Summary of the invention
The purpose of this invention is to provide Ligustrum lucidum Ait, it can suppress influenza neuraminidase hydrolysis cell surface sialic acid, cause influenza virus not combine and to enter in the cell with cell surface receptor, and reduce influenza virus in intracellular generation, duplicating thereby suppress influenza virus effectively pointedly, more importantly is to the invention provides the side reaction drawback that a kind of Ligustrum lucidum Ait can overcome existing medicine.
The molecular formula of described Ligustrum lucidum Ait provided by the invention: C
33H
40O
18, molecular weight: 724.2, the structural formula of described Ligustrum lucidum Ait is as follows:
Described Ligustrum lucidum Ait adopts following extracting method to obtain: get leaf of Turpinia pomifera (Roxb) D O. or Glossy Privet Fruit, add 30%~90% alcohol reflux 1~3 time of 5~15 times of amounts, each refluxing extraction 1~3 hour filters; Merging filtrate reclaims ethanol, and the aqueous solution is collected the wash-out part by the macroporous resin column wash-out, concentrating under reduced pressure, drying, the total glycosides of mixing of Ligustrum lucidum Ait, through separating, merge Ligustrum lucidum Ait stream part, crystallization obtains the pure product of Ligustrum lucidum Ait.
Preferably, wherein the aqueous solution is by macroporous resin column, and water, 5%~10% ethanol, 30%~55% ethanol, 0.8~1.2% sodium hydroxide solution wash-out are collected 30%~55% ethanol elution part, concentrating under reduced pressure, drying respectively.
Preferably, wherein said Ligustrum lucidum Ait adopts following extracting method to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 12 times of amount 70% alcohol reflux 1.5 hours, filter, filter residue adds 10 times of amount 70% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid, reclaim ethanol, the aqueous solution is by macroporous resin column, and water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution wash-out are collected 45% ethanol elution part respectively, concentrating under reduced pressure, drying.
Preferably, wherein said Ligustrum lucidum Ait adopts following extracting method to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 10 times of amount 65% alcohol reflux 2 hours, filter, filter residue adds 8 times of amount 65% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid, reclaim ethanol, the aqueous solution is by macroporous resin column, and water, 8% ethanol, 40% ethanol, 1.1% sodium hydroxide solution wash-out are collected 40% ethanol elution part respectively, concentrating under reduced pressure, drying.
Preferably, wherein said Ligustrum lucidum Ait adopts following extracting method to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 15 times of amount 60% alcohol reflux 2 hours, filter, filter residue adds 12 times of amount 60% alcohol reflux 2 hours again, filters, and merges extracted twice liquid, reclaim ethanol, the aqueous solution is by macroporous resin column, and water, 6% ethanol, 35% ethanol, 1.2% sodium hydroxide solution wash-out are collected 35% ethanol elution part respectively, concentrating under reduced pressure, drying.
Preferably, wherein said Ligustrum lucidum Ait adopts following extracting method to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 9 times of amount 65% alcohol reflux 2.5 hours, filter, filter residue adds 8 times of amount 65% alcohol reflux 2 hours again, filters, and merges extracted twice liquid, reclaim ethanol, the aqueous solution is by macroporous resin column, and water, 5% ethanol, 40% ethanol, 0.9% sodium hydroxide solution wash-out are collected 40% ethanol elution part respectively, concentrating under reduced pressure, drying.
Preferably, wherein said Ligustrum lucidum Ait adopts following extracting method to obtain: get Glossy Privet Fruit, add 12 times of amount 70% alcohol reflux 1.5 hours, filter, filter residue adds 10 times of amount 70% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid, reclaim ethanol, the aqueous solution is by macroporous resin column, and water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution wash-out are collected 45% ethanol elution part respectively, concentrating under reduced pressure, drying.
Preferably, wherein said Ligustrum lucidum Ait adopts following extracting method to obtain: get Glossy Privet Fruit, add 10 times of amount 65% alcohol reflux 2 hours, filter, filter residue adds 8 times of amount 65% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid, reclaim ethanol, the aqueous solution is by macroporous resin column, and water, 8% ethanol, 40% ethanol, 0.8% sodium hydroxide solution wash-out are collected 40% ethanol elution part respectively, concentrating under reduced pressure, drying.
