CN1969923A - Antivirus extract of isatis root, its extraction method, use and content assaying method - Google Patents
Antivirus extract of isatis root, its extraction method, use and content assaying method Download PDFInfo
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Abstract
The invention discloses an extracting method of antiviral active material based on Cruciferae wade root and leaf as raw material, which is characterized by the following: the extract is adsorbed by large-hole resin, which possesses clemastanin B and active compound with general formula (I); the extract can make antiviral oral or non-oral agent with fitful excipient, which utilizes HPLC method to test content.
Description
One, technical field
The present invention relates to the Chinese medicine technical field, exactly it to relate to a kind of be the content assaying method that raw material extracts antiviral activity composition the extracting method of the extract with antivirus action and the Radix Isatidis medical material from crucifer pine blue root (Radix Isatidis), leaf.
Two, background technology
Dry root---" Radix Isatidis " of cruciferae isatis Isatis indigotica Fort., heat-clearing and toxic substances removing is arranged, removing heat from blood sore-throat relieving function, be usually used in viral disease and bacterial infective diseases clinically in the modern times, especially at the anti-virus aspect determined curative effect, multiple diseases such as the influenza that numerous bibliographical information Radix Isatidis cause for virus, hepatitis B, herpes simplex, viral myocarditis, hemorrahgic fever with renal syndrome all have treatment or preventive effect (Gao Xiuzhi preferably clinically.Radix Isatidis pharmacology and clinical practice overview. Chinese medicine research, 2001,17 (12): 57-58).Radix Isatidis is a broad-spectrum natural antiviral drug comparatively.
The external in recent years concern to the Isatis indigotica Fort. plant is that on the anti-tumor activity of indoles alkaloid, the research of antiviral ingredients does not then appear in the newspapers.Big quantity research has been done in domestic composition and pharmacology aspect to Radix Isatidis, The Chemical Constituents mostly is the alcohol extraction position, the composition that has separated and identified reaches (Liu Yunhai more than 60 kinds, the state of Qin is big, the fourth level, Deng. Radix Isatidis chemical constitution study (I) [J]. Chinese herbal medicine, 2001,32 (12): 1057-1060), but antiviral active component of Radix Isatidis and material base are not illustrated yet, applied clinically at present Radix Isatidis preparation mostly is the indefinite plant crude extract of composition, and curative effect and quality all are difficult to be guaranteed.
2005 editions " in the Chinese pharmacopoeia, Radix Isatidis and single medicinal material preparation thereof still do not have the assay item, quality Control during Radix Isatidis medical material and preparation are produced also anxious to be solved (Chinese Pharmacopoeia Commission. Chinese Pharmacopoeia (version was an one in 2005) [M]. Chemical Industry Press, 2005:142).
Three, summary of the invention
The object of the present invention is to provide the antiviral extract of Radix Isatidis is effective site and active component and extraction, isolation and purification method and purposes, and foundation is the Radix Isatidis medical material of index and the content assaying method of preparation with the active component.
The invention discloses a kind of root with cruciferae isatis (Radix Isatidis), leaf is the antivirus extract that raw material extracts, contain reactive compound lariciresinol-4 in this extract, 4 '-two-O-β-D-pyranglucoside and a reactive compound with general formula (I) structure.Antiviral activity composition lariciresinol-4,4 '-two-O-β-D-pyranglucoside can utilize the method for HPLC to carry out assay, and this assay method can be used as the standard of medical material and quality of the pharmaceutical preparations control.
Radix Isatidis extract disclosed in this invention, can develop becomes definite ingredients, the sure new antiviral drug of curative effect.
A kind of Radix Isatidis extract with antivirus action, it is characterized in that this extract is that root with cruciferae isatis is that Radix Isatidis, leaf are that raw material was extracted and can be the antiviral effective site of Radix Isatidis by the mixture of the adsorbed chemical constituent of macroporous resin; Contain reactive compound lariciresinol-4 in this extract, 4 '-two-O-β-D-pyranglucoside (clemastanin B) and a reactive compound with following general formula (I) structure:
Wherein: R1 is hydrogen aryl or the heteroaryl that contains 4～6 carbon atoms, and hetero atom wherein is selected from N, O, S; R1 also comprises by 1 or 2 hydrogen aryl and heteroaryls that following group replaces respectively: CH3, CH3O, CH2OH, COOH, COOCH3, OH; R2 is selected from following group: hydrogen, (CH2) nCH3, (n=2～8), 2～6 interconnective pentoses or hexose base.
The purposes of the extract of above-mentioned Radix Isatidis is the preparation for preparing antiviral drugs as antivirus effective position, and this effective site can separately or add an amount of excipient, makes the medicine of peroral dosage form or non-peroral dosage form with conventional preparation method; Comprise and add to this extract in the Pharmaceutical composition or the compound preparation of this extract participation prescription; Simultaneously comprise that also this extract prepares the purposes of health product, health food aspect.
