553, 100 mg/ml, serving as a positive control drug of the test;

(43) Oseltamivir phosphate: provided by the National Institutes for Food and Drug Control, product batch number: 101096-200901, 100 mg/piece, serving as a positive control drug of the test;

(44) Phillygenin: white powder, produced by Dalian Fusheng Natural Drug Development Co., Ltd., measured through high-performance liquid chromatography by two detectors such as an UV detector and an evaporative light scattering detector by virtue of an area normalization method, having purity of 99.2%.

(45) The drugs are all dissolved in purified water, filtered, degermed to be split charged for later use at 4 C. and serve as to-be-detected drugs in the test.

(46) (2) Cell Strains

(47) Vero strains (African green monkey kidney cells): collected by School of Basic Medicine in Jilin University.

(48) (3) Virus Strains

(49) {circle around (1)} Influenza virus strains, parainfluenza virus strains and respiratory syncytial virus (RSV) strains: purchased from Institute of Virology in Chinese Academy of Preventive Medicine; {circle around (2)} Coxsackie virus B3 (CVB3) strains: purchased from Wuhan Institute of Virology of Chinese Academy of Sciences; {circle around (3)} Coxsackie virus A16 (CoxA16) strains, enterovirus EV71 strains: purchased from Sendai State Hospital; denovirus {circle around (4)} adenovirus (AdV): purchased from Institute of Pediatrics in the First Hospital of Norman Bethune Medical University; and {circle around (5)} hcrps simplex virus I (HSV-1): purchased from the National Institutes for Food and Drug Control.

(50) (4) Main Equipment and Reagents

(51) Biosafety cabinets: BHC-1300IIA/B3, AIRTECH; CO2 incubators: MCO-18AIC, SANYO; inverted microscopes: CKX41, OLYMPUS; electronic analytical balances: AR1140/C, DHAUS; culture media: DMEM, HyClone; fetal calf serum: HyClone; trypsin: Gibco; MTT: Sigma; DMSO: Tianjin Beilian Fine Chemicals Development Co., Ltd.

(52) 1.2 Test Method

(53) (1) Cell Preparation

(54) Performing subculture on Vero cells for 1-2 days to form sheets and have clear boundaries, performing trypsin digestion when stereoscopic impression and diopter are high, completely absorbing digestive juice when needle tip shaped holes occur on cells surfaces, blowing away the cells with milliliters of culture solution, counting, diluting to the quantity of 510.sup.7 cells/L with the culture solution (DMEM containing 10% fetal calf serum), inoculating in a 96-well culture plate, and enabling the cells to grow to a single layer.

(55) (2) Drug Toxicity Determination

(56) Cytotoxicity test: drugs are diluted with a maintenance solution (DMEM containing 2% fetal calf serum) according to concentrations shown in Table 1-1, and are used for cytotoxicity determination.

(57) TABLE-US-00001 TABLE 1-1 Cytotoxicity test concentrations of drugs (unit: g/L) Concentration gradient Drugs Gradient 1 Gradient 2 Gradient 3 Gradient 4 Gradient 5 Gradient 6 Gradient 7 Gradient 8 Compound I 5 2.5 1.25 0.625 0.3125 0.15625 0.078125 0.039063 Compound II 5 2.5 1.25 0.625 0.3125 0.15625 0.078125 0.039063 Compound III 5 2.5 1.25 0.625 0.3125 0.15625 0.078125 0.039063 Ribavirin 5 2.5 1.25 0.625 0.3125 0.15625 0.078125 0.039063 Oseltamivir phosphate 2 1 0.5 0.25 0.125 0.0625 0.03125 0.015625 Phillygenin 5 2.5 1.25 0.625 0.3125 0.15625 0.078125 0.039063

