WO2016161951A1 - 一种连翘脂素葡萄糖醛酸衍生物，制备方法及其应用 - Google Patents

一种连翘脂素葡萄糖醛酸衍生物，制备方法及其应用 Download PDF

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WO2016161951A1
WO2016161951A1 PCT/CN2016/078688 CN2016078688W WO2016161951A1 WO 2016161951 A1 WO2016161951 A1 WO 2016161951A1 CN 2016078688 W CN2016078688 W CN 2016078688W WO 2016161951 A1 WO2016161951 A1 WO 2016161951A1
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forsythia
preparation
virus
water
glucuronic acid
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PCT/CN2016/078688
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English (en)
French (fr)
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富力
樊宏宇
姜人武
张羽
王凯乾
刘正贤
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富力
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Priority to US15/565,249 priority Critical patent/US10407455B2/en
Priority to AU2016245659A priority patent/AU2016245659B2/en
Priority to RU2017138476A priority patent/RU2684100C1/ru
Priority to KR1020177032383A priority patent/KR102104234B1/ko
Priority to CA2982200A priority patent/CA2982200C/en
Priority to EP16776126.1A priority patent/EP3281945B1/en
Priority to JP2017553114A priority patent/JP6505250B2/ja
Publication of WO2016161951A1 publication Critical patent/WO2016161951A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin

Definitions

  • the invention belongs to the technical field of medicines, in particular to a preparation method of forsythiaside glucuronide derivatives and an antiviral effect.
  • the forsythiaside glucuronide derivative is an active ingredient extracted from the dried leaves of Forsythiasuspensa (Thunb.) vahl of the Oleaceae family.
  • Forsythia has a history of two thousand years and was first seen in the Shennong Bencao Classic. "Shen Nong's Herbal Classic” uploaded: "The main cold heat, swollen sores, heat, sputum.”
  • Murakami Since Murakami first obtained oleanolic acid from Forsythia fruit, more than 60 kinds of ingredients in Forsythia and its genus have been reported. They mainly contain steroids, phenylethyl alcohol and its steroids, lignans, Flavonoids and some compounds such as alcohols, esters, ethers and ketones.
  • the lignan components in Forsythia are mainly xylanol and diepoxylignan.
  • Nishibe et al. obtained arctigenin, mataresinol, arctiin and matairesinoside from forsythia.
  • Tsukamoto et al. obtained phillygenin, (+)-pinoresinol, phillyrin, (+)-pinoresnol- ⁇ -D-glucoside and (+)-epinoresinol-4-O-glucoside from the fruit of Forsythia.
  • the object of the present invention is to provide a novel antiviral drug source, forsythiagine glucuronic acid derivative, a preparation method thereof and an antiviral application thereof, and the forsythiaside glucuronic acid derivative provided by the present invention has antibiotic resistance
  • the function of the virus can be used for preparing medicines or health care products for treating and preventing influenza virus; the preparation method of forsythiaside glucuronic acid derivatives is simple in process, convenient in operation, and suitable for industrialized scale production.
  • the present invention provides a forsythiaglycine glucuronic acid derivative having the general formula: (I):
  • R 1 H
  • R 2 C n H 2n+1
  • R 2 C n H 2n+1
  • Another aspect of the present invention provides a method for preparing a forsythin glucuronic acid derivative, comprising the steps of:
  • the number of times of heating extraction in the step 1) is preferably two.
  • the weight ratio of the forsythia leaf to the extraction solvent water during each decoction extraction is from 1:6 to 10, preferably from 1:8 to 10.
  • the weight ratio of the forsythia leaf to the extraction solvent water is 1:9-10; the weight of the forsythia leaf and the solvent water extracted during the second decoction The ratio is 1:8.
  • the method further comprises the step of concentrating the aqueous extract of Forsythia suspensa obtained in the step 1) to prepare a forsythia concentrate, and then performing the macroporous resin column separation treatment.
  • the ratio of the volume of the forsythia syrup obtained after the concentration treatment to the weight of the forsythia leaf is from 1 to 5:1, preferably from 2 to 2.5:1.
  • the macroporous resin column chromatography in the step 2) comprises the following steps: firstly, the forsythia aqueous extract is injected into the macroporous resin column, followed by elution with water as an eluent, and then mass percentage is used.
  • the ethanol solution with a concentration of 3-50% was eluted as an eluent, and finally eluted with anhydrous ethanol as an eluent.
  • the ethanol eluate was collected to obtain a forsythia-macroporous resin eluent.
  • the ratio of the weight of the forsythia leaf in the aqueous extract of Forsythia suspensa to the volume of the macroporous resin is from 1:0.8 to 2.5, preferably 1:1.
  • the ratio of the column diameter of the macroporous resin column to the column height of the resin is 1:5 to 10, preferably 1:5 to 7, more preferably 1:5.5 to 5.9.
  • the macroporous adsorption resin described in the step 2) selects one of X-5, AB-8, NK-2, NKA-2, NK-9, D3520, D101, WLD, preferably X-5, AB-8.
  • the ratio of the amount of water to the column volume of the macroporous resin column is 2-4:1, preferably 4:1; and the concentration by mass is 3-50%.
  • the ratio of the amount of the ethanol solution having a mass percentage of 3-50% to the column volume of the macroporous resin column is 2-8:1, preferably 4-8:1.
  • the ratio of the amount of the anhydrous ethanol solution to the column volume of the macroporous resin column is 2-8:1, preferably 4- 8:1, further preferably 8:1.
  • silica gel column chromatography treatment in the step 3 comprises the following steps:
  • the silica gel column in step B) was selected as a reverse phase silica gel column.
  • the filler in the reversed-phase silica gel column is selected from C18 reverse phase silica gel; and the particle size is 5-10 ⁇ m.
  • the inverted silica gel column has a column inner diameter of 10 to 100 mm and a length of 100 to 300 mm; preferably, the column inner diameter is 22.2 mm and the length is 250 mm.
  • the silica gel column chromatography selects a high performance liquid chromatography column.
  • a mixed liquid of methanol (A) and water (B) is selected in the mobile phase during the silica gel column chromatography.
  • the ratio of the volume of methanol to water is from 8:2 to 10:1.
  • the column temperature during the silica gel column chromatography is 20 to 35 ° C; the flow rate is 4 to 30 ml/min.
  • the mobile phase elutes the sample in an isocratic or gradient elution manner during silica gel column chromatography.
  • the chromatographic conditions of the silica gel column chromatography are as follows: C18 reverse phase silica gel as a filler ( ⁇ 22.2 ⁇ 250 mm, 10 ⁇ m); methanol (A)-water (B) as a mobile phase; gradient elution [0 min (30% A) ⁇ 25 min (50% A) ⁇ 50 min (50% A); flow rate was 4.0 mL/min; column temperature was 20 ° C; detection wavelength was 273 nm.
  • the chromatographic conditions of the silica gel column chromatography are as follows: C18 reverse phase silica gel as a filler ( ⁇ 22.2 ⁇ 250 mm, 10 ⁇ m); methanol (A)-water (B) as a mobile phase; gradient elution: 0 ⁇ 25min, methanol 30% ⁇ 50%; 25 ⁇ 50min, methanol 50% ⁇ 50%, flow rate 4ml / min, column temperature is 20 ° C, UV detection wavelength is 273nm.
  • the forsythin glucuronic acid derivatives prepared by the above methods are compounds I, II, and III, and their structural confirmation analysis is as follows:
  • the method of the invention is purified by a macroporous adsorption resin column to remove most impurities, and the macroporous adsorption resin can be recycled and reused, thereby reducing the production cost and increasing the service life of the next half preparative column.
  • the process is simple and easy to operate, has high extraction efficiency, low pollution, high purity of the obtained product, and easy industrialized production.
  • a further aspect of the present invention provides the use of the above-described forsythiaside glucuronic acid derivative for the preparation of a medicament for preventing or/and treating influenza virus.
  • the present invention provides a pharmaceutical or nutraceutical composition having an antiviral effect containing a forsythiaside glucuronic acid derivative.
  • the pharmaceutical composition comprises a forsythiaside glucuronic acid derivative of the invention, and a pharmaceutically acceptable adjuvant.
  • the preparation method of forsythiaside glucuronide derivatives and the use thereof in the prevention and treatment of influenza virus drugs are not limited to such compounds, and include the use of such compounds as a mother core, by synthesis, fermentation, etc.
  • the derivative produced are not limited to such compounds, and include the use of such compounds as a mother core, by synthesis, fermentation, etc. The derivative produced.
  • a pharmaceutically acceptable excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, carrier, pH adjuster, ionic strength modifier, sustained release or controlled release agent, encapsulating material or other formulation excipient.
  • the carrier used may be adapted to the corresponding administration form, and may be formulated into an injection, a lyophilized powder for injection, a spray, an oral solution, an oral suspension, a tablet, a capsule, or the like, which is known to those skilled in the art.
