CN101890037B - Nuezhenoside and hyperin composition and application thereof for preparing medicine - Google Patents
Nuezhenoside and hyperin composition and application thereof for preparing medicine Download PDFInfo
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- NULBHTHMVOCGOE-ZBCCAYPVSA-N C[C@@H]([C@@H]([C@H]([C@H]1O)O)O)O[C@H]1OC[C@H]([C@H]([C@@H]([C@H]1O[C@@H]([C@@H]([C@@H]2O)O)O[C@@H](C)[C@@H]2O)O)O)O[C@H]1Oc(cc1OC(c(cc2)ccc2O)=C2)cc(O)c1C2=O Chemical compound C[C@@H]([C@@H]([C@H]([C@H]1O)O)O)O[C@H]1OC[C@H]([C@H]([C@@H]([C@H]1O[C@@H]([C@@H]([C@@H]2O)O)O[C@@H](C)[C@@H]2O)O)O)O[C@H]1Oc(cc1OC(c(cc2)ccc2O)=C2)cc(O)c1C2=O NULBHTHMVOCGOE-ZBCCAYPVSA-N 0.000 description 1
Abstract
The invention relates to a nuezhenoside and hyperin composition which is prepared from 20%-80% of nuezhenoside and 80%-20% of hyperin in proportion by weight. The composition can be used as a neuraminidase inhibitor for preventing and treating flu and be prepared into preparations acceptable pharmaceutically.
Description
Technical field
The present invention relates to a kind of nuezhenoside, hyperin compositions and preparation medicinal usage thereof.
Background technology
Influenza virus is serious day by day at present; And influenza virus and respiratory tract disease and the systemic disease that causes are closely related; China is one of influenza country occurred frequently, and is not only populous, and living habit also helps the relay of influenza virus; The number of times of falling ill for each person every year reaches 0.3～0.7 time not to be waited, and key population reaches 2～4 times.There is serious threat in influenza to the mankind, New Development kind influenza virus especially, and it not only causes other infected by microbes of Secondary cases, and can directly cause organ to destroy and the allergy causing death.But the method and the treatment means that do not have efficacious therapy influenza virus and disease (influenza) thereof at present as carry out the inflammatory reaction treatment to clinical symptoms, are cured the symptoms, not the disease.The method of present modal prevention and treatment influenza virus is to use influenza virus vaccine inoculation, non-specific anti virus herb or Western medicine oseltamivir phosphate (oseltamivir phosphate capsule)-neuraminidase inhibitor.Yet influenza virus vaccine prophylactic immunization drawback is that the inoculation crowd is selective, is not that everybody can inoculate.In addition, the influenza virus vaccine protective rate is not high, and guard time is also lacked (3～6 months).Though most of non-specific anti virus herbs lay claim to antivirus action at present, be not to influenza virus, and antivirus action mechanism are unclear.At many kind treatment affection of exogenous wind-cold of our former studies, the Chinese prescription of the flu that doctor trained in Western medicine is thought does not have the effect of direct resisiting influenza virus.Briefly, the Chinese prescription of not every treatment flu all has the effect of clear and definite inhibition influenza virus.Western medicine " oseltamivir phosphate (oseltamivir phosphate capsule; (3R; 4R, 5S)-amino-3 (1-third the 2-ethoxyethyl acetate)-1-cyclohexene-1 carboxylic acid, ethyl ester phosphate of 4-acetamide-5-) " be neuraminidase inhibitor, influenza virus is got into cell has specific inhibitory effect; But not only cost an arm and a leg, and have that some patients take that the back vomiting occurs, feels sick, side reactions such as insomnia, headache, stomachache, diarrhoea, dizziness, fatigue, nasal obstruction, pharyngalgia and cough.Oseltamivir phosphate capsule is that the esterase that is positioned at liver and intestinal in vivo is converted into active metabolite and plays the neuraminidase effect that suppresses, and also will influence its drug effect if patient's liver and intestinal organ dysfunction are undesired.In addition, the renal insufficiency patient also wants careful usefulness.