Above-mentioned Radix Isatidis extract with antivirus action, its extraction step is as follows:
(1) Radix Isatidis medical material hot water extraction, water extract gets the water extract-alcohol precipitation supernatant through concentrating with ethanol precipitation;
(2) with supernatant concentration after macroporous resin adsorption, with the alcohol flushing resin post, collect eluent;
(3) antivirus effective position will be got after eluent concentrating under reduced pressure, the drying.
The Radix Isatidis medical material of above-mentioned steps (1) is dry decoction pieces or powder, 60～100 ℃ of water extraction doubly measuring with medical material amount 4-20 1～4 time, each 0.5h～2h concentrates behind the merge extractive liquid,, adding 95% ethanol to ethanol final concentration in extracting solution is 30～80%, leave standstill filter supernatant.
With supernatant sucking filtration or centrifugal, concentrate behind the filtrate recycling ethanol in the above-mentioned steps (2),, collect ethanol elution with the alcohol flushing resin post of 10-90% through macroporous resin adsorption.
The chemical constituent lariciresinol-4 that contains in the above-mentioned antivirus extract, 4 '-two-O-β-D-pyranglucoside can utilize the method for HPLC to carry out assay.The mobile phase of measuring is selected a kind of or a certain proportion of several mixing in water, methanol, the acetonitrile isochromatic spectrum reagent for use; The ultraviolet detection wavelength is between 200nm～400nm.It is to be the pharmaceutical preparation of primary raw material or the quality control standard of compound medicinal formulation with these medical materials in the medical material in source and the prescription that this assay method can be used for the Isatis indigotica Fort. platymiscium.
Contain chemical constituent lariciresinol-4 in the above-mentioned antivirus extract, 4 '-two-O-β-D-pyranglucoside, its extracting method is: get Radix Isatidis medicinal material drying decoction pieces or powder, with 10～95% alcohol-water solution reflux, extract,, the extracting solution concentrating under reduced pressure is got extractum; Extractum is soluble in water under the heating in water bath situation, by macroporous resin, collects the ethanol elution part, concentrating under reduced pressure, and drying gets crude extract; This crude extract separates through silica gel column chromatography, with lariciresinol-4,4 '-two-O-β-pure product of D-pyranglucoside product TLC are in contrast followed the tracks of the inspection knowledge, there is stream part of same blob to merge with reference substance, after reclaiming solvent, through solvent recrystallization repeatedly, obtain lariciresinol-4,4 '-two-O-β-D-pyranglucoside.Heavy in the optional water of brilliant solvent, methanol, ethanol, propanol, butanols, amylalcohol, acetone, butanone, ethyl acetate one or more.
Include the reactive compound of general formula (I) structure in the above-mentioned antivirus extract, its extracting method is: Radix Isatidis medicinal material drying decoction pieces or powder with 10～95% alcohol-water solution reflux, extract,, get extractum with the extracting solution concentrating under reduced pressure; Extractum is soluble in water under the heating in water bath situation, by macroporous resin, collect the ethanol elution part, concentrating under reduced pressure, dry, obtain crude extract, this crude extract separates through silica gel column chromatography, collects to have the flow point merging that contains this chemical compound, after reclaiming solvent, through solvent recrystallization repeatedly, heavy in the optional water of brilliant solvent, methanol, ethanol, propanol, butanols, amylalcohol, acetone, butanone, ethyl acetate one or more, the chemical compound of (I) structure that obtains having general formula.The chemical compound and the derivant thereof that contain the general formula (I) for the treatment of effective dose are used separately, or with the compositions of other medicines, have the antiviral drugs purposes.
Lariciresinol in the Radix Isatidis medical material-4,4 '-two-O-β-D-pyranglucoside Determination on content method is as follows:
(1) preparation of reference substance solution: precision takes by weighing lariciresinol-4, and 4 '-two-O-β-D-diglucoside reference substance is an amount of, adds methanol and progressively is diluted to the solution that every 1ml contains 0.02mg;
(2) preparation of confession test agent solution: precision takes by weighing the powder 1g that Radix Isatidis is crossed 40 mesh sieves, place the 25ml volumetric flask, add 60% methanol constant volume to 25ml, 60 ℃ of supersound extraction 1h, add 60% methanol again to scale, with the centrifugal 10min of speed of 3500r/min, after supernatant is crossed 0.45 μ m microporous filter membrane, get need testing solution, standby;
(3) chromatograph is measured: accurate respectively above-mentioned reference substance solution and each 10 μ l of need testing solution of drawing, and inject chromatograph of liquid and measure, promptly get the content of measuring.