  • Formulations such as enteric-coated tablets, pills, powders, granules, sustained release or delayed release.
  • the 4-way of the first aspect of the invention Methyl forsythgin glucuronide is administered by injection or by digestive tract.
  • the pharmaceutical composition of the present invention is preferably an injection or a preparation for digestive administration, that is, it is suitable for injection and digestive tract. Excipients for mode administration are particularly preferred.
  • “administered by the digestive tract” as used herein refers to a mode of administration of a pharmaceutical preparation through the digestive tract of a patient, including oral, intragastric, and enema administration, preferably oral, as may be known to those skilled in the art.
  • the auxiliary materials are formulated into an oral solution, an oral suspension, a tablet, a capsule, an enteric-coated tablet, a pill, a powder, a granule, a sustained release or a delayed release release; wherein the preparation for injection administration is mainly an injection and a powder injection.
  • the novel compound forsythiagine glucuronic acid derivative of the invention has antiviral effect and can be used for preparing antiviral, antipyretic, analgesic, anti-inflammatory drugs or health care products, for example, for example, for example,
  • the preparation method has the advantages of easy control of process conditions, high quality controllability, high yield, low energy consumption, environmental protection, and is suitable for industrial large-scale production.
  • Forsythia leaves are crushed and passed through a 20 mesh sieve to obtain forsythia leaf powder, then water (10 kg) is added to the forsythia leaf (1 kg), mixed uniformly, heated, and subjected to the first decoction treatment.
  • the ratio of the weight of water to the leaves of Forsythia suspensa is 10:1, heated and boiled, and extracted by boiling for 2 hours, followed by filtration to obtain a first extract, the first dregs;
  • the aqueous extract of Forsythia suspensa is placed in a vacuum rotary evaporator for concentration under reduced pressure, and the solvent is recovered to obtain a forsythia concentrate (2L).
  • the ratio of the weight of the forsythia leaf to the volume of the forsythia sinensis concentrate It is 1:2, spare.
  • the column size in the high performance liquid chromatograph It is ⁇ 22.2 ⁇ 250mm, C18 reverse phase silica gel packing, particle size is 10um, loading amount is 500mg, mobile phase is methanol-water solution, gradient elution condition is 0 ⁇ 25min, methanol is 30% ⁇ 50%; 25 ⁇ 50min, methanol 50% ⁇ 50%, flow rate 4ml / min, column temperature is 20 ° C, UV detection wavelength is 273nm, respectively, retention retention time of 25.5 ⁇ 27.5min, 30.5 ⁇ 32.5min, 35.5 ⁇ 37.5min of the fraction;
  • the contents of the obtained compounds I, II and III were examined by HPLC.
  • the HPLC conditions were as follows: instrument: Water 515 pump, 2487 detector; column: Kromasil RP-C18; mobile phase: acetonitrile: 0.1% phosphoric acid aqueous solution (13 : 87); detection wavelength: 230 nm; flow rate: 1.0 ml/min.
  • the compound I was found to be 99.6% by HPLC, the purity of the compound II was 98.1%, and the purity of the compound III was 98.3%.
  • Compound I is a white solid, melting point: 111 ° C; soluble in water, ethanol.
  • the material was developed on a TLC plate (chromic solution was chloroform/methanol 3:1, Rf was 0.25), and a spray of 10% H 2 SO 4 -ethanol reagent exhibited a purplish red color.
  • Compound II is a white solid, melting point: 113 ° C; soluble in water, ethanol.
  • the material was developed on a TLC plate (chromic solution was chloroform/methanol 3:1, Rf was 0.32), and a spray of 10% H 2 SO 4 -ethanol reagent appeared purple.
  • Compound III is a white solid, m.p.: 119 ° C; dissolved in water, ethanol. It was developed on a TLC plate (chromic solution was chloroform/methanol 3:1, Rf was 0.36), and a 10% H 2 SO 4 -ethanol reagent was sprayed to give a purple-red color.
  • Forsythia leaves are crushed and passed through a 20 mesh sieve to obtain forsythia leaf powder, then water (9 kg) is added to the forsythia leaf (1 kg), mixed uniformly, heated, and subjected to the first decoction treatment.
  • the ratio of the weight of water to the leaves of Forsythia suspensa is 9:1, heating is boiled, and extracted by boiling for 2.5 hours, and then filtered to obtain a first extract, the first dregs;
  • the aqueous extract of Forsythia suspensa is placed in a vacuum rotary evaporator for concentration under reduced pressure, and the solvent is recovered to obtain a Forsythia sinensis concentrate (2.5 L), the weight of the forsythia leaf and the volume of the Forsythia sinensis concentrate.
  • the ratio is 1:2.5, spare.
  • the forsythia concentrate is applied to a macroporous resin column for macroporous resin column separation treatment, wherein the macroporous adsorption resin selects X-5 macroporous adsorption resin, and the macroporous adsorption resin column has large pores.
  • the column volume of the adsorbent resin is 1 L (the diameter of the column is 60 mm, the height is 500 mm, and the height of the resin is 354 mm), and the ratio of the volume of the resin in the resin column to the weight of the forsythia leaf (dry weight) is 1:1 (ie, if The dry weight of the leaf is 1 kg, the volume of the macroporous resin is 1 L; if the dry weight of the drug is 1 g, the volume of the macroporous resin is 1 ml), and the supernatant after the concentration is completely poured into the resin column, the column volume is 8 times.
  • the column size in the high performance liquid chromatograph It is ⁇ 22.2 ⁇ 250mm, C18 reverse phase silica gel packing, particle size is 10um, loading amount is 800mg, mobile phase is methanol-water solution, gradient elution condition is 0 ⁇ 25min, methanol is 30% ⁇ 50%; 25 ⁇ 50min, methanol 50% ⁇ 50%, flow rate 4ml / min, column temperature is 20 ° C, UV detection wavelength is 273nm, respectively, retention retention time of 25.5 ⁇ 27.5min, 30.5 ⁇ 32.5min, 35.5 ⁇ 37.5min of the fraction;
  • the content of the prepared compounds A, B, and C was examined by HPLC, and the HPLC detection conditions were as follows: Water 515 pump, 2487 detector; column: Kromasil RP-C18; mobile phase: acetonitrile: 0.1% aqueous phosphoric acid (13:87); detection wavelength: 230 nm; flow rate: 1.0 ml/min.
  • the compound A was found to be 99.3% by HPLC, the purity of the compound B was 98.4%, and the purity of the compound C was 98.5%.
  • the physicochemical properties, mass spectrometry, and nuclear magnetic resonance data of the compounds A, B, and C prepared in Example 2 were the same as those of the compounds I, II, and III prepared in Example 1, respectively.
  • Oseltamivir phosphate China National Institute for the Control of Pharmaceutical and Biological Products, product batch number: 101096-200901, 100mg / support as a positive control drug for this test;
  • the above drugs are all dissolved in purified water, filtered, sterilized and dispensed, and stored at 4 ° C for the test.
  • Vero African Green Monkey Kidney Cell
  • Vero African Green Monkey Kidney Cell
  • RSV respiratory syncytial virus
  • Biological safety cabinet BHC-1300 IIA/B3, AIRTECH; CO2 incubator: MCO-18AIC, SANYO; inverted microscope: CKX41, OLYMPUS; electronic analytical balance: AR1140/C, DHAUS; medium: DMEM, HyClone; fetal bovine serum :HyClone; Trypsin: Gibco; MTT: Sigma; DMSO: Tianjin Beilian Fine Chemicals Development Co., Ltd.
  • Vero cells were subcultured for 1-2 days to make them into slices. The boundary was clear. When the stereoscopic effect and the refractive power were strong, trypsin digestion was performed. After the cell surface appeared a pinpoint-like pore, the digestive juice was aspirated, and several milliliters of the culture solution was blown away. The cells were counted and diluted with a culture solution (DMEM containing 10% fetal bovine serum) to about 5 ⁇ 10 7 /L, and then seeded in a 96-well culture plate until the cells were grown into a single layer.
  • DMEM containing 10% fetal bovine serum
  • Cytotoxicity test The drug was diluted with a maintenance solution (DMEM containing 2% fetal bovine serum) at a concentration shown in Table 1-1 for cytotoxicity assay.
  • DMEM fetal bovine serum
  • the virus can be quantified by using this pathological effect.
  • MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide was added to each well.
  • the Chinese chemical name was 3-(4,5-dimethylthiazole). -2)-2,5-diphenyltetrazolium bromide, trade name: thiazole blue. It is a yellow color dye.
  • 20 ⁇ L of solution (5 mg ⁇ mL -1 ), continue to incubate for 4 h, aspirate each well. The solution was added with 100 ⁇ L of DMSO per well, shaken for 5 min, and the OD value was measured at 492 nm to calculate the cell survival rate.