Traditional Chinese medicine is recognized leaf of Turpinia pomifera (Roxb) D O. and is had heat-clearing and toxic substances removing, relieving sore throat and diminishing swelling, promoting blood circulation and stopping pain.Be used for the tonsillitis sore throat, laryngopharynx swelling and pain, sore swollen toxin falls and pounces on the pain of injury.Nuezhenoside and hyperin are one of main components of leaf of Turpinia pomifera (Roxb) D O..The Fructus Ligustri Lucidi function is a nourishing the liver and kidney, and black hair makes eye bright.Be mainly used in vertigo and tinnitus; Soreness of the waist and knees, early whitening of beard and hair, poor vision; Be used to treat chronic tracheitis, hepatitis, hyperlipidemia, diabetes, climacteric syndrome, infertility, atherosclerosis etc. in recent years, nuezhenoside is one of main component of Fructus Ligustri Lucidi more.Herba Hyperici Monogyni has soothing liver-QI for relieving depression, clearing away heat-damp and promoting diuresis, reducing swelling and alleviating pain.It is not smooth to be used for feelings will, and the stagnation of QI is gloomy, arthralgia, dysuria.Hyperin is one of main component of Herba Hyperici Monogyni, and hyperin is to myocardial ischemia-reperfusion, and cerebral ischemia re-pouring, cerebral infarction all show good protective action, also have analgesia, antioxidation, protection cardiac muscle, liver effect.Do not find that leaf of Turpinia pomifera (Roxb) D O. and contained nuezhenoside and hyperin, Fructus Ligustri Lucidi and contained nuezhenoside, Herba Hyperici Monogyni and contained hyperin thereof thereof thereof have the effect that suppresses influenza infection, duplicates, and do not find that it has the effect that suppresses neuraminidase yet.
Summary of the invention
The purpose of this invention is to provide nuezhenoside, hyperin compositions; It can suppress influenza neuraminidase hydrolysis cell surface sialic acid; Cause influenza virus not combine with cell surface receptor and to get in the cell; And reduce influenza virus in intracellular generation, and duplicate thereby suppress influenza virus effectively pointedly, more importantly be that the present invention provides a kind of nuezhenoside, hyperin compositions can overcome the side reaction drawback of existing medicine.
Nuezhenoside provided by the invention, hyperin compositions, the molecular formula of said nuezhenoside: C
33H
40O
18, molecular weight: 724.2, the structural formula of said nuezhenoside is following:
The molecular formula of said hyperin: C
21H
20O
12, molecular weight: 464.38, the structural formula of said hyperin is following:
Wherein said nuezhenoside, hyperin compositions are processed by following weight proportion: nuezhenoside 80%～20%, hyperin 20%～80%.
Preferably, wherein said nuezhenoside, hyperin compositions are processed by following weight proportion: nuezhenoside 75%～25%, hyperin 25%～75%.
Preferably, wherein said nuezhenoside, hyperin compositions are processed by following weight proportion: nuezhenoside 60%～40%, hyperin 40%～60%.
Preferably, wherein said nuezhenoside, hyperin compositions are processed by following weight proportion: nuezhenoside 55%～45%, hyperin 45%～55%.
Preferably, wherein said nuezhenoside, hyperin compositions are processed by following weight proportion: nuezhenoside 50%, hyperin 50%.
On the other hand; The present invention also provides the method for distilling of nuezhenoside, hyperin compositions; Wherein said nuezhenoside, hyperin are extracted acquisition from leaf of Turpinia pomifera (Roxb) D O., said method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., add 30%～90% alcohol reflux 1～3 time of 5～15 times of amounts; Each reflux, extract, 1～3 hour filters; Merging filtrate reclaims ethanol, and aqueous solution is collected the eluting part through the macroporous resin column eluting; Concentrating under reduced pressure, drying, the mixing total glycosides of nuezhenoside, hyperin, through separating; Merge nuezhenoside, hyperin stream part respectively, crystallization obtains nuezhenoside, the pure article of hyperin.
Preferably, wherein said nuezhenoside, hyperin adopt following method for distilling to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 12 times of amount 70% alcohol reflux 1.5 hours; Filter, filtering residue adds 10 times of amount 70% alcohol reflux 1.5 hours again, filters; Merge extracted twice liquid, reclaim ethanol, aqueous solution is through the macroporous resin column eluting; Collect the eluting part, concentrating under reduced pressure, drying.
Preferably, wherein the D101 macroporous resin column of aqueous solution through having handled well distinguished water, 5%～10% ethanol, 30%～55% ethanol, 1% sodium hydroxide solution eluting; Collect 30%～55% ethanol elution part, concentrating under reduced pressure, drying; Get the mixing total glycosides of nuezhenoside, hyperin,, merge nuezhenoside, hyperin stream part respectively through silica gel column chromatography, Sephadex LH-20 column chromatography for separation; Crystallization obtains the pure article of pure article of nuezhenoside and hyperin.
Another object of the present invention has provided the purposes of the medicine of nuezhenoside, the prevention of hyperin preparation of compositions and treatment influenza and complication disease thereof.