Preferably, wherein said Ligustrum lucidum Ait adopts following extracting method to obtain: get Glossy Privet Fruit, add 15 times of amount 60% alcohol reflux 2 hours, filter, filter residue adds 12 times of amount 60% alcohol reflux 2 hours again, filters, and merges extracted twice liquid, reclaim ethanol, the aqueous solution is by macroporous resin column, and water, 6% ethanol, 35% ethanol, 1.2% sodium hydroxide solution wash-out are collected 35% ethanol elution part respectively, concentrating under reduced pressure, drying.
Preferably, wherein said Ligustrum lucidum Ait adopts following extracting method to obtain: get Glossy Privet Fruit, add 9 times of amount 65% alcohol reflux 2.5 hours, filter, filter residue adds 8 times of amount 65% alcohol reflux 2 hours again, filters, and merges extracted twice liquid, reclaim ethanol, the aqueous solution is by macroporous resin column, and water, 5% ethanol, 40% ethanol, 1% sodium hydroxide solution wash-out are collected 40% ethanol elution part respectively, concentrating under reduced pressure, drying.
Preferably, wherein the D101 macroporous resin column wash-out of the aqueous solution by having handled well mixes total glycosides through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merges Ligustrum lucidum Ait stream part, and crystallization obtains the pure product of Ligustrum lucidum Ait.
The purposes that the present invention also provides Ligustrum lucidum Ait to be used to prepare prevention and to treat the medicine of influenza and complication disease thereof.
Preferably, Ligustrum lucidum Ait is used to suppress influenza virus.
Preferably, Ligustrum lucidum Ait is used to suppress influenza virus FM1.
Preferably, Ligustrum lucidum Ait is used to suppress neuraminidase.
Preferably, its complication is meant renal failure.
Preferably, its complication is meant injury of spleen or/and injury of lung.
Ligustrum lucidum Ait of the present invention can be to obtain Ligustrum lucidum Ait crude product or Ligustrum lucidum Ait monomeric compound as extracting in leaf of Turpinia pomifera (Roxb) D O., the Glossy Privet Fruit from the various medicinal materials that contain Ligustrum lucidum Ait.
At last, the present invention also provides a kind of Ligustrum lucidum Ait preparation, and said preparation is a main active ingredient with the above-mentioned Ligustrum lucidum Ait of the present invention.
Experimental data proves, Ligustrum lucidum Ait of the present invention is effectively to suppress the neuraminic acid enzyme component, Ligustrum lucidum Ait is along with the using dosage size variation, 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, Ligustrum lucidum Ait can be by suppressing influenza virus surface neuraminidase, and then the inhibition influenza virus enters the cell the inside, inhibition has entered the influenza virus of cell the inside and has duplicated, propagation, thereby reduced the infection of influenza virus pair cell, growth, and prevention and treatment influenza and complication thereof, can also suppress influenza neuraminidase hydrolysis cell surface sialic acid, cause influenza virus not combine and to enter in the cell with cell surface receptor, and reduce influenza virus in intracellular generation, more importantly be to the invention provides the side reaction drawback that a kind of Ligustrum lucidum Ait can overcome 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 Ligustrum lucidum Ait has prevention and treats the good result that the influenza virus sexuality is emitted.
In sum, Ligustrum lucidum Ait provided by the invention and uses thereof brings significant technique effect.
Embodiment
Embodiment 1
Get leaf of Turpinia pomifera (Roxb) D O. 1000g, add 12 times of amount 70% alcohol reflux 1.5 hours, filter, filter 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 the aqueous solution by having handled well, water respectively, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution wash-out, collect 45% ethanol elution part, concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Ligustrum lucidum Ait, through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Ligustrum lucidum Ait stream part, crystallization obtains Ligustrum lucidum Ait (3.5g, purity: pure product 98.7%), through respectively with the UV of Ligustrum lucidum Ait standard substance, IR, ESI-MS;
1H-NMR,
13C-NMR has relatively determined the structure of Ligustrum lucidum Ait.