  • the cell viability was subjected to Probit regression analysis to calculate the maximum non-toxic concentration (TC 0 ) and half toxicity concentration (TC 50 ) of the drug against Vero cells.
  • viruses were serially diluted 10-fold to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 different dilutions , and sequentially seeded in a single layer of Vero cells in 96-well culture. On the plate, 100 ⁇ L per well, 6 wells per dilution, and a normal cell control group. Incubate at 37 ° C, 5% CO 2 for 2 h, discard the virus solution, then add 100 ⁇ L of cell maintenance solution per well, and incubate at 37 ° C, 5% CO 2 .
  • the cytopathic effect was observed under the microscope on the third day, and the results were recorded on the 7th to 8th day, so that the highest dilution of 50% cell hole positive lesions was used as the end point, and the virus titer was calculated by the karber method.
  • TCID 50 50% tissue cell infection
  • ⁇ pi the sum of the percentage of lesions per dilution
  • the OD value was measured at 492 nm using the MTT colorimetric method to calculate the drug antiviral efficiency (ER%).
  • ANOVA was used to compare the significant differences in the antiviral efficacy of each drug in the SPSS 18.0 statistical software.
  • ER% (mean OD value of drug treatment group - average OD value of virus control group) / (average OD value of cell control group - average OD value of virus control group) ⁇ 100%
  • TC 0 maximum non-toxic concentration
  • TC 50 half toxicity concentration
  • Table 1-3 show that 33-hydroxy-Forsyricin-8-O- ⁇ -D-glucuronide (Compound I), 9-hydroxy-Forsyricin-8-O- ⁇ -D- Glucuronide (Compound II) and 33,34-Methylenedioxy-Fortrans-8-O- ⁇ -D-glucuroniduronic acid glucuronide (Compound III) against influenza
  • the inhibition rate and effective rate of virus, parainfluenza virus, herpes simplex virus type I (HSV-I) and enterovirus EV71 were both over 90%, which was significantly different from the virus control group and was statistically significant.
  • 33-hydroxy-Forsythin-8-O- ⁇ -D-glucuronide, 9-hydroxy-Forsyricin-8-O- ⁇ -D-glucuronide and 33,34-Asia Methyldioxy-Fortrans-8-O- ⁇ -D-glucuroniduronic acid glucuronide is superior to forsythiastrobin and ribavirin in the antiviral efficacy of various viruses. And the advantages of oseltamivir phosphate.
  • the above drugs are all dissolved in purified water, filtered, sterilized and dispensed, and stored at 4 ° C for the test.
  • influenza virus and the parainfluenza virus were diluted 10 times to a virus solution having a concentration of 10 -1 , 10 -2 , 10 -3 , 10 -4 , and 10 -5 .
  • 120 Kunming mice 60 influenza virus and parainfluenza virus groups were randomly divided into 6 groups. The mice were lightly anesthetized with ether and infected with different dilutions of virus solution 0.03mL/only. At the same time, a blank control was set, and the physiological suspension was used instead of the viral suspension. Death and survival were observed and observed daily until 14 days after infection. The death within 24 hours of infection was non-specific death, not counted, and the Karber method calculated the LD50 of the virus solution.
  • mice were randomly divided into 48 groups, 10 rats in each group, for lung index and lung index of 33-hydroxy-Forsyricin-8-O- ⁇ -D-glucuronide in mice infected with influenza virus. The test for inhibition rate was tested in three replicates, and 80 mice were taken each time. Another 480 mice were randomly divided into 48 groups, 10 in each group, for the hemagglutination titer of 33-hydroxy-Forsylin-8-O- ⁇ -D-glucuronide on lung suspension virus. The test was performed, and the test was repeated three times, and 80 mice were taken each time.
  • the 33-hydroxy-Forsylin-8-O- ⁇ -D-glucuronide group, ribavirin and oseltamivir phosphate control group were given conventional intragastric administration on the day before infection, 33- The high, medium and low doses of hydroxy-Forsylin-8-O- ⁇ -D-glucuronide were 10.0, 5.0, 2.5 mg/kg, respectively, and the dose of ribavirin-positive drug was 58.5 mg. /kg, the dose of oseltamivir phosphate positive drug was 19.5mg/kg, and the dose of forsythiaside group was 13.0mg/kg once a day for 5d, and the virus control group was given the same volume of physiology. brine.
  • the lungs of each group of mice on the 5th day after treatment were taken and homogenized by a homogenizer at a low temperature.
  • the physiological saline was diluted to 10% of the lung tissue suspension, and the supernatant was centrifuged and diluted to 0.2 ml/
  • the holes were dropped on the titration plate, 0.2 ml of 1% chicken red blood cell suspension was added to each well, and the mixture was allowed to stand at room temperature for 30 min, and the blood coagulation titer was observed.
  • the end point of red blood cell agglutination (++) is expressed as the titer of the suspension dilution factor.
  • the Kunming mice in the experimental group were infected with different concentrations of influenza virus and parainfluenza virus in 30 ⁇ L, and the mice in the third group (virus concentration 10 -1 group, 10 -2 group, 10 -3 group) before the third day of infection. There were different degrees of symptoms: shrub, shivering, and decreased diet. On the 5th day, the mice walked and waved; on the 6th day, the mice in the highest virus concentration group began to die, and the other groups continued on the 7th day after infection. Death has occurred. After the end of 14 days, the number of deaths of each group of mice was counted. The results are shown in Tables 1-4 and 1-5 below. The LD 50 of the influenza virus was calculated to be a dilution of 10 - 2.9 and the LD 50 of the parainfluenza virus was a dilution of 10 - 2.5 .
  • the Karber method calculates the LD 50 of the virus.
  • the LogLD 50 of the influenza virus is as follows:
  • the Karber method calculates the LD 50 of the virus.
  • the LogLD 50 of the parainfluenza virus is as follows:
  • the blood protein titer (InX) of the infected model group was 31.64 and 32.06, respectively.
  • Different concentrations of 33-hydroxy-Forsylin-8-O- ⁇ -D-glucose After 5 days of treatment with aldonic acid, the blood coagulation titer of lung tissue decreased, and the difference was significant compared with the infection model group (P ⁇ 0.01).
  • the hemagglutination titer of influenza and parainfluenza virus in the middle and high dose groups of 33-hydroxy-Forsyricin-8-O- ⁇ -D-glucuronide was significantly lower than that of the model group, and the inhibition rate was higher than that of the model group.
  • the difference was significant (P ⁇ 0.05, p ⁇ 0.01).
  • Tables 1-8 and 1-9 The test results are shown in Tables 1-8 and 1-9.
  • Compounds II and III are the same as compound I, and have obvious inhibitory effects on influenza virus and parainfluenza virus and mouse viral pneumonia, which can significantly reduce lung index and hemagglutination titer, lung histopathology. There was also a significant improvement, which was significantly different from the virus model control group.