Radix Isatidis extract is the application of effective site in the preparation antiviral drugs.
Lariciresinol-4,4 '-two-O-β-D-pyranglucoside or the application of reactive compound in the preparation antiviral drugs with general formula (I) structure.
Four, the specific embodiment
The present invention is further illustrated with experimental example by the following examples, but the present invention is not subject to these
Embodiment.
Embodiment 1 antivirus extract of isatis root is the extracting method of effective site
The dry decoction pieces 2kg of Radix Isatidis extracts 2h, 1.5h with alcohol-water solution respectively, filters, and merges extracted twice liquid and is concentrated into 4kg, filters.Under the stirring state, in extracting solution, add 95% ethanol, to ethanol content 60-80%.Standing over night, sucking filtration, filtrate is reclaimed solvent, is concentrated into 2kg.Above-mentioned isatis root extract by macroporous resin adsorption, is used the different concentration ethanol eluting respectively repeatedly, collects ethanol elution, and concentrate drying obtains refining effective site 17g.
Embodiment 2 different places of production Radix Isatidis medical material lariciresinols-4,4 '-two-O-β-D-pyranglucoside assay
2.1 instrument and reagent
Tianjin, island CBM-10A highly effective liquid phase chromatographic system; The SPD-10A UV-detector; CLASS SAILNET Driver work station.
Lariciresinol-4,4 '-two-O-β-D-pyranglucoside reference substance is accredited as lariciresinol-4,4 '-two-O-β-D-diglucoside (clemastanin B) by separating voluntarily in the Radix Isatidis medical material through UV, IR, NMR, MS etc.Verify as single component through HPLC and TLC, it is 99.2% that HPLC peak area normalization method detects purity.
The Radix Isatidis medical material is available from the main place of production such as Jiangsu, Anhui, Hebei, through being accredited as Isatis indigotica Fort. (Isatisindigotica Fort.) dry root.Reagent such as methanol, acetonitrile is chromatographically pure.
2.2 method and result
2.2.1 assay method
The preparation precision of reference substance solution takes by weighing lariciresinol-4, and 4 '-two-O-β-D-diglucoside reference substance is an amount of, adds methanol and progressively is diluted to the solution that every 1ml contains 0.02mg.
Preparation precision for test agent solution takes by weighing Isatis Root (crossing 40 mesh sieves) 1g, place the 25ml volumetric flask, add 60% methanol constant volume to 25ml, 60 ℃ of supersound extraction 1h, add 60% methanol again to scale, after centrifugal (3500r/min) 10min, supernatant cross 0.45 μ m microporous filter membrane, get need testing solution, standby.
Accurate respectively above-mentioned reference substance solution and each the 10 μ l of need testing solution of drawing of algoscopy inject chromatograph of liquid and measure, and promptly get the content of measuring.
2.2.2 linear relationship
Reference substance is good in 5.5 μ g/mL～110.0 μ g/mL scope internal linear relation.
2.2.3 precision experiment
Same reference substance solution continuous sample introduction 7 times, its RSD are 2.89%.
2.2.4 stability test
Same as test agent solution, measured once every 2 hours, the result shows that this sample solution is stable in 12 hours, RSD is 2.87%.
2.2.5 average recovery test
Precision takes by weighing the Jiangsu of known content and produces the about 1g of Isatis Root, and totally 6 parts, every part of adding and medical material content reference substance about equally, the accurate title calmly.By carrying out sample treatment and mensuration under " preparation of need testing solution " item, calculate recovery rate gets lariciresinol-4, and the average recovery rate of 4 '-two-O-β-D-pyranglucoside is 98.80%, and RSD is 0.60%.
2.2.6 replica test
The result shows that this method repeatability is good, and RSD is 2.79%.
2.2.7 main product ground sample determination
Get each place of production sample 1g of Radix Isatidis respectively, the accurate title, decide, and by handling and measure under " preparation of need testing solution " item, the results are shown in Table 1.
Table 1 Radix Isatidis main product ground sample size measurement result (n=3)
| The place of production | Content (mg/g) |
| Anguo Anhui, Hebei, Ding County, Hebei, Shangqiu, Henan Yanbian, Jilin Province Hubei, Haozhou Fang County Lianyungang of Jiangsu | 0.3461 0.4124 0.3656 0.4407 0.6192 0.2138 0.5240 |
Embodiment 3 standard substance lariciresinols-4, the preparation method of 4 '-two-O-β-D-pyranglucoside
3.1 preparation method
Isatis Root 1kg, 80% methanol aqueous solution reflux, extract, 2 times, the extracting solution concentrating under reduced pressure gets extractum 65g.Extractum is dissolved under the heating in water bath situation in the 1000ml water, by macroporous resin, collects ethanol elution partly, and concentrating under reduced pressure, drying get 12g.This sample separates through silica gel column chromatography, with lariciresinol-4,4 '-two-O-β-pure product of D-pyranglucoside product TLC are in contrast followed the tracks of the inspection knowledge, with reference substance part merging of same blob stream is arranged, after reclaiming solvent, through water, twice recrystallization of methanol, get standard substance lariciresinol-4,4 '-two-O-β-D-pyranglucoside 120mg respectively.