(58) Dripping the drugs of different concentrations in Table 1-1 on single-layer Vero cells, wherein 0.2 ml of drug is filled in each well, and each concentration occupies 6 complex wells; additionally setting 6-well normal control (normal control without drugs) and 6-well blank control (culture solution), culturing in 5% CO.sub.2 incubators at 37 C., observing CPE (cytopathic effect) through inverted microscopes every day (in in-vitro experiments, cell viruses are killed by cell culture and inoculation, and phenomena that the cells are rounded, die and drop from bottle walls and the like can be observed by microscopes within a certain time, called the cytopathic effect. The cytopathic effect refers to cellular degeneration caused by tissue culture cells infected by viruses. Virus quantitation can be performed by utilizing the cytopathic effect); recording the CPE; adding 20 L (5 mg.mL.sup.1) of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide which has a trade name of thiazolyl blue and is a yellow dye) solution into each well within 72 hours, continuously incubating for 4 hours, sucking the culture solution in each well, adding 100 L of DMSO into each well, oscillating for 5min, measuring an OD value at 492 nm, and calculating cell viability; performing Probit regression analysis on the cell viability in SPSS 18.0 statistical software, and calculating the maximum non-cytotoxic concentration (TC.sub.0) and semi-cytotoxic concentration (TC.sub.50).

(59) (3) Determination of TCID.sub.50 of Various Viruses

(60) Performing 10-time gradient decreasing dilution on the various viruses to reach different degrees of dilution such as 10.sup.1, 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, and 10.sup.6, sequentially inoculating the viruses on a single-layer Vero cell 96-well culture plate, wherein the amount of viruses in each well is 100 L, and each degree of dilution exists in 6 wells; setting a normal cell control group, incubating in 5% CO.sub.2 at 37 C. for 2 hours, removing the virus solution, adding 100 L of cell maintenance solution into each well, and culturing in 5% CO.sub.2 at 37 C.; starting to observe cytopathic effect results from the third day under the microscope, judging results on the 7-8.sup.th day and making a record, taking the highest degree of dilution capable of enabling 50% of cell wells to produce positive lesions as an endpoint, and calculating virus titers by using a karber method.

(61) The formula is:

(62) $\mathrm{Log}{\mathrm{TCID}}_{50}=\mathrm{XM}+\frac{1}{2}d\text{-}d\frac{\mathrm{Pi}}{100}$

(63) TCID.sub.50: 50% tissue cell infection amount

(64) XM: logarithm of the highest concentration dilution degree of the viruses

(65) d: logarithm of dilution degree coefficient (multiple)

(66) pi: sum of lesion percentages of various degrees of dilution

(67) (4) Influences of Drugs on Virus-Iinduced CPE

(68) Taking a culture plate grown with single-layer cells, sucking and removing the culture solution, inoculating the cells by a virus attack amount corresponding to 100TCID50, adsorbing in 5% CO.sub.2 incubators at 37 C. for 2 hours, adding each drug solution of a specific concentration (nearly the maximum non-cytotoxic concentration), culturing at each concentration in 6 complex wells according to the volume of 200 L/well; setting ribavirin Injection and oseltamivir phosphate as positive drug control groups, simultaneously setting a normal control group (without any virus or drug) and a virus control group (a control group added with viruses without any drug), and observing the influences of the drugs on the virus-induced CPE; measuring an OD value under the wavelength of 492 nm by using an MTT colorimetric method, calculating antiviral effective rates (ER %) of the drugs; and comparing obvious differences among the antiviral effective rates of the various drugs in the SPSS 18.0 statistical software by using an ANOVA method.
ER %=(OD mean value of the drug treatment groupOD mean value of the virus control group)/(OD mean value of the cell control groupOD mean value of the virus control group)100%