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Abstract

一种通式(Ⅰ)所示的连翘脂素葡萄糖醛酸衍生物。其中：R 1＝H，R 2＝C nH 2n+1，R 3＝C nH 2n+1或R 1＝C nH 2n+1，R 2＝C nH 2n+1，R 3＝H或R 1-R 2＝-CH 2-，R 3＝C nH 2n+1；n＝1～30。还涉及该化合物的制备方法及以该化合物为活性成分的药物组合物，以及化合物在抗病毒病症中的应用。

Description

一种连翘脂素葡萄糖醛酸衍生物，制备方法及其应用 技术领域
本发明属于药物技术领域，尤其涉及连翘脂素葡萄糖醛酸苷类衍生物制备方法及在抗病毒方面的作用。
背景技术
连翘脂素葡萄糖醛酸苷类衍生物，是从木犀科(Oleaceae)连翘属植物连翘(Forsythiasuspensa(Thunb.)vahl)的干燥叶中提取的有效成分。连翘作为中药应用，已有两千年历史，始见于《神农本草经》。《神农本草经》上载：“主寒热，痈肿恶疮，结热，鼠瘘瘰疬。”
Murakami首次从连翘果实中得到齐墩果酸以来，连翘及其同属植物中已有六十余种成分见诸报道、其主要含萜类、苯乙醇及其甙类、木脂素类、黄酮类和一些醇、酯、醚和酮等类化合物。
连翘属中的木脂素成分主要为木脂内酷和双环氧木脂素。1978年，Nishibe等从连翘中分得了arctigenin，mataresinol，arctiin和matairesinoside。1985年，Tsukamoto等从连翘果实中分得了phillygenin，(+)-pinoresinol，phillyrin，(+)-pinoresnol-β-D-glucoside和(+)-epinoresinol-4-O-glucoside。1997年，刘冬雷等又从连翘果实中分得了(+)松脂素单甲基醚-β-D-葡萄糖营(pinoresinolmonomethylether-β-D-gluco-side)。近来生合成研究表明，连翘属中木脂素前体是松伯醇，已知连翘属中木脂素及其苷的3，3’位全是甲氧基取代，4，4’位可以是羟基，甲氧基取代或与糖成苷。
三种连翘脂素葡萄糖醛酸苷类衍生物系首次从连翘叶中发现，葡萄糖醛酸衍生物具有良好的活性。例如青蒿素葡萄糖醛酸衍生物(Efficient Preparations of the β-Glucuronides of Dihydroartemisinin and Structural Confirmation of the Human Glucuronide Metabolite.Paul M.O′Neill，Feodor Scheinmann，Andrew V. Stachulski，James L.Maggs，and B.Kevin Park.J.Med.Chem.，2001，44(9)，pp 1467-1470)、依达拉奉葡萄糖醛酸衍生物(Synthesis of the metabolites of a free radical scavenger edaravone(MCI-186，RadicutTM).Kazutoshi Watanabe，Masao Taniguchi，Masaki Shinoda.Redox Report，Vol.8，No.3，2003，157-161)、风车子素A-1葡萄糖醛酸衍生物(Regio-and Stereospecific Synthesis of Mono-β-d-Glucuronic Acid Derivatives of Combretastatin A-1.Rajendra P.Tanpure，Tracy E.Strecker，David J.Chaplin，Bronwyn G.Siim，Mary Lynn Trawick and Kevin G.Pinney.J.Nat.Prod.，2010，73(6)，pp 1093-1101)、白藜芦醇葡萄糖醛酸衍生物(WANG LAIXI；Heredia，A.；Song，HJ；ZHANG ZHAOJUN；YU BIAO；Davis，C.；Redfield，R.Resveratrol glucuronides as the metabolites of resveratrol in humans：Characterization，synthesis，and anti-HIV activity.J.Pharm.Sci.2004，93(10)，2448-2457)和姜黄素葡萄糖醛酸衍生物(K.S.Psrvathy，M.Sc.University of Mysore.2009)等等。因此我们研究了三种连翘脂素葡萄糖醛酸苷类衍生物的制备方法，并进行了药理研究。
发明内容
本发明的目的是提供一种新的抗病毒药物来源，连翘脂素葡萄糖醛酸衍生物、其制备方法和其抗病毒的应用，本发明提供的连翘脂素葡萄糖醛酸衍生物具有抗病毒的作用，可用于制备用于治疗和预防流感病毒的药物或保健品；连翘脂素葡萄糖醛酸衍生物的制备方法工艺简单，操作方便，适合工业化放大生产。
为实现本发明的目的，本发明一方面提供一种结构通式如式(I)所示的连翘脂素葡萄糖醛酸衍生物：
Figure PCTCN2016078688-appb-000001
其中：R1＝H，R2＝CnH2n+1，R3＝CnH2n+1或R1＝CnH2n+1，R2＝CnH2n+1，R3＝H或R1-R2＝-CH2-，R3＝CnH2n+1；n＝1～30。
其中，所述取代基的n＝1。
本发明另一方面提供一种连翘脂素葡萄糖醛酸衍生物的制备方法，包括如下顺序进行的步骤：
1)将连翘叶与提取溶剂水混合后加热，进行煎煮提取2-3次，收集、合并提取液，获得连翘水提液；
2)将连翘水提液采用大孔树脂柱进行分离处理，收集、合并洗脱液，得到连翘树脂柱洗脱液；
3)对连翘树脂柱洗脱液进行干燥处理，然后进行硅胶柱层析处理，分段收集洗脱液，洗脱液分别干燥，即得。
其中，步骤1)中所述加热提取次数优选为2次。
特别是，每次煎煮提取过程中，所述连翘叶与提取溶剂水的重量配比为1∶6-10，优选为1∶8-10。
尤其是，第一次煎煮过程中，所述连翘叶与提取溶剂水的重量配比为1∶9-10；第二次煎煮过程中，所述连翘叶与提取溶剂水的重量配比为1∶8。
特别是，还包括将步骤1)获得的连翘水提液进行浓缩处理，制成连翘浓缩液后再进行所述的大孔树脂柱分离处理。
尤其是，浓缩处理后获得的连翘浓缩液的体积与连翘叶的重量之比为1-5∶1，优选为2-2.5∶1。
其中，步骤2)中所述大孔树脂柱层析处理包括以下顺序进行的步骤：首先将连翘水提液注入大孔树脂柱，接着采用水为洗脱剂进行洗脱，然后采用质量百分比浓度为3-50％的乙醇溶液为洗脱剂进行洗脱，最后采用无水乙醇为洗脱剂进行洗脱，收集无水乙醇洗脱液，获得连翘-大孔树脂洗脱液。
特别是，大孔树脂柱层析过程中，连翘叶水提液中连翘叶的重量与大孔树脂体积之比为1∶0.8-2.5，优选为1∶1。
尤其是，大孔树脂柱分离处理过程中，大孔树脂柱的柱直径与树脂的柱高之比为1∶5-10，优选为1∶5-7，进一步优选为1∶5.5-5.9。
其中，步骤2)中所述的大孔吸附树脂选择X-5、AB-8、NK-2、NKA-2、NK-9、D3520、D101、WLD中的一种，优选为X-5、AB-8。
特别是，采用水为洗脱剂进行洗脱过程中，水的用量与大孔树脂柱的柱体积之比为2-4∶1，优选为4∶1；采用质量百分比浓度为3-50％的乙醇溶液为洗脱剂进行洗脱过程中，质量百分比浓度为3-50％的乙醇溶液的用量与大孔树脂柱的柱体积之比为2-8∶1，优选为4-8∶1，进一步优选为8∶1；采用无水乙醇溶液为洗脱剂进行洗脱过程中，无水乙醇溶液的用量与大孔树脂柱的柱体积之比为2-8∶1，优选为4-8∶1，进一步优选为8∶1。
其中，步骤3)中所述硅胶柱层析处理包括如下顺序进行的步骤：
A)对连翘树脂柱洗脱液进行浓缩、干燥处理，制得连翘粗品；
B)将连翘粗品用水溶解后注入硅胶柱，进行硅胶柱层析处理，分段收集洗脱液，洗脱液分别干燥，即得。
特别是，步骤B)中所述硅胶柱选择反相硅胶柱。
其中，所述反相硅胶柱中填料选择C18反相硅胶；粒径5～10μm。
特别是，所述反相硅胶柱的柱内径10～100mm、长度100～300mm；优选为柱内径22.2mm、长度250mm。
其中，所述硅胶柱层析选择高效液相色谱柱。
特别是，所述硅胶柱层析过程中流动相选择甲醇(A)和水(B)的混合液。
其中，甲醇与水的体积之比为8∶2-10∶1。
特别是，硅胶柱层析过程中柱温20～35℃；流速4～30ml/min。
特别是，硅胶柱层析过程中流动相以等度或梯度洗脱的方式洗脱样品。
其中，梯度洗脱[0min(30％A)→25min(50％A)→50min(50％A)；流速为4.0mL/min；柱温20℃；检测波长为273nm。
特别是，所述硅胶柱层析的色谱条件如下：以C18反相硅胶为填充剂(Φ22.2×250mm，10μm)；甲醇(A)-水(B)为流动相；梯度洗脱[0min(30％A)→25min(50％A)→50min(50％A)；流速为4.0mL/min；柱温20℃；检测波长为273nm。
特别是，所述硅胶柱层析的色谱条件如下：以C18反相硅胶为填充剂(Φ22.2×250mm，10μm)；甲醇(A)-水(B)为流动相；梯度洗脱：0～25min，甲醇30％～50％；25～50min，甲醇50％～50％，流速4ml/min，柱温为20℃，紫外检测波长为273nm。