3.2 the structural confirmation of standard substance
Press " preparation method " following gained monomer through 13CNMR, 1HNMR analyzes, and spectral data is as follows: 1H-NMR (DMSO-d6), and δ: 6.88 (d, J=1.5, H-2), 7.02 (d, J=8.6, H-5), 6.77 (dd, J=8.6,1.5, H-6), 4.72 (d, J=6.5, H-7), 2.20 (m, H-8), 3.47 (d, J=8.0, H-α), 3.65 (d, J=8.0, H-9), 6.82 (d, J=1.5, H-2 '), 6.97 (d, J=8.6,1.5, H-5 '), (6.68 dd, J=6.5, H-6 '), 2.46 (d, J=11.0, H-7 ' α), 2.84 (dd, J=11.0,3.8, H-7 ' β), 2.61 (m, H-8 '), 3.56 (t, J=7.0, H-9 ' α), 3.90 (t, J=7.0, H-9 ' β), 3.74 (s ,-OCH3), 4.84 (d, J=5.0, β-glu-1 "), 4.86 (d, J=5.5, β-glu-1 ).1C-NMR(DMSO-d6)，δ：137.8(C-1)，110.2(C-2)，149.0(C-3)，145.7(C-4)，115.2(C-5)，118.0(C-6)，81.8(C-7)，52.7(C-8)，58.8(C-9)，134.8(C-1’)，113.2(C-2’)，149.0(C-3’)，145.0(C-4’)，115.5(C-5’)，120.5(C-6’)，32.4(C-7’)，42.0(C-8’)，72.1(C-9’)，55.8(OCH3)，100.4(C-1”)，100.3(C-1)，73.4(C-2”、2)，77.1(C-3’3)，69.9(C-4’、4)，77.2(C-5”、5)，60.9(C-6”、6)。The contrast of above data and document is in full accord, is lariciresinol-4,4 '-two-O-β-D-pyranglucoside (lariciresinol-4,4 '-bis-O-β-D-glucopyranoside), i.e. clemastanin B.
3.3 the purity test of standard substance
The TLC method is checked: precision takes by weighing standard substance weight, adds methanol and makes the solution that every 1ml contains standard substance 10mg, as need testing solution.Precision is drawn need testing solution 4,6,8,10 μ l, puts in same to contain on the silica gel G plate, and be developing solvent with chloroform-methanol (7: 3), n-butyl alcohol-acetic acid-water (4: 1: 5) respectively, launch, develop the color with 10% phosphomolybdic acid ethanol solution solution.
The result shows, prepared lariciresinol-4, and 4 '-two-O-β-D-pyranglucoside standard substance launch under two kinds of different solvents systems, after 10% phosphomolybdic acid ethanol solution heating colour developing, all are single speckle.
Embodiment 4 has the extracting method of the reactive compound I of general formula (I) structure
Isatis Root 1kg, alcohol-water solution extracts 2 times, and the extracting solution concentrating under reduced pressure gets extractum 65g.Extractum is dissolved under the heating in water bath situation in the 1000ml water, by macroporous resin, collects ethanol elution partly, and concentrating under reduced pressure, drying get 12g.This sample separates through silica gel column chromatography, and the pure product of identifying through structure of general formula (I) chemical compound product TLC are in contrast followed the tracks of inspection and known, and has same blob stream part to merge with reference substance, reclaim solvent after, through recrystallizing methanol, general formula (I) chemical compound 20mg.
The extracorporeal antivirus effect pharmacodynamic experiment of embodiment 5 Radix Isatidis antivirus effective positions and noval chemical compound
5.1 material
5.1.1 be subjected to the reagent thing:
(1) separates the effective site that obtains in the example one
(2) has the compound monomer of summary of the invention formula of (I) structure.
(3) positive control drug: acyclovir (ACV), Hubei Qianjiang Pharmaceutical Co., Ltd., lot number: 20040802.
5.1.2 virus
Human simple herpesvirus I type (HSV-1), II type (HSV-2).
5.1.3 cell strain
Vero cell (African green monkey kidney cell) is HSV type virus sensitive cells.