Embodiment 2
Get leaf of Turpinia pomifera (Roxb) D O. 1000g, add 10 times of amount 65% alcohol reflux 2 hours, filter, filter residue adds 8 times of amount 65% alcohol reflux 1.5 hours again, filters, merge extracted twice liquid, reclaim ethanol, the D101 macroporous resin column of the aqueous solution by having handled well, water respectively, 8% ethanol, 40% ethanol, 1.1% sodium hydroxide solution wash-out, collect 40% ethanol elution part, concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Ligustrum lucidum Ait, through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Ligustrum lucidum Ait stream part, crystallization obtains Ligustrum lucidum Ait (3.2g, purity: pure product 98.6%), through respectively with the UV of Ligustrum lucidum Ait standard substance, IR, ESI-MS;
1H-NMR,
13C-NMR has relatively determined the structure of Ligustrum lucidum Ait.
Embodiment 3
Get leaf of Turpinia pomifera (Roxb) D O. 1000g, add 15 times of amount 60% alcohol reflux 2 hours, filter, filter residue adds 12 times of amount 60% alcohol reflux 2 hours again, filters, merge extracted twice liquid, reclaim ethanol, the D101 macroporous resin column of the aqueous solution by having handled well, water respectively, 6% ethanol, 35% ethanol, 1.2% sodium hydroxide solution wash-out, collect 35% ethanol elution part, concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Ligustrum lucidum Ait, through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Ligustrum lucidum Ait stream part, crystallization obtains Ligustrum lucidum Ait (2.9g, purity: pure product 98.8%), through respectively with the UV of Ligustrum lucidum Ait standard substance, IR, ESI-MS;
1H-NMR,
13C-NMR has relatively determined the structure of Ligustrum lucidum Ait.
Embodiment 4
Get leaf of Turpinia pomifera (Roxb) D O. 1000g, add 9 times of amount 65% alcohol reflux 2.5 hours, filter, filter residue adds 8 times of amount 65% alcohol reflux 2 hours again, filters, merge extracted twice liquid, reclaim ethanol, the D101 macroporous resin column of the aqueous solution by having handled well, water respectively, 5% ethanol, 40% ethanol, 0.9% sodium hydroxide solution wash-out, collect 40% ethanol elution part, concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Ligustrum lucidum Ait, through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Ligustrum lucidum Ait stream part, crystallization obtains Ligustrum lucidum Ait (2.7g, purity: pure product 98.7%), through respectively with the UV of Ligustrum lucidum Ait standard substance, IR, ESI-MS;
1H-NMR,
13C-NMR has relatively determined the structure of Ligustrum lucidum Ait.
Embodiment 5
Get Glossy Privet Fruit 1000g, add 12 times of amount 70% alcohol reflux 1.5 hours, filter, filter 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 the aqueous solution by having handled well, water respectively, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution wash-out, collect 45% ethanol elution part, concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Ligustrum lucidum Ait, through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Ligustrum lucidum Ait stream part, crystallization obtains Ligustrum lucidum Ait (18.5g, purity: pure product 98.7%), through respectively with the UV of Ligustrum lucidum Ait standard substance, IR, ESI-MS;
1H-NMR,
13C-NMR has relatively determined the structure of Ligustrum lucidum Ait.
Embodiment 6
Get Glossy Privet Fruit 1000g, add 10 times of amount 65% alcohol reflux 2 hours, filter, filter residue adds 8 times of amount 65% alcohol reflux 1.5 hours again, filters, merge extracted twice liquid, reclaim ethanol, the D101 macroporous resin column of the aqueous solution by having handled well, water respectively, 8% ethanol, 40% ethanol, 0.8% sodium hydroxide solution wash-out, collect 40% ethanol elution part, concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Ligustrum lucidum Ait, through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Ligustrum lucidum Ait stream part, crystallization obtains Ligustrum lucidum Ait (19.2g, purity: pure product 98.6%), through respectively with the UV of Ligustrum lucidum Ait standard substance, IR, ESI-MS;
1H-NMR,
13C-NMR has relatively determined the structure of Ligustrum lucidum Ait.