通过上述方法制备而成的连翘脂素葡萄糖醛酸衍生物为化合物I、II、III，其结构确认分析如下：
化合物I：33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷
ESI-MS：m/z 533.1658[M-H]-，分子量为：534；分子式为：C26H30O12。
核磁共振氢谱(400MHz，d6-DMSO)：δ(ppm)：12.0(1H，s，COOH)，7.119-7.099(1H，d，J＝8.0Hz，Ar-H)，6.530-6.943(2H，d，J＝4.0Hz，Ar-H)，6.872(3H，s，Ar-H)，5.39(2H，s，J＝4.8Hz)，5.23(1H，d，J＝4.8Hz)，5.1(1H，d，J＝4.8Hz)，4.800(1H，d，J＝4.8Hz)，4.374-4.388(1H，d，J＝9.6Hz)，4.105-4.085(1H，d，J＝8.0Hz)，4.005-3.982(1H，d，J＝9.2Hz)，3.75(8H，d，J＝8.4Hz)，3.422(1H，t，J＝8.7Hz)，3.08(1H，t，J＝8.1Hz)，2.85(1H，d，J＝7.2Hz)；
核磁共振碳谱(100MHz，d6-DMSO)：δ(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)。
Figure PCTCN2016078688-appb-000002
化合物II：9-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷
ESI-MS m/z 533.1641[M-H]-，分子量为：534；分子式为：C26H30O12。
核磁共振氢谱(400MHz，d6-DMSO)：δ(ppm)：12.0(1H，s，COOH)，7.119-7.099(1H，d，J＝8.0Hz，Ar-H)，6.530-6.943(2H，d，J＝4.0Hz，Ar-H)，6.872(3H，s，Ar-H)，5.39(2H，s，J＝4.8Hz)，5.23(1H，d，J＝4.8Hz)，5.1(1H，d，J＝4.8Hz)，4.800(1H，d，J＝4.8Hz)，4.374-4.388(1H，d，J＝9.6Hz)，4.105-4.085(1H，d，J＝8.0Hz)，4.005-3.982(1H，d，J＝9.2Hz)，3.75(8H，d，J＝8.4Hz)，3.422(1H，t，J＝8.7Hz)，3.08(1H，t，J＝8.1Hz)，2.85(1H，d，J＝7.2Hz)；
核磁共振碳谱(100MHz，d6-DMSO)：δ(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)。
Figure PCTCN2016078688-appb-000003
化合物III：33，34-亚甲基二氧-连翘脂素-8-O-β-D-葡萄糖醛酸苷
ESI-MS m/z 531.4933[M-H]-，分子量为：532；分子式为：C26H28O12。
核磁共振氢谱(400 MHz，d6-DMSO)：δ(ppm)：12.0(1H，s，COOH)，7.119-7.099(1H，d，J＝8.0Hz，Ar-H)，6.530-6.943(2H，d，J＝4.0Hz，Ar-H)，6.872(3H，s，Ar-H)，6.12(2H，s)，5.39(2H，s，J＝4.8Hz)，5.23(1H，d，J＝4.8Hz)，5.1(1H，d，J＝4.8Hz)，4.800(1H，d，J＝4.8Hz)，4.374-4.388(1H，d，J＝9.6Hz)，4.105-4.085(1H，d，J＝8.0Hz)，4.005-3.982(1H，d，J＝9.2Hz)，3.75(8H，d，J＝8.4Hz)，3.422(1H，t，J＝8.7Hz)，3.08(1H，t，J＝8.1Hz)，2.85(1H，d，J＝7.2Hz)；
核磁共振碳谱(100MHz，d6-DMSO)：δ(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)。
Figure PCTCN2016078688-appb-000004
本发明方法通过大孔吸附树脂柱纯化，去除绝大多数杂质，大孔吸附树脂可以回收再利用，从而降低了生产成本，增加下一步半制备色谱柱使用寿命。该工艺简便易操作、提取效率高、污染小、所得产品纯度高、易于实现工业化生产。
本发明又一方面提供上述连翘脂素葡萄糖醛酸衍生物的抗病毒应用。
本发明再一方面提供上述连翘脂素葡萄糖醛酸衍生物在制备预防或/和治疗流感病毒药物中的用途。
其中，本发明提供含连翘脂素葡萄糖醛酸衍生物的具有抗病毒功效的药物或保健品组合物。
特别是，所述药物组合物，其包括本发明连翘脂素葡萄糖醛酸衍生物，以及药学上可接受的辅料。
连翘脂素葡萄糖醛酸苷类衍生物的制备方法及在预防和治疗流感病毒药物中的用途，同时并不限于该类化合物，还包括以此类化合物为母核，通过合成，发酵等方法所制备的衍生物。
在本文中，药学上可接受的辅料指无毒固态、半固态或液态填充剂、稀释剂、载体、pH调节剂、离子强度调节剂、缓释或控释剂、包裹材料或其他制剂辅料。所用载体可与相应的给药形式相适应，可使用本领域技术人员所知晓的辅料配成注射剂、(注射用)冻干粉、喷雾剂、口服溶液、口服混悬液、片剂、胶囊、肠溶片、丸剂、粉剂、颗粒剂、持续释放或延迟释释放等制剂。优选本发明第一方面的4-去 甲基连翘脂素葡萄糖醛酸苷通过注射或经消化道方式给药，因此，本发明的药物组合物优选为注射剂或经消化道给药的制剂，即适于配制成注射和经消化道方式给药的辅料特别优选的。其中，“经消化道给药”在本文中指药物制剂通过患者消化道的给药方式，包括口服、灌胃给药和灌肠给药等，优选是口服，如可使用本领域技术人员所知晓的辅料配成口服溶液、口服混悬液、片剂、胶囊、肠溶片、丸剂、粉剂、颗粒剂、持续释放或延迟释释放等制剂；其中，注射给药的制剂主要是针剂和粉针剂。
本发明的新的化合物连翘脂素葡萄糖醛酸衍生物具有抗病毒的功效，可用于制备抗病毒、解热、镇痛、抗炎药物或保健品，连翘脂素葡萄糖醛酸衍生物的制备方法操作工艺条件容易控制，质量可控性强，得率高，耗能低，环保，适宜工业化大规模生产。
具体实施方式
以下通过实施例进一步描述本发明，但这些实施例仅是说明本发明，而不应理解为对本发明范围的任何限制。
实施例1
1、煎煮处理
1-1)连翘叶粉碎并过20目筛，得连翘叶粉，接着向连翘叶(1kg)中加入水(10kg)，混合均匀后加热，进行第一次煎煮处理，加入的水与连翘叶的重量之比为10∶1，加热沸腾，煎煮提取2h后进行过滤，获得第一提取液，第一药渣；
1-2)向第一药渣中加入水(8kg)，加热沸腾，进行第二次煎煮处理，加入的水与连翘叶的重量之比为8∶1，煎煮提取时间1h后进行过滤，获得第二提取液，药渣(弃去)；
1-3)合并第一、第二提取液，制得连翘水提液；
1-4)将连翘水提液置于减压旋转蒸发器中进行减压浓缩处理，回收溶剂，获得连翘浓缩液(2L)，连翘叶的重量与连翘浓缩液的体积之比为1∶2，备用。
2、大孔树脂柱层析
2-1)将连翘浓缩液上样于大孔树脂柱上，进行大孔树脂柱分离处理，其中，大 孔吸附树脂选择AB-8型大孔吸附树脂，大孔吸附树脂柱内大孔吸附树脂的柱体积为1L(层析柱的直径60mm，高500mm，内装树脂高度为354mm)，树脂柱内树脂的体积与连翘叶重量(干重)之比为1∶1(即如果连翘叶干重1公斤，大孔树脂的体积是1L；如果药材干重1g，则大孔树脂的体积为1ml)，待浓缩后的上清液完全流入树脂柱后，先用4倍柱体积(即4L)的水洗涤，弃去洗脱液；接着用8倍柱体积(即8L)的质量百分比浓度为3％的乙醇溶液洗脱，弃去洗脱液；然后再用8倍柱体积(即8L)的无水乙醇洗脱，收集洗脱液，得连翘-大孔树脂洗脱液；
2-2)将连翘-大孔树脂洗脱液置于旋转蒸发器中进行减压浓缩处理，回收溶剂，浓缩残留物进行干燥，得连翘粗品62g；
3、硅胶柱层析
3-1)称取连翘粗品0.5g，加入适量(2.5ml)水，搅拌溶解，备用；
3-2)采用高效液相色谱仪(日本岛津SCL-10AVP半制备型高效液相色谱仪，LC-8A泵，SPD-20A监测器)对连翘粗品进行层析处理，分离纯化连翘粗品，将溶解后的连翘粗品注入(即上样于)半制备型高效液相色谱仪中，采用甲醇-水溶液为洗脱液进行梯度洗脱，其中：高效液相色谱仪中色谱柱尺寸为Φ22.2×250mm，C18反相硅胶填料为，粒径为10um，上样量500mg，流动相为甲醇-水溶液，梯度度洗脱条件为，0～25min，甲醇30％～50％；25～50min，甲醇50％～50％，流速4ml/min，柱温为20℃，紫外检测波长为273nm，分别收集保留时间在25.5～27.5min，30.5～32.5min，35.5～37.5min的流份；
3-3)将收集的3个流份分别进行减压浓缩，真空干燥，分别得化合物I(70.5mg)，化合物II(53.2mg)，化合物III(46.6mg)。
采用HPLC法检查制得的化合物I、II、III的含量，HPLC检测条件为：仪器：Water 515泵，2487检测器；色谱柱：Kromasil RP-C18；流动相：乙腈：0.1％磷酸水溶液(13∶87)；检测波长：230nm；流速：1.0ml/min。