Preferably, nuezhenoside, hyperin compositions are used to suppress influenza virus.
Preferably, nuezhenoside, hyperin compositions are used to suppress influenza virus FM1.
Preferably, nuezhenoside, hyperin compositions are used to suppress neuraminidase.
Preferably, its complication is meant renal failure.
Preferably, its complication is meant splenic trauma or/and injury of lung.
Nuezhenoside of the present invention, hyperin can be from the various medical materials that contain nuezhenoside, hyperin, to obtain nuezhenoside, hyperin bullion or nuezhenoside, hyperin monomeric compound as extracting in leaf of Turpinia pomifera (Roxb) D O., Fructus Ligustri Lucidi, the Herba Hyperici Monogyni.
At last, the present invention also provides a kind of nuezhenoside, hyperin composite preparation, and said preparation is a main active with the above-mentioned nuezhenoside of the present invention, hyperin compositions.
Experimental data proves; Nuezhenoside of the present invention, hyperin compositions are effectively to suppress the neuraminic acid enzyme component, and nuezhenoside, hyperin compositions are along with the using dosage size variation, and it suppresses the active ability of neuraminidase; Be also corresponding the changing of height of neuraminic acid enzyme inhibition rate; And one-tenth positive correlation, nuezhenoside, hyperin compositions can be through suppressing influenza virus surface neuraminidase, and then suppress that influenza virus gets into the cell the inside, the influenza virus that suppresses to have got into the cell the inside duplicates, breeds; Thereby infection, the growth of influenza virus pair cell have been reduced; And prevention and treatment influenza and complication thereof, can also suppress influenza neuraminidase hydrolysis cell surface sialic acid, cause influenza virus not combine with cell surface receptor and to get in the cell; And reduce influenza virus in intracellular generation, more importantly be that the present invention provides a kind of nuezhenoside, hyperin compositions can overcome the side reaction drawback of existing medicine.
And medical science and study of pharmacy personnel can't not do the inhibition influenza infection, duplicate in advance, or under the prerequisite of the experiment of inhibition neuraminidase, learn that in advance nuezhenoside, hyperin compositions have prevention and treat the good result that the influenza virus sexuality is emitted.
In sum, nuezhenoside provided by the invention, hyperin composition and use thereof bring significant technique effect.
The specific embodiment
Embodiment 1
The molecular formula of said nuezhenoside provided by the invention: C
33H
40O
18, molecular weight: 724.2, the structural formula of said nuezhenoside is following:
The molecular formula of said hyperin provided by the invention: C
21H
20O
12, molecular weight: 464.38, the structural formula of said hyperin is following:
Wherein said nuezhenoside, hyperin are extracted acquisition from leaf of Turpinia pomifera (Roxb) D O., said method for distilling is following: get Folium Isatidis 100000g, add 12 times of amount 70% alcohol reflux 1.5 hours; Filter, filtering residue adds 10 times of amount 70% alcohol reflux 1.5 hours again, filters; Merge extracted twice liquid, reclaim ethanol, the D101 macroporous resin column of aqueous solution through having handled well; Water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution eluting are collected 45% ethanol elution part, concentrating under reduced pressure respectively; Drying, the mixing total glycosides of 30% above nuezhenoside, hyperin, through silica gel column chromatography, Sephadex LH-20 column chromatography for separation; Merge nuezhenoside, hyperin stream part respectively, crystallization obtains nuezhenoside (320g; Purity: 98.8%) and hyperin (65g, purity: pure article 98.6%), through respectively with the UV of nuezhenoside, hyperin standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of above-mentioned two chemical compounds.
In addition, nuezhenoside provided by the invention, hyperin also can be extracted acquisition respectively, like following listed examples.
Embodiment 2
Get Fructus Ligustri Lucidi 1000g, add 12 times of amount 70% alcohol reflux 1.5 hours, filter, filtering residue adds 10 times of amount 70% alcohol reflux 1.5 hours again; Filter, merge extracted twice liquid, reclaim ethanol, the D101 macroporous resin column of aqueous solution through having handled well; Water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution eluting are collected 45% ethanol elution part, concentrating under reduced pressure, drying respectively; Get the mixing total glycosides of 30% above nuezhenoside,, merge nuezhenoside stream part, crystallization through silica gel column chromatography, Sephadex LH-20 column chromatography for separation; Obtain nuezhenoside (18.5g, purity: pure article 98.7%), through respectively with the UV of nuezhenoside standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of nuezhenoside.