Embodiment 7
Get Glossy Privet Fruit 1000g, add 15 times of amount 60% alcohol reflux 2 hours, filter, filter residue adds 12 times of amount 60% alcohol reflux 2 hours again, filters, merge extracted twice liquid, reclaim ethanol, the D101 macroporous resin column of the aqueous solution by having handled well, water respectively, 6% ethanol, 35% ethanol, 1.2% sodium hydroxide solution wash-out, collect 35% ethanol elution part, concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Ligustrum lucidum Ait, through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Ligustrum lucidum Ait stream part, crystallization obtains Ligustrum lucidum Ait (17.9g, purity: pure product 98.8%), through respectively with the UV of Ligustrum lucidum Ait standard substance, IR, ESI-MS;
1H-NMR,
13C-NMR has relatively determined the structure of Ligustrum lucidum Ait.
Embodiment 8
Get Glossy Privet Fruit 1000g, add 9 times of amount 65% alcohol reflux 2.5 hours, filter, filter residue adds 8 times of amount 65% alcohol reflux 2 hours again, filters, merge extracted twice liquid, reclaim ethanol, the D101 macroporous resin column of the aqueous solution by having handled well, water respectively, 5% ethanol, 40% ethanol, 1% sodium hydroxide solution wash-out, collect 40% ethanol elution part, concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Ligustrum lucidum Ait, through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Ligustrum lucidum Ait stream part, crystallization obtains Ligustrum lucidum Ait (16.7g, purity: pure product 98.7%), through respectively with the UV of Ligustrum lucidum Ait standard substance, IR, ESI-MS;
1H-NMR,
13C-NMR has relatively determined the structure of Ligustrum lucidum Ait.
Experimental data 1: Ligustrum lucidum Ait is to the active restraining effect of neuraminidase
Get the Ligustrum lucidum Ait that embodiment 1 preparation method is obtained, add suitable quantity of water and make dissolving, use tiring of neuraminidase inhibitor identification kit mensuration Ligustrum lucidum Ait inhibition neuraminidase (N1) and see Table 1.
(1). typical curve is prepared: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases and detects damping fluid; 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 damping fluid; B. every hole adds 10 μ l H5N1 neuraminidases again; C. every hole adds 0~10 μ l Ligustrum lucidum Ait 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 typical 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 fluorometric assay after ℃ hatching 20~30min.Excitation wavelength is 360nm, and emission wavelength is 440nm.
(4). calculate the inhibition per-cent of sample according to typical curve, and calculate the IC50 of Ligustrum lucidum Ait after doing concentration curve for the H5N1 neuraminidase for the H5N1 neuraminidase.The inhibiting rate IC50 that Ligustrum lucidum Ait reaches neuraminidase is 0.33g/L.See Table 1.
Table 1. Ligustrum lucidum Ait suppresses the activity of neuraminidase
Can be clear that according to above-mentioned experimental result:
(1). Ligustrum lucidum Ait can extract effective inhibition neuraminic acid enzyme component;
(2). Ligustrum lucidum Ait is 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 becomes positive correlation;
(3). by above-mentioned experiment as seen, Ligustrum lucidum Ait can be by suppressing influenza virus surface neuraminidase, and then suppress that influenza virus enters the cell the inside, the influenza virus that suppresses to have entered 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 Ligustrum lucidum Ait has prevention and treats the good result that the influenza virus sexuality is emitted.
Experimental data 2: Ligustrum lucidum Ait is to the restraining effect of influenza virus infected chicken embryo
Get the Ligustrum lucidum Ait that embodiment 1 preparation method is obtained, use influenza virus A-prime mouse lung adapted strain (FM1) and (H1N1) identify that Ligustrum lucidum Ait suppresses the ability that the FM1 influenza virus is duplicated and suppresses in the chicken embryo.(1). FM1 influenza virus liquid is inoculated in 10d no-special pathogen in the age chick embryo allantoic cavity, and every embryo 0.2ml is hatched 72h for 37 ℃, observes and calculate half chicken embryo infective dose (EID50).(2). Ligustrum lucidum Ait adopts the toxic action of chicken embryo, stroke-physiological saline solution is done to be inoculated in 10d no-special pathogen in the age chick embryo allantoic cavity behind the serial dilution to Ligustrum lucidum Ait, every embryo 0.2ml, each concentration is inoculated 6 embryos, hatch for 37 ℃, observe chicken embryonic development developmental state, can survive the peak concentration of 96h as the TD of medicine with the chicken embryo.(3). Ligustrum lucidum Ait restraining effect to influenza virus in the chicken embryo adopts, and the influenza virus liquid of 0.1ml mixes with different dilution Ligustrum lucidum Aits, and 37 ℃ of effect 2h are inoculated in 10d no-special pathogen in age chick embryo allantoic cavity, and every winding kind 6 embryos are hatched 72h for 37 ℃.The virus attack amount is 50EID50, establishes virus control, stroke-physiological saline solution normal control simultaneously, calculates the median effective dose (ED50) of Ligustrum lucidum Ait to viral inhibition.
