CN101879197B - Turpinia Montana leaf ethanol reflux extractive, preparation method and application thereof - Google Patents
Turpinia Montana leaf ethanol reflux extractive, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a turpinia montana leaf ethanol reflux extractive which is obtained by extracting turpinia montana and ethanol. The specific extract method is as follows: getting turpinia Montana leaves, adding 30-90% of ethanol with the volume of 5-15 times of the volume of the turpinia Montana leaves, carrying out reflux extraction for 1-3 times, and filtering, wherein the time of each reflux extraction is 1-3 hours; and combining the filter liquors, decompressing and concentrating the combine filter liquor into a thick paste, and drying to obtain the turpinia montana leaf ethanol reflux extractive. The extractive can serve as a neuraminidase inhibitor to be used for preventing and curing influenza, and can be prepared into a pharmaceutically acceptable dosage form.
Description
Technical field
The present invention relates to a kind of turpinia Montana leaf ethanol reflux extractive.
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.
Therefore; Specific aim suppresses influenza virus and duplicates, suppresses influenza neuraminidase hydrolysis cell surface sialic acid; Thereby cause influenza virus not combine with cell surface receptor and to get in the cell, and the minimizing influenza virus will be the cause of disease that treatment influenza virus sexuality is emitted in intracellular generation.
Summary of the invention
The purpose of this invention is to provide a kind of turpinia Montana leaf ethanol reflux extractive; Suppress influenza neuraminidase hydrolysis cell surface sialic acid; Cause influenza virus not combine with cell surface receptor and to get in the cell; Duplicating thereby can effectively suppress influenza virus, more importantly is the side reaction drawback that overcomes medicine.
Another object of the present invention has provided a kind of method for preparing of turpinia Montana leaf ethanol reflux extractive.
Another object of the present invention has provided the purposes of the medicine of turpinia Montana leaf ethanol reflux extractive preparation prevention and treatment influenza and complication disease thereof;
Another object of the present invention has provided the pharmaceutical composition that contains turpinia Montana leaf ethanol reflux extractive.
Turpinia Montana leaf ethanol reflux extractive of the present invention; It obtains through Turpinia pomifera(Roxb) D O. is added ethanol extraction, and concrete 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 volumes; Each reflux, extract, 1～3 hour filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Preferably, concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., add 40%～85% alcohol reflux 2～3 times of 5～13 times of volumes, each reflux, extract, 1～2.5 hour filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Preferred, concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., add 50%～80% alcohol reflux 2～3 times of 5～11 times of volumes, each reflux, extract, 1～2 hour filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Preferred, concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., add 55%～75% alcohol reflux 2～3 times of 5～10 times of volumes, each reflux, extract, 1～1.5 hour filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Preferred, concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 60～75% ethanol of 6～9 times of volumes as extracting solvent; Reflux, extract, 2 times, each 2 hours, merge extractive liquid; Filter, be evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Preferably, concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 65% ethanol of 7 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid, filtered, and was evaporated to thick paste or dry turpinia Montana leaf ethanol reflux extractive.
Preferred, concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 70% ethanol of 8 times of volumes as extracting solvent; Reflux, extract, 2 times, each 2 hours, merge extractive liquid; Filter, be evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
The purposes that the present invention also provides turpinia Montana leaf ethanol reflux extractive to be used to prepare prevention and to treat the medicine of influenza and complication disease thereof.
Preferably, turpinia Montana leaf ethanol reflux extractive is used to suppress influenza virus.
Preferably, turpinia Montana leaf ethanol reflux extractive is used to suppress influenza virus FM1.
Preferably, turpinia Montana leaf ethanol reflux extractive is used to suppress neuraminidase.
Preferably, its complication is meant renal failure.
Preferably, its complication is meant splenic trauma or/and injury of lung.
The present invention also provides a kind of turpinia Montana leaf ethanol reflux extractive preparation, and said preparation is main active with the turpinia Montana leaf ethanol reflux extractive.
Turpinia Montana leaf ethanol reflux extractive of the present invention can extract effective inhibition neuraminic acid enzyme component; Turpinia Montana leaf ethanol reflux extractive is along with the using dosage size variation; 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; Turpinia Montana leaf ethanol reflux extractive 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, thus infection, the growth of influenza virus pair cell reduced, and prevention and treatment influenza and complication thereof.
The specific embodiment
Turpinia Montana leaf ethanol reflux extractive of the present invention; It obtains through Turpinia pomifera(Roxb) D O. is added ethanol extraction, and concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., add 5～15 times of amount 30%～90% alcohol reflux 1～3 time; Each reflux, extract, 1～3 hour filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Embodiment 1
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., add 13 times of amount 45% alcohol reflux 3 times, each reflux, extract, 2.5 hours filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Embodiment 2
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., add 11 times of amount 50% alcohol reflux 3 times, each reflux, extract, 2 hours filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Embodiment 3
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., add 12 times of amount 70% alcohol reflux 3 times, each reflux, extract, 2 hours filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Embodiment 4
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., add 9 times of amount 65% alcohol reflux 3 times, each reflux, extract, 2 hours filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Embodiment 5
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., add 8 times of amount 70% alcohol reflux 2 times, each reflux, extract, 2 hours filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Embodiment 6
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., add 7 times of amount 65% alcohol reflux 2 times, each reflux, extract, 2 hours filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Embodiment 7
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., add 9 times of amount 68% alcohol reflux 2 times, each reflux, extract, 2 hours filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Embodiment 8
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., add 5 times of amount 85% alcohol reflux 1 time, reflux, extract, 1 hour filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Experimental data 1, extraction process optimization research
Leaf of Turpinia pomifera (Roxb) D O. mainly contains chemical compounds such as flavonoid, organic acid; Flavone compound is preferable with the ethanol extraction effect, therefore, we with concentration of alcohol (A), add four of amount of alcohol (B), extraction time (C), extraction time (D) as the investigation factor; Each factor is established three levels, presses L9 (3
4) orthogonal table carries out orthogonal experiment design (table 1, table 2) extraction process is optimized research, investigating index is the total flavones extraction ratio.