5.2. experimental technique
5.2.1 the toxic action of medicine pair cell
Get the 96 porocyte culture plates that grow up to cell monolayer, abandoning supernatant, the cell culture fluid 0.2ml that will contain the variable concentrations medicine adds cell, 4 multiple holes of each concentration, and 37 ℃ of 5%CO2 cultivate 72h, every hole adds the MTT solution of 20 μ L5mg/ml, continue to cultivate 4h, the supernatant that inclines adds 0.2mL DMSO, 570-630nm double wave regular way is surveyed the O.D value, calculates cell survival rate and half toxic concentration (TC50).With maximal non-toxic concentration is initial concentration, and the several concentration of doubling dilution are carried out the antiviral experiment.
Cell survival rate=(the average O.D value of the medicine group/average O.D value of cell matched group) * 100%
5.2.2 antiviral experiment
If the contrast of blank cell, viral infection contrast, acyclovir (ACV) positive control and be subjected to reagent thing group, totally 4 groups.Get the cell that grows up to monolayer, abandoning supernatant, add 20 μ L 100TCID50 virus liquid, absorption 1h, the virus of not adsorbing with PBS liquid flush away, add the cell culture fluid 0.2mL that contains the variable concentrations medicine, 4 multiple holes of each concentration, 37 ℃ of 5%CO2 cultivate 72h, and every hole adds the MTT solution of 20 μ L 5mg/ml, continue to cultivate 4h, the supernatant that inclines adds 0.2mL DMSO, and 570nm (630nm is as the reference wavelength) double wave regular way is surveyed the O.D value, calculate viral suppression ratio, medicine suppresses the drug level (IC50) of virocyte pathological changes to 50%, and calculates therapeutic index (TI).
Virus suppression ratio=(the average O.D value of the experimental group-average O.D value of virus control group)/(the average O.D value of the cell matched group-average O.D value of virus control group) * 100%
Therapeutic index (TI)=half toxic concentration (TC50)/half-inhibition concentration (IC50)
5.3. experimental result
5.3.1 the toxicity test of medicine pair cell
After measured, the TC50 value of effective site and newization and thing is respectively: 10.00mg/mL and 0.662mg/mL.The results are shown in Table 1.
Table 1 is subjected to reagent thing pair cell toxicity test
| Group | Drug level (mg/mL) | The OD value (x ± s) | Cell survival rate (%) | TC50(mg/mL) |
| The contrast of position administration group cell | 10.000 5.000 2.500 1.250 0.625 | 0.914±0.102 0.840±0.079 1.039±0.258 1.469±0.137 1.448±0.019 1.647±0.079 | 55.49 51.02 63.08 89.19 87.09 | 10.00 |
| The contrast of compound administration group cell | 2.000 1.000 0.500 0.250 0.125 | 0.260±0.044 0.332±0.067 1.198±0.050 1.532±0.080 1.818±0.151 1.695±0.027 | 15.36 19.56 70.64 90.39 107.22 | 0.662 |
5.3.2 antiviral experiment
Radix Isatidis effective site and noval chemical compound demonstrate stronger anti-HSV virus function to virus infected cell.Measure cytoactive and calculate viral suppression ratio through mtt assay and be respectively 53.21%, 57.31%, approaching with the positive control drug acyclovir; Therapeutic index (TI) is respectively 8.3,7.1.
Table 2 Radix Isatidis effective site and noval chemical compound are to the inhibitory action of HSV virus
| Group | Drug level (mg/mL) | The OD value (x ± s) | Virus suppression ratio (%) | ||
| HSV-1 | HSV-2 | HSV-1 | HSV-2 | ||
| Effective site positive controls infection group and viral infection group cell matched group | 4.800 2.400 1.200 0.600 0.300 0.150 0.200 | 0.436±0.347 0.788±0.068 1.100±0.098 0.883±0.050 0.853±0.059 0.733±0.087 1.186±0.037 0.611±0.023 1.531±0.055 | 0.299±0.051 1.025±0.049 0.881±0.049 0.885±0.062 0.819±0.073 0.762±0.061 0.972±0.050 0.634±0.006 1.555±0.122 | -19.01 19.30 53.21 29.55 26.37 13.26 62.55 | -36.44 42.45 26.77 27.20 20.10 13.91 36.73 |
| Noval chemical compound positive controls infection group and viral infection group cell matched group | 0.500 0.250 0.125 0.063 0.031 0.016 0.200 | 0.479±0.074 0.792±0.262 0.647±0.129 0.474±0.081 0.536±0.026 0.759±0.134 1.014±0.067 0.506±0.015 1.427±0.019 | 0.422±0.065 0.835±0.367 1.023±0.343 0.861±0.333 0.868±0.392 0.944±0.367 1.137±0.011 0.482±0.022 1.426±0.022 | -2.93 31.11 15.34 -3.47 3.26 27.50 55.14 | -6.43 37.35 57.31 40.14 40.92 48.94 69.36 |
The interior resisting virus pharmacodynamic experiment of embodiment 6 Radix Isatidis antivirus effective positions
6.4.1 be subjected to the reagent thing
(1) separates the effective site that obtains in the example one
(2) positive control drug, ribavirin tablet, Meidakang Pharmaceutical Co., Ltd., Sichuan Prov., lot number: 051101
6.4.2 experiment is with viral
Influenza A virus A/PR8/34 (H1H1) is provided by Chinese preventive medicine zoo virus institute.