Embodiment 3
Get Herba Hyperici Monogyni 1000g, add 12 times of amount 70% alcohol reflux 1.5 hours, filter, filtering residue adds 10 times of amount 70% alcohol reflux 1.5 hours again; Filter, merge extracted twice liquid, reclaim ethanol; The D101 macroporous resin column of aqueous solution through having handled well, water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution eluting are collected 45% ethanol elution part respectively; Concentrating under reduced pressure, drying, the mixing total glycosides of 30% above hyperin; Through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge hyperin stream part, crystallization; Obtain hyperin (8.5g, purity: pure article 98.6%), through respectively with the UV of hyperin standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of hyperin.
Embodiment 4
Get Fructus Ligustri Lucidi 1000g, add 10 times of amount 65% alcohol reflux 2 hours, filter, filtering residue adds 8 times of amount 65% alcohol reflux 1.5 hours again; Filter, merge extracted twice liquid, reclaim ethanol, the D101 macroporous resin column of aqueous solution through having handled well; Water, 8% ethanol, 40% ethanol, 1% sodium hydroxide solution eluting are collected 40% ethanol elution part, concentrating under reduced pressure, drying respectively; Get the mixing total glycosides of 30% above nuezhenoside,, merge nuezhenoside stream part, crystallization through silica gel column chromatography, Sephadex LH-20 column chromatography for separation; Obtain nuezhenoside (19.2g, purity: pure article 98.6%), through respectively with the UV of nuezhenoside standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of nuezhenoside.
Embodiment 5
Get Herba Hyperici Monogyni 1000g, add 15 times of amount 60% alcohol reflux 2 hours, filter, filtering residue adds 9 times of amount 60% alcohol reflux 2 hours again; Filter, merge extracted twice liquid, reclaim ethanol; The D101 macroporous resin column of aqueous solution through having handled well, water, 8% ethanol, 40% ethanol, 1% sodium hydroxide solution eluting are collected 40% ethanol elution part respectively; Concentrating under reduced pressure, drying, the mixing total glycosides of 30% above hyperin; Through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge hyperin stream part, crystallization; Obtain hyperin (9.2g, purity: pure article 98.5%), through respectively with the UV of hyperin standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of hyperin.
Embodiment 6
The method for distilling of pressing embodiment 1 gets nuezhenoside, hyperin compositions with nuezhenoside (14g) and the mixed of hyperin (6g) with 7: 3 behind stirring, grinding, the mix homogeneously.
Embodiment 7
Press the method for distilling of embodiment 1,, get nuezhenoside, hyperin compositions behind stirring, grinding, the mix homogeneously nuezhenoside (10g) that extracts and the mixed of hyperin (10g) with 5: 5.
Embodiment 8
Press the method for distilling of embodiment 1,, get nuezhenoside, hyperin compositions behind stirring, grinding, the mix homogeneously nuezhenoside (6g) that extracts and the mixed of hyperin (14g) with 3: 7.
Embodiment 9
Press the method for distilling of embodiment 1,, get nuezhenoside, hyperin compositions behind stirring, grinding, the mix homogeneously nuezhenoside (8g) that extracts and the mixed of hyperin (12g) with 4: 6.
Embodiment 10
Press the method for distilling of embodiment 1,, get nuezhenoside, hyperin compositions behind stirring, grinding, the mix homogeneously nuezhenoside (12g) that extracts and the mixed of hyperin (8g) with 6: 4.
Provide some preferred embodiments of nuezhenoside of the present invention and hyperin mass percent below in conjunction with table 1; But the two the content of composition of the present invention is not limited to listed numerical value in this table; For a person skilled in the art, fully can be in table rationally summarize and reasoning on the basis of listed numerical range.
Table 1: nuezhenoside, hyperin account for the percentage by weight (%) of compositions
Experimental data 1: nuezhenoside, hyperin compositions are to the active inhibitory action of neuraminidase
Get nuezhenoside, hyperin compositions that embodiment 6 method for preparinies are obtained, add suitable quantity of water and make dissolving, application neuraminidase inhibitor identification kit is measured nuezhenoside, the hyperin compositions suppresses tiring of neuraminidase (N1) and sees table 2.
(1). standard curve is prepared: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases and detects buffer; B. every hole adds 0,1,2,5,7.5,10 μ l H5N1 neuraminidases more respectively; C. every hole adds 0～20 μ l Milli-Q water again.