(69) 1.3 Test results

(70) (1) TCID.sub.50 of Various Viruses

(71) $\mathrm{Parainfluenza}\mathrm{virus}\text{:}\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+50}{100}=-4$$\mathrm{Influenza}\mathrm{virus}\text{:}\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+50}{100}=-4$${\mathrm{CVB}}_3\text{:}\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+100+50}{100}=-5$$\mathrm{HSV}\text{-}1\text{:}\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+100+30}{100}=-4.8$$\mathrm{AdV}\text{:}\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+50}{100}=-4$$\mathrm{RSV}\text{:}\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+100+50}{100}=-5$$\mathrm{CoxA}16\text{:}\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+100+50}{100}=-5$$\mathrm{EV}71\text{:}\mathrm{Log}{\mathrm{TCID}}_{50}=-2+0.5-\frac{100+100+100+50}{100}=-5$

(72) (2) Drug Toxicity Determination Results

(73) 1) Determination of drugs on cytotoxicity

(74) The maximum non-cytotoxic concentration (TC.sub.0) and semi-cytotoxic concentration (TC.sub.50) of the various drugs on the Vero cells are shown in Table 1-2.

(75) TABLE-US-00002 TABLE 1-2 Cytotoxicity test results of drugs (unit: g/L) Drug Compound Compound Oseltamivir Concentration Compound I II III Ribavirin phosphate Phillygenin Maximum 0.106 0.103 0.0906 0.063 0.27 0.012 non-cytotoxic concentration Semi-cytotoxic 0.482 0.412 0.435 1.382 0.834 0.293 concentration

(76) 2) Protective Effect Results of Drugs on Virus-Induced CPE

(77) See effective rates of drugs on various viruses and one-way ANOVA (Analysis of Variance) results in Table 1-3 in detail.

(78) TABLE-US-00003 TABLE 1-3 Statistical table for antiviral effective rates (ER %) of drugs Drug Virus Compound Compound Oseltamivir Compound I II III Ribavirin phosphate Phillygenin Influenza virus 95.81** 92.74** 91.35** 56.44** 82.19** 88.17** Parainfluenza virus 96.24** 90.13** 92.22** 95.13** 92.11** 94.06** CoxA16 100.00** 99.25** 100.00** 0.72 2.94 97.03** RSV 83.21** 81.14** 86.18** 52.15* 39.22 81.41** HSV-I 91.23** 93.35** 96.06** 64.65** 63.24** 85.36** ADV 21.26* 20.46* 22.17* 0.47 12.12 14.25* EV71 52.15 54.32 51.64 4.03** 48.65 32.27 CVB3 10.16 11.18 9.63 12.14 1.53 2.29 Notes: compared with the virus control group, *P < 0.05, **P < 0.01; and compared with the phillygenin, .sup.#P < 0.05, .sup.##P < 0.01.

(79) The results in the Table 1-3 show that 33-Hydroxy phillygenin-8-O--D-glucuronide (compound I), 9-Hydroxy phillygenin-8-O--D-glucuronide (compound II) and 33,34-Methylenedioxy phillygenin-8-O--D-glucuronide (compound III) have inhibition rates and effective rates exceeding 90% on the influenza virus, the parainfluenza virus, the herpes simplex virus I (HSV-I) and enterovirus EV71, have obvious differences from the virus control group and have statistical significances. The 33-Hydroxy phillygenin-8-O--D-glucuronide, 9-Hydroxy phillygenin-8-O--D-glucuronide and 33,34-Methylenedioxy phillygenin-8-O--D-glucuronide have antiviral curative effects on multiple viruses superior to the advantages of the phillygenin, Ribavirin and Oseltamivir phosphate.

(80) Test Case 2 In-Vivo Antiviral Test

(81) 2.1 Test Material

(82) (1) Laboratory Animals

(83) Kunming mice: weight of 18-22 g, half male and half female, purchased from Laboratory Animal Center in Dalian Medical University, quality certificate number: SCXK (13)2012-0003.