6.4.3 experimental technique and result
(1) influenza A virus increases poison through Embryo Gallus domesticus, is more than 640 through blood clotting result of the test blood clotting titre, can be used for infection animal in the body.
(2) virus infected mice half death (LD50) titration after measured, LD50 is 10-1.71 as a result.
(3) Radix Isatidis effective site is to the protective effect of influenza A virus A/PR8/34 infecting mouse
Get 80 of ICR mices, body weight 18-22g, male and female half and half are divided 5 groups by the following method at random, 16 every group.
I normal control group, equivalent NS
II virus control group, equivalent NS
III ribavirin group, 50mg/kg
IV effective site administration group, 0.15g/kg
V effective site administration group, 0.30g/kg
More than each treated animal all adopt gastric infusion, once a day, successive administration 5 days.Beginning administration next day, except that the normal control group, respectively organize mice under light anaesthesia, with the allantoic fluid of blood clotting titre more than 640 to mice collunarium infective virus, every Mus 50ul, observe zoogenetic infection incidence and mortality, mortality in the record 14d, and the natural law of relatively surviving.The results are shown in Table 3.
Table 3 Radix Isatidis antivirus effective position is to the protective effect of influenza a virus infection mice
| Group | Number of animals (only) | Dosage (g/kg) | The survival natural law |
| Normal group infection group and viral infection group ribavirin group effective site group effective site group | 16 16 16 16 16 | Equivalent NS equivalent NS 0.05 0.10 0.20 | 5.68±3.33 11.06±3.98* 9.13±3.15* 10.50±3.72* |
Experimental result shows that the Radix Isatidis effective part extract can obviously prolong influenza a virus infection mice survival natural law, with the virus model group significant difference is arranged relatively, and curative effect and ribavirin are suitable.
The preparation of embodiment 7 Radix Isatidis antiviral capsules
Get the prepared refining effective site 200g of Radix Isatidis of method among the embodiment 1, add the about 300g of starch, the preparation granule, drying incapsulates, and every dress 0.5g makes about 1000, promptly altogether.
The preparation of embodiment 8 Radix Isatidis antiviral injections
The dry decoction pieces 500g of Radix Isatidis extracts 2h, 1.5h with appropriate solvent respectively, filters, and merges extracted twice liquid and is concentrated into 1kg, filters.Under the stirring state, in extracting solution, add 95% ethanol, to suitable concentration.Standing over night, sucking filtration, filtrate is reclaimed solvent, is concentrated into 500g.Above-mentioned isatis root extract by macroporous resin adsorption, is used the different concentration ethanol eluting respectively repeatedly, collects ethanol elution, merges, be concentrated into 350～500ml, cold preservation filters, and it is 8.0～8.5 that filtrate is regulated pH value with ammonia solution, stir evenly, cold preservation 48 hours adds heat extraction ammonia, add the injection water to 900ml, cold preservation filters, it is 7.0～7.5 that filtrate is regulated pH value with 1% sodium hydroxide solution, and cold preservation filters, filtrate is with mannitol 10g. and add injection water adjustment total amount to 1000ml, filters, promptly.
The preparation of embodiment 9 Radix Isatidis antiviral eye drops
The dry decoction pieces 500g of Radix Isatidis extracts 2h, 1.5h with appropriate solvent respectively, filters, and merges extracted twice liquid and is concentrated into 1kg, filters.Under the stirring state, in extracting solution, add 95% ethanol, to suitable concentration.Standing over night, sucking filtration, filtrate is reclaimed solvent, is concentrated into 500g.Above-mentioned isatis root extract by macroporous resin adsorption, is used the different concentration ethanol eluting respectively repeatedly, collects ethanol elution, merge, be concentrated into 350～500ml, cold preservation, filter, it is 8.0～8.5 that filtrate is regulated pH value with ammonia solution, stirs evenly, cold preservation 48 hours adds heat extraction ammonia, adds the 1g ethyl hydroxybenzoate, adding distil water is to 1000ml, and sterilization is with G4 sintered filter funnel, the unconcerned filtration of 0.22um filter, embedding, promptly.