(2). the preparation of sample detection: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases and detects buffer; B. every hole adds 10 μ l H5N1 neuraminidases again; C. every hole adds 0～10 μ l nuezhenoside, hyperin composition sample again; D. every hole adds 0～10 μ l Milli-Q water again.
(3). detect step:
A. vibrate the about 1min of mixing;
B.37 ℃ hatch 2min inhibitor and H5N1 neuraminidase are fully interacted, the sample of doing standard curve is also hatched together;
C. every hole adds 10 μ l neuraminidase fluorogenic substrates;
D. vibrate the about 1min of mixing again;
E.37 carry out fluoremetry after ℃ hatching 20～30min.Excitation wavelength is 360nm, and emission wavelength is 440nm.
(4). calculate the inhibition percentage ratio of sample according to standard curve, and do and calculate nuezhenoside, hyperin compositions IC50 behind the concentration curve for the H5N1 neuraminidase for the H5N1 neuraminidase.The suppression ratio IC50 that nuezhenoside, hyperin compositions reach neuraminidase is 0.338g/L.See table 2.
Table 2. nuezhenoside, hyperin compositions suppress the activity of neuraminidase
Can be clear that according to above-mentioned experimental result:
(1). nuezhenoside, hyperin compositions can extract effective inhibition neuraminic acid enzyme component;
(2). nuezhenoside, hyperin compositions are along with the using dosage size variation, and it suppresses the active ability of neuraminidase, and promptly the height of neuraminic acid enzyme inhibition rate is also corresponding changes, and become positive correlation;
(3). visible by above-mentioned experiment; Nuezhenoside, hyperin compositions can be through suppressing influenza virus surface neuraminidase; And then suppress that influenza virus gets into the cell the inside, the influenza virus that suppresses to have got into the cell the inside duplicates, breeds; Thereby reduced infection, the growth of influenza virus pair cell, and prevention and treatment influenza and complication thereof.
Medical science and study of pharmacy personnel can't not do the inhibition influenza infection, duplicate in advance, or under the prerequisite of the experiment of inhibition neuraminidase, learn that in advance nuezhenoside, hyperin compositions have prevention and treat the good result that the influenza virus sexuality is emitted.
Experimental data 2: nuezhenoside, hyperin compositions infect the inhibitory action of Embryo Gallus domesticus to influenza virus
Get nuezhenoside, hyperin compositions that embodiment 6 method for preparinies are obtained, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify that nuezhenoside, hyperin compositions suppress the ability that the FM1 influenza virus is duplicated and suppresses in Embryo Gallus domesticus.(1). FM1 influenza virus liquid is inoculated in 10d no-special pathogen in age chick embryo allantois intracavity, and every embryo 0.2ml is hatched 72h for 37 ℃, observes and calculate half Embryo Gallus domesticus infective dose (EID50).(2). nuezhenoside, hyperin compositions adopt the toxic action of Embryo Gallus domesticus; Physiological saline solution is done to be inoculated in 10d no-special pathogen in age chick embryo allantois intracavity behind the serial dilution to nuezhenoside, hyperin compositions; Every embryo 0.2ml, each concentration is inoculated 6 embryos, hatches for 37 ℃; Observe Embryo Gallus domesticus growth promoter situation, can survive the Cmax of 96h as the TD of medicine with Embryo Gallus domesticus.(3). nuezhenoside, the hyperin compositions inhibitory action to influenza virus in Embryo Gallus domesticus adopts; 0.1ml influenza virus liquid and different dilution nuezhenoside, hyperin compositions mix; 37 ℃ of effect 2h; Be inoculated in 10d no-special pathogen in age chick embryo allantoic cavity, every winding kind 6 embryos are hatched 72h for 37 ℃.The virus attack amount is 50EID50, establishes virus control, physiological saline solution normal control simultaneously, calculates nuezhenoside, the hyperin compositions median effective dose (ED50) to viral inhibition.
(1) the .FM1 influenza virus is calculated through the Reed-Muench method the virulence of Embryo Gallus domesticus, and its EID50 is 10
-5.07(2). after nuezhenoside, hyperin compositions are inoculated in Embryo Gallus domesticus, its growth promoter and normal control group basically identical.The 96h Embryo Gallus domesticus is all survived.Embryo Gallus domesticus gives nuezhenoside, hyperin compositions stock solution is not seen chicken embryo death, so can think that TD0 is 4.6g/L.(3). nuezhenoside, the hyperin compositions inhibitory action to influenza virus in Embryo Gallus domesticus is seen table 3.