(84) (2) Drugs

(85) {circle around (1)} The compound I prepared in the embodiment 1 of the present invention, that is, 33-Hydroxy phillygenin-8-O-D-glucuronide;

(86) {circle around (2)} Ribavirin injection: colorless transparent liquid, produced by Henan Runhong Pharmaceutical Co., Ltd., product batch number: 1206261, SFDA approval number: H19993553, 100 mg/ml, serving as a positive control drug of the test;

(87) {circle around (3)} Oseltamivir phosphate: provided by the National Institutes for Food and Drug Control, product batch number: 101096-200901, 100 mg/piece, serving as a positive control drug of the test;

(88) {circle around (4)} Phillygenin: white powder, produced by Dalian Fusheng Natural Drug Development Co., Ltd., measured through high performance liquid chromatography by two detectors (such as an UV detector and an evaporative light scattering detector) by virtue of an area normalization method, having purity of 99.2%.

(89) The drugs are all dissolved in purified water, filtered, degermed to be split charged for later use at 4 C. and serve as to-be-detected drugs in the test.

(90) (2) Detecting Instruments and Reagents

(91) TABLE-US-00004 Instrument Name Model Manufacturer Quantitative PCR 7300 ABI insturment PCR instrument ES-60J Shenyang Longteng Electronic Weighing Instrument Co., Ltd. Electronic FA1004 Shenyang analytical balance Longteng Co., Ltd. CO.sub.2 incubator HG303-5 Nanjing Experimental Instrument Plant Clean bench SW-CJ-IF Suzhou Antai Technology Co., Ltd. Inverted microscope CKX41 Olympus Instrument 80 C. ultralow TECON- Australia temperature refrigerator 5082 Water bath HZS-H Harbin Donglian oscillator Co., Ltd. ELIASA TECAN Australia A-5082 Spectrophotometer 7550 type Japan

(92) 2.2 Test Method

(93) (1) Determination of influenza virus and parainfluenza virus on half lethal dose of mice Performing 10-time gradient dilution on the influenza virus and parainfluenza virus (cell lysis buffer) to obtain virus solutions with concentrations of 10.sup.1, 10.sup.2, 10.sup.3, 10.sup.4 and 10.sup.5; taking 120 Kunming mice, 60 influenza viruses and 60 parainfluenza viruses, respectively randomly dividing into 6 groups, lightly anesthetizing the mice with ethyl ether, performing nasal inhalation to infect the virus solutions of different degrees of dilution at a dose of 0.03 mL per mouse; simultaneously setting blank control, replacing virus suspension with normal saline, taking death and survival as observation indexes, observing the mice every day within 14 days after infection, not counting mice suffering from nonspecific death within 24 hours after infection, and calculating LD50 of the virus solution by a Karber method, wherein the calculation formula is: Log LD.sub.50=XM+1/2 ddPi/100 [wherein: TCID.sub.50: the half lethal dose; XM: logarithm of the highest concentration dilution degree of the viruses; d: logarithm of dilution degree coefficient (multiple); pi: sum of lesion percentages of various degrees of dilution].

(94) (2) Study of 33-Hydroxy phillygenin-8-O--D-glucuronide for resisting pneumonia caused by infection of influenza virus and parainfluenza virus

(95) 1) Laboratory Animals and Groups

(96) taking 960 Kunming mice at an age of four weeks for carrying out two tests; taking 480 mice, randomly dividing the mice into 48 groups at a quantity of 10 mice in one group to be used for test of determining the 33-Hydroxy phillygenin-8-O--D-glucuronide on lung indexes and lung index inhibition rates of the mice infected by the influenza virus, and repeatedly testing for 3 times, wherein 80 mice are taken each time; taking the other 480 mice, randomly dividing the mice into 48 groups at a quantity of 10 mice in one group to be used for test of determining the 33-Hydroxy phillygenin-8-O--D-glucuronide on lung suspension virus hemagglutination titers, and repeatedly testing for 3 times, wherein 80 mice are taken each time.

(97) 2) Infection Method

(98) Putting a ball of degreasing cotton into a 200-300 mL of beaker, pouring a proper amount of ethyl ether (just enabling the degreasing cotton to be wetted), backing off the beaker filled with the degreasing cotton, putting a mouse for anesthetizing, then the mouse is extremely excited, turning up the mouse when the mouse is obviously weak, performing nasal inhalation to infect the influenza virus and parainfluenza virus at a dose of 0.03 ml per naris, and replacing the virus suspension with the normal saline in the normal control group.