The preparation of embodiment 10 Radix Isatidis antiviral tablets
Get the prepared refining effective site 200g of Radix Isatidis of method among the embodiment 1, add the about 300g of starch, mixing, 95% ethanol wet method prepares granule, and drying is pressed into 1000, promptly.
The preparation of embodiment 11 Radix Isatidis antiviral granules
Get the prepared refining effective site 200g of Radix Isatidis of method among the embodiment 1, with sucrose 400g, dextrin 260g, mixing, 95% ethanol wet method prepares granule, and drying is made about 1000 bags, promptly.
The preparation of embodiment 12 Radix Isatidis antivirus oral liquids
The dry decoction pieces 500g of Radix Isatidis extracts 2h, 1.5h with appropriate solvent respectively, filters, and merges extracted twice liquid and is concentrated into 1kg, filters.Under the stirring state, in extracting solution, add 95% ethanol, to suitable concentration.Standing over night, sucking filtration, filtrate is reclaimed solvent, is concentrated into 500g.Above-mentioned isatis root extract by macroporous resin adsorption, is used the different concentration ethanol eluting respectively repeatedly, collects ethanol elution, merges, be concentrated into 350～500ml, add correctives (sucrose 400g), antiseptic (sodium benzoate 3g), the dissolving after-filtration, add water and be adjusted to 1000ml, fill, promptly.
Claims (10)
1. Radix Isatidis extract with antivirus action, it is characterized in that this extract is that root with cruciferae isatis is that Radix Isatidis, leaf are that raw material was extracted and can be the antiviral effective site of Radix Isatidis by the mixture of the adsorbed chemical constituent of macroporous resin; Contain reactive compound lariciresinol-4 in this extract, 4 '-two-O-β-D-pyranglucoside (clemastanin B) and a reactive compound with following general formula (I) structure:
Wherein: R1 is hydrogen aryl or the heteroaryl that contains 4～6 carbon atoms, and hetero atom wherein is selected from N, O, S; R1 also comprises by 1 or 2 hydrogen aryl and heteroaryls that following radicals replaces respectively: CH3, CH3O, CH2OH, COOH, COOCH3, OH; R2 is selected from following group: hydrogen, (CH2) nCH3, (n=2～8), 2～6 interconnective pentoses or hexose base.
2. the Radix Isatidis extract that has antivirus action according to claim 1, it is characterized in that: it is that root with Isatis indigotica Fort. is that Radix Isatidis, leaf are the preparation that is prepared antiviral drugs by the adsorbed chemical constituent of macroporous resin as antivirus effective position that raw material extracts, this effective site can separately or add an amount of excipient, make the antiviral drugs of peroral dosage form or non-peroral dosage form with the preparation method of routine, perhaps add to this extract in the Pharmaceutical composition or the compound preparation of this extract participation prescription, perhaps make health product, health food.
3. one kind is extracted the described method with Radix Isatidis extract of antivirus action of claim 1, and its extraction step is as follows:
(1) Radix Isatidis medical material hot water extraction, water extract gets the water extract-alcohol precipitation supernatant through concentrating with ethanol precipitation;
(2) with supernatant concentration after macroporous resin adsorption, with the alcohol flushing resin post, collect eluent;
(3) be effective site with getting antivirus extract after eluent concentrating under reduced pressure, the drying.
4. the extracting method of Radix Isatidis extract according to claim 3, it is characterized in that the described Radix Isatidis medical material of step (1) is dry decoction pieces or powder, 60～100 ℃ of water extraction doubly measuring with medical material amount 4-20 1～4 time, each 0.5h～2h, concentrate behind the merge extractive liquid,, adding 95% ethanol to ethanol final concentration in extracting solution is 30～80%, leave standstill filter supernatant.
5. the extracting method of Radix Isatidis extract according to claim 3 is characterized in that in the step (2) supernatant sucking filtration or centrifugal is concentrated behind the filtrate recycling ethanol through macroporous resin adsorption, with the alcohol flushing resin post of 10-90%, collects ethanol elution.
6. Radix Isatidis extract as claimed in claim 1, it is characterized in that described lariciresinol-4, the extracting method of 4 '-two-O-β-D-pyranglucoside is: get Radix Isatidis medicinal material drying decoction pieces or powder, with 10～95% alcohol-water solution reflux, extract,, the extracting solution concentrating under reduced pressure is got extractum; Extractum is soluble in water under the heating in water bath situation, by macroporous resin, collects the ethanol elution part, concentrating under reduced pressure, and drying gets crude extract; This crude extract separates through silica gel column chromatography, with lariciresinol-4,4 '-two-O-β-pure product of D-pyranglucoside product TLC are in contrast followed the tracks of the inspection knowledge, there is stream part of same blob to merge with reference substance, after reclaiming solvent,, obtain lariciresinol-4 through solvent recrystallization repeatedly, 4 '-two-O-β-D-pyranglucoside, described recrystallization solvent is selected one or both in water, methanol, ethanol, propanol, butanols, amylalcohol, acetone, butanone, the ethyl acetate for use.