Table 3. nuezhenoside, hyperin compositions infect the inhibitory action of Embryo Gallus domesticus to influenza virus
Compare with the virus control group:
*P＜0.05
Can know that by table 3 nuezhenoside, hyperin compositions have significant inhibitory effect (P＜0.05), ED at 0.2875～2.3g/L to influenza virus
50Be 0.38g ± 0.16mg/ml, TI is 21.84 ± 3.91.
Experimental data 3: nuezhenoside, hyperin compositions influence the FM1 influenza virus
Get nuezhenoside, hyperin compositions that embodiment 6 method for preparinies are obtained, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify that nuezhenoside, hyperin compositions suppress the ability of FM1 influenza virus virulence.(1) .FM1 adopts cell ID 50, median infective dose (TCID50) micromethod to the toxicity test of Testis et Pentis Canis passage cell (MDCK).(2). nuezhenoside, hyperin compositions adopt the DMEM of serum-free that nuezhenoside, hyperin compositions are done to be inoculated in the mdck cell hole that forms monolayer behind the serial dilution to the toxicity test of mdck cell; Every hole 100 μ l; Each dilution factor repeats 4 holes, establishes the normal cell contrast simultaneously.Culture plate is put 37 ℃, 5%CO
2Cultivate in the incubator, observation of cell pathological changes every day (CPE) is observed 3d continuously, with " +～++ ++ " the record result, press the Reed-Muench method and calculate medicine median toxic concentration (TD50) and maximal non-toxic concentration (TD0).(3). nuezhenoside, hyperin compositions suppress the effect of FM1 influenza virus and measure: mdck cell 5 * 10
5/ ml, every hole 100 μ l, in 96 orifice plates, 37 ℃, the interior cultivation of 5%CO2 incubator are inhaled and remove culture fluid in the hole next day, add 100TCID50 influenza virus liquid, every hole 100 μ l, supernatant is removed in suction behind 37 ℃ of absorption 1h.Wash 2 times with phosphate buffer (PBS); TD0 with medicine is the 1st hole, and the DMEM liquid of reuse serum-free is made serial dilution to nuezhenoside, hyperin compositions, adds respectively in the cell of above-mentioned infective virus; Establish virus control and normal control group simultaneously, 37 ℃, 5%CO
2Cultivate in the incubator, observe the mdck cell characteristics of lesion that influenza virus produces every day, i.e. cell monolayer degeneration becomes circle etc., and 3d calculates 50% of medicine and suppresses pathological changes concentration (IC50) and therapeutic index (TI) continuously.The calculating of TI: TI=TD50/IC50, the TI value is big more, shows that the safety range of medicine is big more.With Kruskal-Walis and Mann-Whitney method of inspection comparative test group and the cytopathic difference of virus control group; Drug dose and suppression ratio that virus infected cell is avoided cytopathy (CPE) takes place are carried out correlation analysis, judge whether amount validity response relation.
(1) the .FM1 influenza virus is calculated through the Reed-Muench method the virulence of mdck cell, and its TCID50 is 10
-4.81(2). the TD0 of nuezhenoside, hyperin compositions mdck cell is respectively 1.2g ± 0.11g/L.(3). after nuezhenoside, hyperin compositions made serial dilution, the 100TCID50 influenza virus is carried out inhibition test, median effective dose IC50 and the TI value of calculating medicine are big or small, and the result sees table 4.
Table 4. nuezhenoside, hyperin compositions are to the IC of FM1 influenza virus
50(g/L) and TI (x ± s)
Can be known that by table 4 IC50 of nuezhenoside, hyperin compositions is low, TI is high.Nuezhenoside, hyperin compositions suppress the cytopathogenic effect of FM1 influenza virus and all strengthen along with the increase of drug dose.Drug dose and medicine are shown that to the correlation analysis that the suppression ratio of CPE carries out the dosage of nuezhenoside, hyperin compositions and medicine are to being tangible positive correlation between the CPE suppression ratio.