(1) the .FM1 influenza virus is calculated through the Reed-Muench method the virulence of chicken embryo, and its EID50 is 10
-5.07
(2). after Ligustrum lucidum Ait was inoculated in the chicken embryo, it grew and normal control group basically identical.96h chicken embryo is all survived.The chicken embryo gives Ligustrum lucidum Ait stoste and does not see chicken embryo death, so can think that TD0 is 3.2g/L.(3). Ligustrum lucidum Ait restraining effect to influenza virus in the chicken embryo sees Table 2.
Table 2. Ligustrum lucidum Ait is to the restraining effect of influenza virus infected chicken embryo
Compare with the virus control group:
*P<0.05
As shown in Table 2, Ligustrum lucidum Ait has significant inhibitory effect (P<0.05), ED at 0.4~1.6g/L to influenza virus
50Be 0.63g ± 0.026g/L, TI is 29.68 ± 2.85.
Experimental data 3: Ligustrum lucidum Ait influences the FM1 influenza virus
Get the Ligustrum lucidum Ait that embodiment 1 preparation method is obtained, use influenza virus A-prime mouse lung adapted strain (FM1) and (H1N1) identify that Ligustrum lucidum Ait suppresses the ability of FM1 influenza virus virulence.(1) .FM1 adopts cell median infective dose (TCID50) micromethod to the toxicity test of dog kidney passage cell (MDCK).(2). Ligustrum lucidum Ait adopts the DMEM of serum-free that Ligustrum lucidum Ait is done to be inoculated in the mdck cell hole that forms individual layer behind the serial dilution to the toxicity test of mdck cell, every hole 100 μ l, each extent of dilution repeats 4 holes, establishes the normal cell contrast simultaneously.Culture plate is put 37 ℃, 5%CO
2Cultivate in the incubator, observation of cell pathology 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). Ligustrum lucidum Ait suppresses the effect of FM1 influenza virus and measures: 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 nutrient solution in the hole next day, add 100TCID50 influenza virus liquid, every hole 100 μ l, supernatant liquor is removed in suction behind 37 ℃ of absorption 1h.Washing 2 times with phosphate buffered saline buffer (PBS), is the 1st hole with the TD0 of medicine, with the DMEM liquid of serum-free Ligustrum lucidum Ait is made serial dilution again, adds respectively in the cell of above-mentioned infective virus, establishes 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. monolayer cell sex change becomes circle etc., and 3d calculates 50% of medicine and suppresses pathology 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 comparison test group and the cytopathic difference of virus control group, drug dose and inhibiting rate 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 Ligustrum lucidum Ait mdck cell is respectively 1.4g ± 0.09g/L.(3). after Ligustrum lucidum Ait 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, the results are shown in Table 3.
Table 3. Ligustrum lucidum Ait is to the IC of FM1 influenza virus
50(g/L) and TI (x ± s)
As shown in Table 3, the IC50 of Ligustrum lucidum Ait is low, the TI height.Ligustrum lucidum Ait suppresses the cytopathogenic effect of FM1 influenza virus all to be strengthened along with the increase of drug dose.Drug dose and medicine are shown that to the correlation analysis that the inhibiting rate of CPE carries out the dosage of Ligustrum lucidum Ait and medicine are to being tangible positive correlation between the CPE inhibiting rate.