经HPLC检测化合物I为99.6％、化合物II的纯度为98.1％、化合物III的纯度为98.3％。
化合物I为白色固体，熔点：111℃；溶于水、乙醇。在TLC板上展开(层析液为氯仿/甲醇3∶1，Rf为0.25)，喷雾10％H2SO4-乙醇试剂呈现紫红色。
ESI-MS：m/z 533.1658[M-H]-，分子量为：534。
核磁共振氢谱(400MHz，d6-DMSO)：δ(ppm)：12.0(1H，s，COOH)，7.119-7.099(1H，d，J＝8.0Hz，Ar-H)，6.530-6.943(2H，d，J＝4.0Hz，Ar-H)，6.872(3H，s，Ar-H)，5.39(2H，s，J＝4.8Hz)，5.23(1H，d，J＝4.8Hz)，5.1(1H，d，J＝4.8Hz)，4.800(1H，d，J＝4.8Hz)，4.374-4.388(1H，d，J＝9.6Hz)，4.105-4.085(1H，d，J＝8.0Hz)，4.005-3.982(1H，d，J＝9.2Hz)，3.75(8H，d，J＝8.4Hz)，3.422(1H，t，J＝8.7Hz)，3.08(1H，t，J＝8.1Hz)，2.85(1H，d，J＝7.2Hz)；
核磁共振碳谱(100MHz，d6-DMSO)：δ(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)。
根据ESI-MS、1H-NMR和13C-NMR的测试数据，确定化合物I为33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷，英文名：33-Hydroxy phillygenin-8-O-β-D-glucuronide，结构式为：
Figure PCTCN2016078688-appb-000005
化合物II为白色固体，熔点：113℃；溶于水、乙醇。在TLC板上展开(层析液为氯仿/甲醇3∶1，Rf为0.32)，喷雾10％H2SO4-乙醇试剂呈现紫红色。
ESI-MS m/z 533.1641[M-H]-，分子量为：534。
核磁共振氢谱(400MHz，d6-DMSO)：δ(ppm)：12.0(1H，s，COOH)，7.119-7.099(1H，d，J＝8.0Hz，Ar-H)，6.530-6.943(2H，d，J＝4.0Hz，Ar-H)，6.872(3H， s，Ar-H)，5.39(2H，s，J＝4.8Hz)，5.23(1H，d，J＝4.8Hz)，5.1(1H，d，J＝4.8Hz)，4.800(1H，d，J＝4.8Hz)，4.374-4.388(1H，d，J＝9.6Hz)，4.105-4.085(1H，d，J＝8.0Hz)，4.005-3.982(1H，d，J＝9.2Hz)，3.75(8H，d，J＝8.4Hz)，3.422(1H，t，J＝8.7Hz)，3.08(1H，t，J＝8.1Hz)，2.85(1H，d，J＝7.2Hz)；
核磁共振碳谱(100MHz，d6-DMSO)：δ(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)。
根据ESI-MS、1H-NMR和13C-NMR的测试数据，确定化合物II为9-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷，英文名：9-Hydroxy phillygenin-8-O-β-D-glucuronide，
结构式为：
Figure PCTCN2016078688-appb-000006
化合物III为白色固体，熔点：119℃；溶于水、乙醇。在TLC板上展开(层析液为氯仿/甲醇3∶1，Rf为0.36)，喷雾10％H2SO4-乙醇试剂呈现紫红色。
ESI-MS m/z 531.4933[M-H]-，分子量为：532。
核磁共振氢谱(400MHz，d6-DMSO)：δ(ppm)：12.0(1H，s，COOH)，7.119-7.099(1H，d，J＝8.0Hz，Ar-H)，6.530-6.943(2H，d，J＝4.0Hz，Ar-H)，6.872(3H，s，Ar-H)，6.12(2H，s)，5.39(2H，s，J＝4.8Hz)，5.23(1H，d，J＝4.8Hz)，5.1(1H，d， J＝4.8Hz)，4.800(1H，d，J＝4.8Hz)，4.374-4.388(1H，d，J＝9.6Hz)，4.105-4.085(1H，d，J＝8.0Hz)，4.005-3.982(1H，d，J＝9.2Hz)，3.75(8H，d，J＝8.4Hz)，3.422(1H，t，J＝8.7Hz)，3.08(1H，t，J＝8.1Hz)，2.85(1H，d，J＝7.2Hz)；
核磁共振碳谱(100MHz，d6-DMSO)：δ(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)。
根据ESI-MS、1H-NMR和13C-NMR的测试数据，确定化合物III为33，34-亚甲基二氧-连翘脂素-8-O-β-D-葡萄糖醛酸苷，英文名：33，34-Methylenedioxy phillygenin-8-O-β-D-glucuronide，
结构式为：
Figure PCTCN2016078688-appb-000007
实施例2
1、煎煮处理
1-1)连翘叶粉碎并过20目筛，得连翘叶粉，接着向连翘叶(1kg)中加入水(9kg)，混合均匀后加热，进行第一次煎煮处理，加入的水与连翘叶的重量之比为9∶1，加热沸腾，煎煮提取2.5h后进行过滤，获得第一提取液，第一药渣；
1-2)向第一药渣中加入水(8kg)，加热沸腾，进行第二次煎煮处理，加入的水 与连翘叶的重量之比为8∶1，煎煮提取时间1h后进行过滤，获得第二提取液，药渣(弃去)；
1-3)合并第一、第二提取液，制得连翘水提液；
1-4)将连翘水提液置于减压旋转蒸发器中进行减压浓缩处理，回收溶剂，获得连翘浓缩液(2.5L)，连翘叶的重量与连翘浓缩液的体积之比为1∶2.5，备用。
2、大孔树脂柱层析
2-1)将连翘浓缩液上样于大孔树脂柱上，进行大孔树脂柱分离处理，其中，大孔吸附树脂选择X-5型大孔吸附树脂，大孔吸附树脂柱内大孔吸附树脂的柱体积为1L(层析柱的直径60mm，高500mm，内装树脂高度为354mm)，树脂柱内树脂的体积与连翘叶重量(干重)之比为1∶1(即如果连翘叶干重1公斤，大孔树脂的体积是1L；如果药材干重1g，则大孔树脂的体积为1ml)，待浓缩后的上清液完全流入树脂柱后，先用8倍柱体积(即8L)的去离子水洗涤，弃去洗脱液；接着用4倍柱体积(即4L)的质量百分比浓度为3％的乙醇溶液洗脱，弃去洗脱液；然后再用8倍柱体积(即8L)的无水乙醇洗脱，收集洗脱液，得连翘-大孔树脂洗脱液；
2-2)将连翘-大孔树脂洗脱液置于旋转蒸发器中进行减压浓缩处理，回收溶剂，浓缩残留物进行干燥，得连翘粗品58g；
3、硅胶柱层析
3-1)称取连翘粗品0.8g，加入适量(1.6ml)水，搅拌溶解，备用；
3-2)采用高效液相色谱仪(日本岛津SCL-10AVP半制备型高效液相色谱仪，LC-8A泵，SPD-20A监测器)对连翘粗品进行层析处理，分离纯化连翘粗品，将溶解后的连翘粗品注入(即上样于)半制备型高效液相色谱仪中，采用甲醇-水溶液为洗脱液进行梯度洗脱，其中：高效液相色谱仪中色谱柱尺寸为Φ22.2×250mm，C18反相硅胶填料为，粒径为10um，上样量800mg，流动相为甲醇-水溶液，梯度度洗脱条件为，0～25min，甲醇30％～50％；25～50min，甲醇50％～50％，流速4ml/min，柱温为20℃，紫外检测波长为273nm，分别收集保留时间在25.5～27.5min，30.5～32.5min，35.5～37.5min的流份；
3-3)将收集的3个流份分别进行减压浓缩，真空干燥，分别得化合物A(104.2mg)，化合物B(74.3mg)，化合物C(58.1mg)。
采用HPLC法检查制得的化合物A、B、C的含量，HPLC检测条件为：仪器： Water 515泵，2487检测器；色谱柱：Kromasil RP-C18；流动相：乙腈：0.1％磷酸水溶液(13∶87)；检测波长：230nm；流速：1.0ml/min。
经HPLC检测化合物A为99.3％、化合物B的纯度为98.4％、化合物C的纯度为98.5％。
实施例2制备的化合物A、B、C的理化特性、质谱、核磁共振数据分别与实施例1制备的化合物I、II、III相同。
试验例1  体外抗病毒试验
1.1试验材料
(1)药物
①受试药物
本发明实施例1制备的连翘脂素葡萄糖醛酸苷类衍生物(即化合物I、II、III)；
②阳性对照药
利巴韦林注射液，无色透明液体，由河南润弘股份有限公司生产，产品批号：1206261，国药准字：H19993553，100mg/ml，作为本次试验阳性对照药物；
磷酸奥司他韦，中国药品生物制品检定所，产品批号：101096-200901，100mg/支作为本次试验阳性对照药物；
连翘脂素，白色粉末，大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器紫外检测器和蒸发光散射检测器面积归一化法测定，其纯度为99.2％。
上述药品均用纯净水溶解，滤过，除菌分装，4℃备用，为本次试验待测药物。
(2)细胞株
Vero(非洲绿猴肾细胞细胞)由吉林大学基础医学院保存细胞株。