7. Radix Isatidis extract as claimed in claim 1, it is characterized in that described extracting method with reactive compound of general formula (I) structure is: Radix Isatidis medicinal material drying decoction pieces or powder, with 10～95% alcohol-water solution reflux, extract,, the extracting solution concentrating under reduced pressure is got extractum; Extractum is soluble in water under the heating in water bath situation, pass through macroporous resin, collect the ethanol elution part, concentrating under reduced pressure, drying, obtain crude extract, this crude extract separates through silica gel column chromatography, collects to have the flow point merging that contains this chemical compound, behind the recovery solvent, through solvent recrystallization repeatedly, this recrystallization solvent is selected one or both in water, methanol, ethanol, propanol, butanols, amylalcohol, acetone, butanone, the ethyl acetate for use.
8. Radix Isatidis extract as claimed in claim 1, it is characterized in that described lariciresinol-4,4 '-two-O-β-D-pyranglucoside can utilize the method for HPLC to carry out assay, and the mobile phase of mensuration is selected one or both mixing in water, methanol or the acetonitrile chromatorgaphy reagent for use; The ultraviolet detection wavelength is between 200nm～400nm; The concrete steps of its content assaying method are as follows:
(1) preparation of reference substance solution: precision takes by weighing lariciresinol-4, and 4 '-two-O-β-D-diglucoside reference substance is an amount of, adds methanol and progressively is diluted to the solution that every 1ml contains 0.02mg;
(2) preparation of confession test agent solution: precision takes by weighing the powder 1g that Radix Isatidis is crossed 40 mesh sieves, place the 25ml volumetric flask, add 60% methanol constant volume to 25ml, 60 ℃ of supersound extraction 1h, add 60% methanol again to scale, with the centrifugal 10min of speed of 3500r/min, after supernatant is crossed 0.45 μ m microporous filter membrane, get need testing solution, standby;
(3) measure: accurate respectively above-mentioned reference substance solution and each 10 μ l of need testing solution of drawing, inject chromatograph of liquid and measure, promptly get the content of measuring.
9. lariciresinol-4 according to claim 8, the content assaying method of 4 '-two-O-β-D-pyranglucoside is characterized in that: can be used for the Isatis indigotica Fort. platymiscium is to be the quality control standard of the pharmaceutical preparation of raw material or compound medicinal formulation or health product, health food with these medical materials in the medical material in source and the prescription.
10. Radix Isatidis extract as claimed in claim 1 is effective site or lariciresinol-4,4 '-two-O-β-D-pyranglucoside or the application of reactive compound in the preparation antiviral drugs with general formula (I) structure.
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| CN102331467A (en) * | 2011-07-28 | 2012-01-25 | 江西普正制药有限公司 | Method for detecting quality of south isatis root granules |
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| CN102614206A (en) * | 2011-01-31 | 2012-08-01 | 澳门科技大学 | Application of 7S,8R,8'R-(+)-lariciresinol-4,4'-bi-O-beta-D-glucopyranoside in preparing medicines |
| CN102331467A (en) * | 2011-07-28 | 2012-01-25 | 江西普正制药有限公司 | Method for detecting quality of south isatis root granules |
| CN102391316A (en) * | 2011-10-11 | 2012-03-28 | 泸州医学院 | Disaccharide compound and separation and purification method thereof |
| CN102391316B (en) * | 2011-10-11 | 2013-11-13 | 泸州医学院 | Disaccharide compound and separation and purification method thereof |
| CN104805097A (en) * | 2014-01-23 | 2015-07-29 | 中国人民解放军第二军医大学 | Coding sequences and applications of isatis indigotica fortune pinoresinol reductase protein |
| CN105001275A (en) * | 2015-08-05 | 2015-10-28 | 南京中医药大学 | Alkaloid carbon-glycoside with antiviral activity and antibacterial activity and application thereof |
| CN105001275B (en) * | 2015-08-05 | 2018-02-23 | 南京中医药大学 | With antiviral activity and the alkaloid c-glycosides of antibacterial activity and its application |
| CN115957208A (en) * | 2021-10-08 | 2023-04-14 | 中国医学科学院药物研究所 | Application of (-) -lariciresinol compound in isatis root in resisting hepatitis B virus |
| CN114404463A (en) * | 2022-02-22 | 2022-04-29 | 徐昊 | Preparation process of radix isatidis buccal tablets |
| CN114404463B (en) * | 2022-02-22 | 2023-05-05 | 重庆市国信医药有限公司 | Preparation process of radix isatidis buccal tablet |
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