(99) 3) Administration Method and Administration Dosage

(100) Respectively performing regular intragastric administration on 33-Hydroxy phillygenin-8-O--D-glucuronide groups, ribavirin control groups and oseltamivir phosphate control groups within one day before infection, wherein high, medium and low administration dosages of the 33-Hydroxy phillygenin-8-O--D-glucuronide are respectively 10.0 mg/kg, 5.0 mg/kg and 2.5 mg/kg, an administration dosage of the positive drug ribavirin is 58.5 mg/kg, an administration dosage of the positive drug oseltamivir phosphate is 19.5 mg/kg, and an administration dosage of the phillygenin is 13.0 mg/kg; continuously administrating once a day for 5 days, and performing intragastric administration with the normal saline of the same volume in the virus control groups.

(101) 4) Observation Indexes

(102) {circle around (1)} Lung Index Determination

(103) Inhibiting from food and water for 8 hours on the fifth day after administration of each mouse, weighing, extracting eyeballs, bloodletting, killing the animals, opening the chest to extract the total lung, washing the lung with the normal saline twice, sucking the surface moisture dry by using filter paper, weighing the lung by using an electronic balance, and calculating the lung index and the lung index inhibition rate according to the following formula:
lung index=(mouse lung weight/mouse body weight)100%; lung index inhibition rate=(mean lung index of the infection model group-mean lung index of the test group)/mean lung index of the infection model group100%.

(104) {circle around (2)} Lung Suspension Virus Hemagglutination Titer Determination

(105) Respectively taking lungs of mice in various groups on the fifth day after treatment, grinding the lungs into homogenate by a homogenizer at a low temperature, diluting the homogenate into 10% of lung tissue suspension with the normal saline, centrifuging to take the supernatant, performing doubling dilution, dripping onto a titer plate according to 0.2 ml/well, adding 0.2 ml of 1% chicken erythrocyte suspension into each well, uniformly mixing, standing at a room temperature for 30 minutes, and observing and recording the hemagglutination titer, wherein erythrocyte agglutination (++) time is taken as the endpoint, and the suspension dilution ratio represents the titer.

(106) 2.3 Test Results and Analysis

(107) (1) Determination Results of Influenza Virus and Parainfluenza Virus on Half Lethal Dose of Mice

(108) The Kunming mice in the test groups are respectively subjected to nasal inhalation to be infected with 30 L of influenza virus and parainfluenza virus of different concentrations, on the third day after infection, the mice in the previous 3 groups (groups with respective virus concentrations of 10.sup.1, 10.sup.2 and 10.sup.3) have disease symptoms of different degrees as follows: pilomotor fur, shiver, decreased appetite and the like; on the fifth day, the mice wobble; on the sixth day, mice in the group with the highest virus concentration start to die, and mice in the other various groups start to die in succession from the seventh day after infection. After 14-day observation is finished, the numbers of dead mice in the various groups are counted, and results are shown in the following Table 1-4 and Table 1-5. The LD.sub.50 of the influenza virus is calculated to be the dilution of 10.sup.2.5. and the LD.sub.50 of the parainfluenza virus is calculated to be the dilution of 10.sup.2.5.

(109) TABLE-US-00005 TABLE 1-4 Statistics of test results of influenza virus on half lethal dose Influenza Cumulative Cumulative Cumulative virus group death survival death rate 10.sup.1 Group 9 1 90% 10.sup.2 Group 7 3 70% 10.sup.3 Group 4 6 40% 10.sup.4 Group 3 7 30% 10.sup.5 Group 1 9 10% Blank Group 0 10 0% LD.sub.50 of viruses is calculated by the Karber method. LogLD.sub.50 of the influenza virus is as follows: ${\mathrm{LogLD}}_{50}=\mathrm{XM}+\frac{1}{2}d-d\frac{\mathrm{Pi}}{100}=$1 + 0.5 (80% + 60% + 40% + 20% + 0% + 0%) = 2.9