(3)病毒株
①流感病毒株、副流感病毒株、呼吸道合胞病毒(RSV)株：购于中国预防医学科学院病毒研究所；②柯萨奇病毒B3(CVB3)株：购自中科院武汉病毒所；③柯萨奇病毒A16(CoxA16)株、肠道病毒EV71株：购自日本仙台国立医院；④腺病毒(AdV)：购于白求恩医科大学一院儿科研究室；⑤单纯疱疹病毒I型(HSV-1)：购于中国药品生物制品检定所。
(4)主要设备与试剂
生物安全柜：BHC-1300 IIA/B3，AIRTECH；CO2培养箱：MCO-18AIC，SANYO；倒置显微镜：CKX41，OLYMPUS；电子分析天平：AR1140/C，DHAUS；培养基：DMEM，HyClone；胎牛血清：HyClone；胰蛋白酶：Gibco；MTT：Sigma；DMSO：天津市北联精细化学品开发有限公司。
1.2试验方法
(1)细胞准备
Vero细胞传代培养1-2d，使之成片，界线清晰，立体感及折光度强时，用胰酶消化，待细胞面出现针尖样小孔，吸尽消化液，取数毫升培养液吹散细胞，计数，用培养液(含10％胎牛血清的DMEM)稀释至约5×107个/L后，接种于96孔培养板内，待细胞长成单层。
(2)药物毒性测定
细胞毒性试验：将药物按表1-1所示浓度进行用维持液(含2％胎牛血清的DMEM)稀释，用于细胞毒性测定。
表1-1    药物细胞毒性试验浓度(单位：g/L)
Figure PCTCN2016078688-appb-000008
将表1-1中不同浓度的药品滴加于Vero单层细胞上，每孔0.2ml，每个浓度6个复孔，另设6孔正常对照(不加药物的正常对照组)和6孔空白对照(培养液)，置37℃，5％CO2培养箱中培养，每日置倒置显微镜观察CPE(cytopathic effect，在体外实验中，通过细胞培养和接种杀细胞性病毒，经过一定时间后，可用显微镜观察到细胞变圆，坏死，从瓶壁脱落等现象，称之细胞病变作用。指病毒对组织培养细胞侵染后产生的细胞变性。利用此种病变效应可进行病毒定量。)并记录。72h后，每孔加入MTT(3-(4，5-dimethyl-2-thiazolyl)-2，5-diphenyl-2-H-tetrazolium bromide，汉语化学名为3-(4，5-二甲基噻唑-2)-2，5-二苯基四氮唑溴盐，商品名：噻唑蓝。 是一种黄颜色的染料。)溶液20μL(5mg·mL-1)，继续孵育4h，吸出各孔培养液，每孔加入100μL DMSO，振荡5min，492nm测定OD值，计算细胞存活率。在SPSS18.0统计软件中，将细胞存活率进行Probit回归分析，计算药物对Vero细胞的最大无毒浓度(TC0)和半数毒性浓度(TC50)。
(3)各种病毒TCID50的测定
将各种病毒进行10倍梯度递减稀释为10-1，10-2，10-3，10-4，10-5，10-6不同稀释度，按序接种于单层的Vero细胞96孔培养板上，每孔100μL，每个稀释度6孔，同时设正常细胞对照组。置37℃，5％CO2中孵育2h，弃病毒液，随即每孔加细胞维持液100μL，置37℃，5％CO2中培养。第3天开始在显微镜下观察细胞病变结果，第7-8天判定结果并做好记录，以能使50％细胞孔发生阳性病变的最高稀释度作为终点，用karber法计算病毒滴度。
公式：
Figure PCTCN2016078688-appb-000009
TCID50：50％组织细胞感染量
XM：病毒最高浓度稀释度的对数
d：稀释度系数(倍数)的对数
∑pi：每个稀释度病变百分数的总和
(4)药物对病毒致细胞病变作用的影响
取已长满单层细胞的培养板，吸出并弃去培养液，以100TCID50对应的病毒攻击量接种细胞，37℃，5％CO2培养箱吸附2h，加入特定浓度(最大无毒浓度左右)的各药液，每浓度6个复孔培养，200μL/孔。设利巴韦林注射液和磷酸奥司他韦为阳性药物对照组，同时设正常对照组(不加病毒不加药)和病毒对照组(加病毒但不加药物的对照组)，观察药物对病毒致CPE的影响。72h后，用MTT比色法，在492nm波长下测定OD值，计算药物抗病毒有效率(ER％)。在SPSS 18.0统计软件中用ANOVA法比较各药物抗病毒有效率之间的显著性差异。
ER％＝(药物处理组平均OD值-病毒对照组平均OD值)/(细胞对照组平均OD值-病毒对照组平均OD值)×100％
1.3试验结果
(1)各种病毒的TCID50
Figure PCTCN2016078688-appb-000010
(2)药物毒性测定结果
1)药物对细胞毒性的测定
各药物对Vero细胞的最大无毒浓度(TC0)、半数毒性浓度(TC50)见表1-2。
表1-2    药物细胞毒性实验结果(单位：g/L)
Figure PCTCN2016078688-appb-000011
2)药物对病毒致细胞病变保护作用结果
药物抗各种病毒的有效率及ANOVA法单因素方差分析结果，详见表1-3。
表1-3    药物抗病毒有效率(ER％)统计表
Figure PCTCN2016078688-appb-000012
注：与病毒对照组相比，*P＜0.05，**P＜0.01；与连翘脂素比，#P＜0.05，##P＜0.01。
表1-3结果显示，33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷(化合物I)，9-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷苷(化合物II)和33，34-亚甲基二氧-连翘脂素-8-O-β-D-葡萄糖醛酸苷糖醛酸苷醛酸苷(化合物III)对流感病毒、副流感病毒、单纯疱疹病毒I型(HSV-I)、肠道病毒EV71的抑制率及有效率均超过90％，与病毒对照组相比差异明显，具有统计学意义。33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷，9-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷苷和33，34-亚甲基二氧-连翘脂素-8-O-β-D-葡萄糖醛酸苷糖醛酸苷醛酸苷对多种病毒的抗病毒疗效表现均优于连翘脂素、利巴韦林和磷酸奥司他韦的优势。
试验例2  体内抗病毒试验
2.1实验材料
(1)实验动物
昆明小鼠，体重18～22g，雌雄各半性，购自大连医科大学实验动物中心，质量合格证号：SCXK(13)2012-0003。
(2)药物
①本发明实施例1制备的化合物I，即33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷；
②利巴韦林注射液，无色透明液体，由河南润弘股份有限公司生产，产品批号：1206261，国药准字：H19993553，100mg/ml，作为本次试验阳性对照药物；
③磷酸奥司他韦，中国药品生物制品检定所，产品批号：101096-200901，100mg/支，作为本次试验阳性对照药物；
④连翘脂素，白色粉末，大连富生天然药物开发有限公司生产，经高效液相色谱两种检测器(紫外检测器和蒸发光散射检测器)面积归一化法测定，其纯度为99.2％。
上述药品均用纯净水溶解，滤过，除菌分装，4℃备用，为本次试验待测药物。
(2)检测仪器、试剂
Figure PCTCN2016078688-appb-000013
Figure PCTCN2016078688-appb-000014
2.2实验方法
(1)流感病毒和副流感病毒对小鼠半数致死量的测定
将流感病毒和副流感病毒(细胞裂解液)10倍递比稀释为10-1、10-2、10-3、10-4、10-5浓度的病毒液。取昆明种小鼠120只，流感病毒和副流感病毒组各60只，分别随机分成6组，乙醚轻度麻醉小鼠，滴鼻感染不同稀释度病毒液0.03mL/只。同时设空白对照，用生理盐水代替病毒悬液。以死亡和生存为观察指标，每天观察，直至感染后的14天。感染24h内死亡的为非特异死亡，不予统计，Karber法计算病毒液LD50。计算公式：
Figure PCTCN2016078688-appb-000015
[其中：LD50：半数致死量；XM：病毒最高浓度稀释度的对数；d：稀释度系数(倍数)的对数；∑pi：每个稀释度病变百分数的总和]。
(2)33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷抗流感病毒和副流感病毒感染所致肺炎的研究
1)试验动物及分组
取四周龄的昆明小鼠960只，进行2项试验。取小鼠480只，随机分成48组，每组10只，用于33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷对流感病毒感染小鼠肺指数和肺指数抑制率的测定试验，3次重复试验，每次取小鼠80只。另取小鼠480只，随机分成48组，每组10只，用于33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷对肺悬液病毒血凝滴度的测定试验，3次重复试验，每次取小鼠80只。
2)感染方法
在200～300mL大小的烧杯内放入一团脱脂棉，然后倒入适量的乙醚(使脱脂棉变湿即可)，把装有脱脂棉的烧杯倒扣过来，把小鼠放入进行麻醉，见小鼠极度兴奋，明显呈无力样时，将小鼠仰卧，滴鼻感染流感病毒和副流感病毒0.03ml/鼻孔，正常对照组用生理盐水代替病毒悬液。