Experimental data 4: Ligustrum lucidum Ait is to the spleen index and the influence of lung exponential of influenza virus infection FM1 strain in the mouse body
Get the Ligustrum lucidum Ait that embodiment 1 preparation method is obtained, use influenza virus A-prime mouse lung adapted strain (FM1) and (H1N1) identify the dead provide protection of Ligustrum lucidum Ait influenza virus infection FM1 strain in the mouse body.(1). influenza virus FM1 strain virus is inoculated every group of 10 BALB/C mice 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 mouse collunarium is inoculated 20 μ l.Observe the dead mouse situation of 10d, calculating LD50 by the Reed-Muench method is 10
-1.36So determine that the used modeling concentration of experiment is 10LD50.(2). Ligustrum lucidum Ait is to the dead provide protection of influenza virus infection FM1 strain in the mouse body: normal control group, influenza virus FM1 strain virus control group, Ligustrum lucidum Ait 0.25g/L, 0.5mg/L, 1.0g/L, 2.0g/L dosage group etc. are irritated stomach respectively, irritate gastric capacity and only are 0.4ml/.Behind the 3d, each group is only used 10LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ under the slight anesthesia of ether except that the normal control group.The normal control group gives the physiological 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 ratio (mortality ratio=every group of death toll/every group of total mice * 100%), the results are shown in Table 4.(3). Ligustrum lucidum Ait influences influenza virus infection FM1 strain lung exponential in the mouse body: normal control group, influenza virus FM1 strain virus control group, Ligustrum lucidum Ait 0.25g/L, 0.5mg/L, 1.0g/L, 2.0g/L dosage group etc. are irritated stomach respectively, irritate gastric capacity and only are 0.4ml/.After 3 days, each group is only used 1.0LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ under the slight anesthesia of ether except that the normal control group.The normal control group gives the physiological 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 virus infection, put to death mouse on the 8th day, weigh, get lung and claim lung heavy, calculate lung index (lung index=lung quality/physique amount * 100%); In addition, get spleen and claim spleen heavy, calculate spleen index (spleen index=spleen quality/physique amount * 100%), the results are shown in Table 5.
Table 4. Ligustrum lucidum Ait is to the death protection result of influenza virus infection FM1 strain in the mouse body
Annotate: ※ ※ P<0.01VS influenza virus model group
Table 5. Ligustrum lucidum Ait is to the spleen index and the influence of lung exponential of influenza virus infection FM1 strain in the mouse body
Annotate: #P<0.05VS normal control group is annotated: ##P<0.01VS normal control group
※ ※ p<0.001VS influenza virus model group ※ p<0.05VS influenza virus model group
(1). as shown in Table 4, Ligustrum lucidum Ait has significant provide protection (p<0.01) at 1.0~2.0g/L to the influenza virus infecting mouse.
(2). as shown in Table 5, Ligustrum lucidum Ait has significant effect (p<0.01) at 2.0g/L to the lung index inhibiting rate of influenza virus infecting mouse.
Experimental data 5: Ligustrum lucidum Ait is to the influence of renal dysfunction
1) SD rat, 200~240g, Shanghai west pul-Bi Kai laboratory animal responsibility company limited, animal conformity certification number: SCXK (Shanghai) 2003-0002
2) reagent and medicine VITAMIN B4, VITAMIN B4, (content>98% is the import packing, Chinese Shanghai, lot number 20010520), the Ligustrum lucidum Ait that embodiment 1 preparation method is 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 control group at random by body weight, 13~15 every group.Administration experimental group and modeling control group are irritated stomach with VITAMIN B4 and are made chronic renal failure (CRF) model, irritate stomach with VITAMIN B4 320mg/ (kgd), only make the about 2ml/ of suspension with distilled water, totally 20 days; Administration after 20 days, the administration experimental group is irritated stomach with Ligustrum lucidum Ait 4.0g/ (kgd), Ligustrum lucidum Ait is made suspension solution (0.5g/ml) with distilled water, each about 2ml/ only, stomach is irritated in gradation, normal control group and modeling control group are irritated stomach with equal-volume water, and administration was used etherization with rat after 35 days, and every index is observed in the blood sampling of mouse caudal artery.
4) result:
Table 6. Ligustrum lucidum Ait is to the influence of CRF kidney of rats function
Annotate: through the T check, administration Ligustrum lucidum Ait and modeling control group and normal control group be " * " expression P<0.05 relatively, and " * * " represents P<0.01
This experiment is looked sidelong at purine with gland and is irritated after stomach sets up rat CRF model, the modeling control rats is One's spirits are drooping, 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 Ligustrum lucidum Ait, and very remarkable with the model group comparing difference, so Ligustrum lucidum Ait is to the kidney free of toxic effects.