(110) TABLE-US-00006 TABLE 1-5 Statistics of test results of parainfluenza virus on half lethal dose Parainfluenza Cumulative Cumulative Cumulative virus group death survival death rate 10.sup.1 Group 8 2 80% 10.sup.2 Group 6 4 60% 10.sup.3 Group 4 6 40% 10.sup.4 Group 2 8 20% 10.sup.5 Group 0 10 0% Blank Group 0 10 0% LD.sub.50 of viruses is calculated by the Karber method. LogLD.sub.50 of the parainfluenza virus is as follows: ${\mathrm{LogLD}}_{50}=\mathrm{XM}+\frac{1}{2}d-d\frac{\mathrm{Pi}}{100}=$1 + 0.5 (90% + 70% + 40% + 30% + 10% + 0%) = 2.5

(111) (2) Action Results of 33-Hydroxy phillygenin-8-O--D-glucuronide for Resisting Pneumonia Caused by Infection of Influenza Virus and Parainfluenza Virus

(112) {circle around (1)} Lung Index Determination

(113) After the mice are infected by the influenza virus and the parainfluenza virus, the determination results of the mean lung index show that: compared with the infection model group, the concentrations of the 33-Hydroxy phillygenin-8-O--D-glucuronide have a certain protective effects in a range of 2.25-10.0 mg/kg/d, and the lung indexes are obviously decreased; curative effects of high-dose 33-Hydroxy phillygenin-8-O--D-glucuronide groups on the influenza virus and the parainfluenza virus are superior to the curative effects of the phillygenin group (P<0.05).

(114) Test results are shown in Table 1-6 and Table 1-7.

(115) TABLE-US-00007 TABLE 1-6 Influences of compound I on lung indexes and lung index inhibition rates of mice infected with influenza virus (n = 3) Drug Lung Lung index dose index inhibition rate Group (mg/kg/d) (X S) (%) P value Normal control group 0 1.273 0.101 Virus control group 0 1.475 0.026 Ribavirin group 58.5 1.279 0.056 13.84 *<0.05 Oseltamivir phosphate group 19.5 1.176 0.045 19.76 *<0.01 Phillygenin group 13.0 1.208 0.025 11.67 *<0.05 Compound I High-dose 10.0 1.145 0.037 22.82 **<0.01, .sup.#<0.05 group Medium- 5.0 1.187 0.028 20.03 **<0.01, .sup.#<0.05 dose group Low-dose 2.25 1.225 0.034 17.85 *<0.05, >0.05 group Compared with the virus control group, *P < 0.05, **P < 0.01; and compared with the phillygenin group, .sup.#P < 0.05, .sup.##P < 0.01.

(116) TABLE-US-00008 TABLE 1-7 Influences of compound I on lung indexes and lung index inhibition rates of mice infected with parainfluenza virus (n = 3) Lung index Drug Lung inhibition dose index rate Group (mg/kg/d) (X S) (%) P value Normal control group 0 1.303 0.037 Virus control group 0 1.585 0.056 Ribavirin group 58.5 1.337 0.056 15.68 *<0.01 Oseltamivir phosphate group 19.5 1.241 0.046 21.83 *<0.01 Phillygenin group 13.0 1.349 0.046 14.56 *<0.01 Compound I High-dose group 13.0 1.239 0.037 22.26 *<0.01, .sup.#<0.05 Medium-dose group 6.5 1.277 0.054 19.73 *<0.01, .sup.#<0.05 Low-dose group 3.25 1.318 0.028 16.94 *<0.01, >0.05 Compared with the virus control group, *P < 0.05, **P < 0.01; and compared with the phillygenin group, .sup.#P < 0.05, .sup.##P < 0.01.