3)给药方法及给药剂量
33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷组、利巴韦林和磷酸奥司他韦对照组，分别于感染前一天开始常规灌胃给药，33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷高、中、低给药剂量分别为10.0、5.0、2.5mg/kg，利巴韦林阳性药给药剂量为58.5mg/kg，磷酸奥司他韦阳性药给药剂量为19.5mg/kg，连翘脂素组给药剂量为13.0mg/kg，每天一次，连续给药5d，病毒对照组灌服相同体积的生理盐水。
4)观察指标
①肺指数测定
小鼠用药后第5天，先禁食水8小时，称体重后摘眼球放血处死动物，打开胸腔摘出全肺，以生理盐水洗涤两次，用滤纸吸干表面水份，电子天平称肺重，按下列公式计算计算肺指数和肺指数抑制率：
肺指数＝(小鼠肺重/小鼠体重)×100％；肺指数抑制率＝(感染模型组平均肺指数-实验组平均肺指数)/感染模型组平均肺指数×100％。
②肺悬液病毒血凝滴度测定
分别取治疗后第5天的各组小鼠肺，低温下置匀浆器研磨成匀浆，生理盐水稀释为10％的肺组织悬液，离心取上清，倍比稀释，按0.2ml/孔滴于滴定板上，每孔加入0.2ml 1％鸡红细胞悬液，混匀，置室温30min，观察记录血凝滴度。以红细胞凝集(++)时为终点，以悬液稀释倍数表示其滴度。
2.3实验结果及分析
(1)流感病毒和副流感病毒对小鼠半数致死量的测定结果
实验组昆明种小鼠分别被滴鼻感染不同浓度流感病毒、副流感病毒液30μL，感染第3天前3组(病毒浓度为10-1组、10-2组、10-3组)小鼠均出现不同程度的发病症状：耸毛、发抖、饮食减少等；第5天小鼠出现走路摇摆不定；第6天最高病毒 浓度组小鼠开始出现死亡，其余各组于感染后第7天陆续出现死亡现象。观察14天结束后，统计各组小鼠死亡数目，结果见下表1-4、1-5。计算该流感病毒的LD50为稀释度10-2.9，副流感病毒的LD50为稀释度10-2.5。
表1-4    流感病毒半数致死量试验结果统计
Figure PCTCN2016078688-appb-000016
Karber法计算病毒的LD50。流感病毒的LogLD50如下：
Figure PCTCN2016078688-appb-000017
表1-5    副流感病毒半数致死量试验结果统计
Figure PCTCN2016078688-appb-000018
Karber法计算病毒的LD50。副流感病毒的LogLD50如下：
Figure PCTCN2016078688-appb-000019
(2)33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷抗流感病毒和副流感病毒感染所致肺炎的作用结果
①肺指数测定
流感病毒和副流感病毒感染小鼠后，平均肺指数测定结果显示：与感染模型组比较，33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷浓度在2.25～10.0mg/kg/d范围内有 一定保护作用，肺指数均明显降低；33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷高剂量组对流感病毒和副流感病毒的疗效优于连翘脂素组(P＜0.05)。试验结果见表1-6、1-7。
表1-6  化合物I对流感病毒感染小鼠肺指数和肺指数的抑制率(n＝3)
Figure PCTCN2016078688-appb-000020
与病毒对照组比较，*P＜0.05，**P0.01；与连翘脂素组比较，#P＜0.05，##P＜0.01。
表1-7  化合物I对副流感病毒感染小鼠肺指数和肺指数抑制率的影(n＝3)
Figure PCTCN2016078688-appb-000021
与病毒对照组比较，*P＜0.05，**P0.01；与连翘脂素组比较，#P＜0.05，##P＜0.01。
②肺悬液病毒血凝滴度测定
流感病毒和副流感病毒感染小鼠后，感染模型组肺组织病毒血凝滴度(InX)分别为31.64和32.06，不同浓度33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷治疗5天后，肺组织病毒血凝滴度均有所下降，与感染模型组比较，差异有显著性，(P＜0.01)； 其中，33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷中、高剂量组对流感、副流感病毒血凝滴度均明显低于模型组，抑制率均高于连翘脂素组，差异显著(P＜0.05，p＜0.01)。试验结果见表1-8、1-9。
表1-8  化合物I对流感病毒感染小鼠肺悬液血凝滴度的影响(n＝3)
Figure PCTCN2016078688-appb-000022
与病毒对照组比较，*P＜0.05，**P＜0.01；与连翘脂素组比较，#P＜0.05，##P＜0.01。
表1-9  化合物I对副流感病毒感染小鼠肺悬液血凝滴度的影响(n＝3)
Figure PCTCN2016078688-appb-000023
与病毒对照组比较，*P＜0.05，**P＜0.01；与连翘脂素组比较，#P＜0.05，##P＜0.01。
2.4结论
体内抗病毒试验结果显示，33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷在2.25～10mg/kg/d的剂量范围对流感病毒和副流感病毒及所引起的小鼠病毒性肺炎具有较明显的抑制作用，能明显降低其肺指数和血凝滴度，肺组织病理学也有明显改善， 与病毒模型对照组比较差异显著；且33-羟基-连翘脂素-8-O-β-D-葡萄糖醛酸苷中、高剂量组疗效明显优于连翘脂素(*P＜0.05或**P＜0.01)，同时表现出优于利巴韦林和磷酸奥司他韦的趋势。
化合物II、III与化合物I相同，均具有对流感病毒和副流感病毒及所引起的小鼠病毒性肺炎具有较明显的抑制作用，能明显降低其肺指数和血凝滴度，肺组织病理学也有明显改善，与病毒模型对照组比较差异显著。

Claims (10)

  1. 一种连翘脂素葡萄糖醛酸衍生物，结构通式如式(Ⅰ)：
    Figure PCTCN2016078688-appb-100001
    其中：R1＝H，R2＝CnH2n+1，R3＝CnH2n+1或R1＝CnH2n+1，R2＝CnH2n+1，R3＝H或R1-R2＝-CH2-，R3＝CnH2n+1；n＝1～30。
  2. 如权利要求1所述的衍生物，其特征是所述n＝1。
  3. 一种连翘脂素葡萄糖醛酸衍生物的制备方法，其特征是，包括如下顺序进行的步骤：
    1)将连翘叶与提取溶剂水混合后加热，进行煎煮提取2-3次，收集、合并提取液，获得连翘水提液；
    2)将连翘水提液采用大孔树脂柱进行分离处理，收集、合并洗脱液，得到连翘树脂柱洗脱液；
    3)对连翘树脂柱洗脱液进行硅胶柱层析处理，分段收集洗脱液，洗脱液分别干燥，即得。
  4. 如权利要求3所述的制备方法，其特征是步骤1)中每次煎煮过程中，所述连翘叶与提取溶剂水的重量配比为1：6-10。
  5. 如权利要求3或4所述的制备方法，其特征是还包括将步骤1)获得的连翘水提液进行浓缩处理，制成连翘浓缩液后再进行所述的大孔树脂柱分离处理。
  6. 如权利要求5所述的制备方法，其特征是浓缩处理后所制成的连翘浓缩液的体积与连翘叶的重量之比为1-5：1。
  7. 如权利要求3或4所述的制备方法，其特征是步骤3)中所述硅胶柱层析过程中选择C18的反相硅胶填料；色谱柱规格：内径10～100mm、长度10～300mm；填料粒径为5～10μm；流动相以等度或梯度洗脱的方式洗脱。
  8. 如权利要求7所述的制备方法，其特征是所述硅胶柱层析过程中流动相为甲醇与水的混合液，其中，甲醇与水的体积比为8：2～10：1。
  9. 如权利要求1所述连翘脂素葡萄糖醛酸衍生物的抗病毒应用。
  10. 如权利要求1所述连翘脂素葡萄糖醛酸衍生物在制备预防或/和治疗流感病毒药物中的用途。
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AU2016245659A1 (en) 2017-11-23
CN104945452B (zh) 2017-12-05
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CA2982200C (en) 2019-08-20
KR102104234B1 (ko) 2020-04-24
RU2684100C1 (ru) 2019-04-04
US10407455B2 (en) 2019-09-10
EP3281945B1 (en) 2022-08-17
JP6505250B2 (ja) 2019-04-24
CN104945452A (zh) 2015-09-30
AU2016245659B2 (en) 2019-02-21
EP3281945A4 (en) 2019-01-23
US20180057523A1 (en) 2018-03-01
CA2982200A1 (en) 2016-10-13
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