Experimental data 4: nuezhenoside, hyperin compositions are to the spleen index and the exponential influence of lung of influenza virus infection FM1 strain in the mice body
Get nuezhenoside, hyperin compositions that embodiment 6 method for preparinies are obtained, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify the dead protective effect of nuezhenoside, hyperin compositions influenza virus infection FM1 strain in the mice body.(1). influenza virus FM1 strain virus is inoculated every group of 10 BALB/C mices respectively, male and female half and half after doing 10 times of doubling dilutions.After the slight anesthesia of ether, give and different dilution viruses respectively for every group, every mice collunarium is inoculated 20 μ l.Observe the dead mouse situation of 10d, calculating LD50 by the Reed-Muench method is 10
-1.36So confirm that the used modeling concentration of experiment is 10LD50.(2). nuezhenoside, hyperin compositions are to the dead protective effect of influenza virus infection FM1 strain in the mice body: normal control group, influenza virus FM1 strain virus matched group, nuezhenoside, hyperin compositions 5.0mg/ml, 10.0mg/ml, 20.0mg/ml, 40.0mg/ml dose groups etc. are irritated stomach respectively, irritate gastric capacity and only are 0.4ml/.Behind the 3d, each is organized under the slight anesthesia of ether with 10LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ except that the normal control group.The normal control group gives the normal saline with volume simultaneously.4 groups of administration are continued administration, normal control group and influenza virus FM1 strain virus control group administered physiological saline 8 days, and dosage is the same.Day by day observe animal morbidity and record death toll, observed altogether 14 days, calculate mortality rate (mortality rate=every group of death toll/every group of total mice * 100%), the result sees table 5.(3). nuezhenoside, hyperin compositions are to the exponential influence of influenza virus infection FM1 strain lung in the mice body: normal control group, influenza virus FM1 strain virus matched group, nuezhenoside, hyperin compositions 20.0mg/ml, 40.0mg/ml, 80.0mg/ml, 160.0mg/ml dose groups etc. are irritated stomach respectively, irritate gastric capacity and only are 0.4ml/.After 3 days, each is organized under the slight anesthesia of ether with 1.0LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ except that the normal control group.The normal control group gives the normal saline with volume simultaneously.4 groups of administration are continued administration, normal control group and influenza virus FM1 strain virus control group administered physiological saline 8 days, and dosage is the same.Behind viral infection, put to death mice on the 8th day, weigh, get lung and claim that lung is heavy, calculates lung index (lung index=lung quality/body constitution amount * 100%); In addition, get spleen and claim that spleen is heavy, calculate spleen index (spleen index=spleen quality/body constitution amount * 100%), the result sees table 6.
Table 5. nuezhenoside, hyperin compositions are protected the result to the death of influenza virus infection FM1 strain in the mice body
Annotate: ※ ※ P＜0.01VS influenza virus model group
Table 6. nuezhenoside, hyperin compositions are to the spleen index and the exponential influence of lung of influenza virus infection FM1 strain in the mice body
Annotate: #P＜0.05VS normal control group ※ ※ p＜0.001VS influenza virus model group ※ p＜0.05VS influenza virus model group
(1). can know that by table 5 nuezhenoside, hyperin compositions have significant protective effect (p＜0.01) at 1.15～2.3g/L to the influenza virus infecting mouse.
(2). can know that by table 6 nuezhenoside, hyperin compositions have significant effect (p＜0.01) at 2.3g/L to the lung index suppression ratio of influenza virus infecting mouse.
Experimental data 5: nuezhenoside, hyperin compositions are to the influence of renal dysfunction
1) SD rat, 200～240g, Shanghai west pul-Bi Kai laboratory animal responsibility company limited, the animal quality certification number: SCXK (Shanghai) 2003-0002
2) reagent and medicine adenine, adenine, (content＞98% is the import packing, Chinese Shanghai, lot number 20010520), nuezhenoside, hyperin compositions that embodiment 6 method for preparinies are obtained;
3) test method: male SD rat, about body weight 220g, earlier feed 10 days normal growths with normal diet after, be divided into normal control group, administration experimental group and modeling matched group at random by body weight, 13～15 every group.Administration experimental group and modeling matched group are irritated stomach with adenine and are made chronic renal failure (CRF) model, irritate stomach with adenine 320mg/ (kgd), only process the about 2ml/ of suspension with distilled water, totally 20 days; Administration after 20 days; The administration experimental group is irritated stomach with nuezhenoside, hyperin compositions 3.4g/ (kgd), and nuezhenoside, hyperin compositions are processed suspension solution (0.44g/ml) with distilled water, and about at every turn 2ml/ only; Stomach is irritated in gradation; Normal control group and modeling matched group are irritated stomach with equal-volume water, and administration was used etherization with rat after 35 days, and each item index is observed in the blood sampling of Mus arteria caudalis.