(117) {circle around (2)} Lung Suspension Virus Hemagglutination Titer Determination

(118) After the mice are infected by the influenza virus and the parainfluenza virus, lung tissue virus hemagglutination titers (InX) in the infection model groups are respectively 31.64 and 32.06; after the mice are treated with the 33-Hydroxy phillygenin-8-O--D-glucuronide of different concentrations by 5 days, the lung tissue virus hemagglutination titers are slightly decreased; compared with the infection model group, differences are obvious (P<0.01), wherein the virus hemagglutination titers on the influenza virus and the parainfluenza virus in the mediumand high-dose 33-Hydroxy phillygenin-8-O--D-glucuronide groups are obviously lower than that in the model group, the inhibition rates are all higher than the inhibition rate in the phillygenin group, and differences are obvious (P<0.05, p<0.01). Test results are shown in Table 1-8 and Table 1-9.

(119) TABLE-US-00009 TABLE 1-8 Influences of compound I on lung suspension hemagglutination titers of mice infected with influenza virus (n = 3) Drug Hemagglutination Inhibition dose titer rate Group (mg/kg/d) (InX) (%) P value Normal control group 0 0 Virus control group 0 32.06 1.095 Ribavirin group 58.5 21.87 1.050 32.35 **<0.01 Oseltamivir phosphate group 19.5 20.47 1.104 35.26 **<0.01 Phillygenin group 13.0 21.15 1.024 29.15 *<0.01 Compound I High-dose 10.0 19.24 0.513 40.28 **<0.01, .sup.##<0.01 group Medium- 5.0 20.37 0.285 36.32 **<0.01, .sup.#<0.05 dose group Low-dose 2.25 22.16 1.270 31.26 **<0.01, >0.05 group Compared with the virus control group, *P < 0.05, **P < 0.01; and compared with the phillygenin group, .sup.#P < 0.05, .sup.##P < 0.01.

(120) TABLE-US-00010 TABLE 1-9 Influences of compound I on lung suspension hemagglutination titers of mice infected with parainfluenza virus (n = 3) Drug Hemagglutination Inhibition dose titer rate Group (mg/kg/d) (InX) (%) P value Normal control group 0 0 Virus control group 0 33.17 1.190 Ribavirin group 58.5 24.32 1.123 23.25 *<0.01 Oseltamivir phosphate group 19.5 23.24 1.242 31.14 *<0.01 Phillygenin group 13.0 23.63 1.156 27.75 *<0.01 Compound I High- 10.0 19.68 0.638 38.36 *<0.01, .sup.#<0.01 dose group Medium- 5.0 20.47 0.583 36.32 *<0.01, .sup.#<0.05 dose group Low- 2.25 21.62 0.553 33.61 *<0.01, dose group Compared with the virus control group, *P < 0.05, **P < 0.01; and compared with the phillygenin group, .sup.#P < 0.05, .sup.##P < 0.01.

(121) 2.4 Conclusion

(122) In-vivo antiviral test results show that the 33-Hydroxy phillygenin-8-O--D-glucuronide has obvious inhibitory effects on the influenza virus and the parainfluenza virus as well as mouse virus pneumonia caused by the viruses in the dosage range of 2.25-10 mg/kg/d and can achieve effects of obviously decreasing the lung indexes and hemagglutination titers of the mice and obviously improving pulmonary pathology, and compared with the virus model control group, the differences are obvious; and moreover, the curative effects of the medium-and high-dose 33-Hydroxy phillygenin-8-O--D-glucuronide groups are obviously superior to that of the phillygenin (*P<0.05 or **P<0.01), and the medium-and high-dose group have a trend of being superior to the ribavirin and oseltamivir phosphate.

(123) The compounds II and III are the same as the compound I, have obvious inhibitory effects on the influenza virus and the parainfluenza virus as well as the mouse virus pneumonia caused by the viruses, and can achieve effects of obviously decreasing the lung indexes and hemagglutination titers of the mice and obviously improving the pulmonary pathology, and compared with the virus model control group, the differences are obvious.