4) result:
Table 7. nuezhenoside, hyperin compositions are to the influence of CRF kidney of rats function
Annotate: through the T check, nuezhenoside, hyperin compositions experimental group and modeling matched group be " * " expression P＜0.05 relatively, and " * * " representes P＜0.01
This experiment is looked sidelong at purine with gland and is irritated after stomach sets up rat CRF model, modeling control rats lethargy, and body weight obviously reduces; Serum Bun, Ser obviously raise, and Hb obviously descends, and Ret obviously raises; Show the rat impaired renal function; Rat and normal control group difference are not remarkable after giving nuezhenoside, hyperin compositions, and with the model group comparing difference very significantly, so nuezhenoside, hyperin compositions are to the kidney free of toxic effects.
Claims (14)
1. nuezhenoside, hyperin compositions, the molecular formula of said nuezhenoside: C
33H
40O
18, molecular weight: 724.2,
The structural formula of said nuezhenoside is following:
The molecular formula of said hyperin: C
21H
20O
12, molecular weight: 464.38, the structural formula of said hyperin is following:
Wherein said nuezhenoside, hyperin compositions are processed by following weight proportion: nuezhenoside 80%～20%, hyperin 20%～80%.
2. nuezhenoside according to claim 1, hyperin compositions, wherein said nuezhenoside, hyperin compositions are processed by following weight proportion: nuezhenoside 75%～25%, hyperin 25%～75%.
3. nuezhenoside according to claim 2, hyperin compositions, wherein said nuezhenoside, hyperin compositions are processed by following weight proportion: nuezhenoside 60%～40%, hyperin 40%～60%.
4. nuezhenoside according to claim 3, hyperin compositions, wherein said nuezhenoside, hyperin compositions are processed by following weight proportion: nuezhenoside 55%～45%, hyperin 45%～55%.
5. nuezhenoside according to claim 4, hyperin compositions, wherein said nuezhenoside, hyperin compositions are processed by following weight proportion: nuezhenoside 50%, hyperin 50%.
6. claim 1,2,3,4 or 5 described compositionss are used to prepare the purposes of preventing and treating the medicine of influenza disease.
7. purposes as claimed in claim 6 is characterized in that described compositions is used to suppress influenza virus.
8. purposes as claimed in claim 7 is characterized in that described compositions is used to suppress influenza virus FM1.
9. claim 1,2,3, the application of 4 or 5 described compositionss in the medicine of preparation inhibition neuraminidase.
10. claim 1,2,3,4 or 5 described compositionss are used to prepare the purposes of preventing and treating the medicine of influenza disease complication, and its complication is meant renal failure.
11. the purposes that claim 1,2,3,4 or 5 described compositionss are used to prepare prevention and treat the medicine of influenza disease complication, its complication is meant splenic trauma or/and injury of lung.
12. a nuezhenoside, hyperin composite preparation, wherein said preparation is a main active with each described nuezhenoside of claim 3～5, hyperin compositions.
13. the method for distilling of claim 1,2,3,4 or 5 described nuezhenoside, hyperin compositions; Wherein said nuezhenoside, hyperin are extracted acquisition from leaf of Turpinia pomifera (Roxb) D O.; Said method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O.; 30%～90% alcohol reflux 1～3 time that adds 5～15 times of amounts, each reflux, extract, 1～3 hour filters; Merging filtrate reclaims ethanol, and aqueous solution is through the macroporous resin column eluting; Water, 5%～10% ethanol, 30%～55% ethanol, 1% sodium hydroxide solution eluting are collected 30%～55% ethanol elution part, concentrating under reduced pressure respectively; Drying, the mixing total glycosides of nuezhenoside, hyperin, through silica gel column chromatography, SephadexLH-20 column chromatography for separation; Merge nuezhenoside, hyperin stream part respectively, crystallization obtains the pure article of pure article of nuezhenoside and hyperin.
14. method as claimed in claim 13, wherein said nuezhenoside, hyperin adopt following method for distilling to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 12 times of amount 70% alcohol reflux 1.5 hours; Filter, filtering residue adds 10 times of amount 70% alcohol reflux 1.5 hours again, filters; Merge extracted twice liquid, reclaim ethanol, aqueous solution is through the macroporous resin column eluting; Water, 5%～10% ethanol, 30%～55% ethanol, 1% sodium hydroxide solution eluting are collected 30%～55% ethanol elution part, concentrating under reduced pressure respectively; Drying, the mixing total glycosides of nuezhenoside, hyperin, through silica gel column chromatography, Sephadex LH-20 column chromatography for separation; Merge nuezhenoside, hyperin stream part respectively, crystallization obtains the pure article of pure article of nuezhenoside and hyperin.
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| 章光文等.山香圆叶中黄酮类成分研究.《中国中药杂志》.2009,第34卷(第12期),第1603-1604页. * |
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