Table 1 factor level table
Get 27 parts of leaf of Turpinia pomifera (Roxb) D O. medical materials, every group average three parts, totally nine groups, by table 1L9 (3
4) the orthogonal test table design experiment.The result sees table 2.
Table 2L9 (3
4) orthogonal test table
Variance analysis
Orthogonal experiments is carried out variance analysis, and the result sees table 3.
Table 3 is the variance analysis of index with the total flavones extraction ratio
＊F
1-0.05(2，2)＝19.0
＊＊F
1-0.01(2，2)＝99.0
Conclusion: intuitive analysis can know, extraction time (C) and to add amount of alcohol (B) bigger to the extraction effect influence, and concentration of alcohol (A) and extraction time (D) are less to the extraction effect influence.Table 3 The results of analysis of variance has further been verified the intuitive analysis conclusion, and promptly factor C, B have considerable influence to extraction effect successively.According to producing practical situation, analyze in conjunction with the K value, confirm that extraction conditions is: get leaf of Turpinia pomifera (Roxb) D O., add 10 times of amount 70% ethanol and carry 3 times, extracted 2 hours at every turn, promptly get.
Experimental data 2: turpinia Montana leaf ethanol reflux extractive is to the active inhibitory action of neuraminidase
Get the turpinia Montana leaf ethanol reflux extractive that embodiment 4 method for preparinies are obtained, add suitable quantity of water and make dissolving, use tiring of neuraminidase inhibitor identification kit mensuration turpinia Montana leaf ethanol reflux extractive inhibition neuraminidase (N1) and see table 4.
(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 turpinia Montana leaf ethanol reflux extractive 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 calculate the IC50 of turpinia Montana leaf ethanol reflux extractive after doing concentration curve for the H5N1 neuraminidase for the H5N1 neuraminidase.The suppression ratio IC50 that turpinia Montana leaf ethanol reflux extractive reaches neuraminidase is 9.97mg/ml.See table 4.
Table 4. turpinia Montana leaf ethanol reflux extractive suppresses the activity of neuraminidase
Can be clear that according to above-mentioned experimental result:
(1). turpinia Montana leaf ethanol reflux extractive can extract effective inhibition neuraminic acid enzyme component;
(2). turpinia Montana leaf ethanol reflux extractive 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). visible by above-mentioned experiment; Turpinia Montana leaf ethanol reflux extractive 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 turpinia Montana leaf ethanol reflux extractive has prevention and treats the good result that the influenza virus sexuality is emitted.
Experimental data 3: turpinia Montana leaf ethanol reflux extractive infects the inhibitory action of Embryo Gallus domesticus to influenza virus
Get turpinia Montana leaf ethanol reflux extractive, add suitable quantity of water and make dissolving, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify that turpinia Montana leaf ethanol reflux extractive suppresses the ability that the FM1 influenza virus is duplicated and suppresses in Embryo Gallus domesticus.(1). FM1 influenza virus liquid is inoculated in no-special pathogen chick embryo allantois intracavity in 10 day age, and every embryo 0.2ml was hatched 72 hours for 37 ℃, observed and calculate half Embryo Gallus domesticus infective dose (EID50).(2). turpinia Montana leaf ethanol reflux extractive adopts the toxic action of Embryo Gallus domesticus; Physiological saline solution is done to be inoculated in no-special pathogen chick embryo allantois intracavity in 10 day age behind the serial dilution to turpinia Montana leaf ethanol reflux extractive; Every embryo 0.2ml, each concentration is inoculated 6 embryos, hatches for 37 ℃; Observe Embryo Gallus domesticus growth promoter situation, the Cmax that can survive 96 hours with Embryo Gallus domesticus is as the TD of medicine.(3). turpinia Montana leaf ethanol reflux extractive inhibitory action to influenza virus in Embryo Gallus domesticus adopts; 0.1ml influenza virus liquid and different dilution turpinia Montana leaf ethanol reflux extractives mix; 37 ℃ act on 2 hours; Be inoculated in no-special pathogen chick embryo allantoic cavity in 10 day age, every winding kind 6 embryos were hatched 72 hours for 37 ℃.The virus attack amount is 50EID50, establishes virus control, physiological saline solution normal control simultaneously, calculates the median effective dose (ED50) of turpinia Montana leaf ethanol reflux extractive 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 turpinia Montana leaf ethanol reflux extractive is inoculated in Embryo Gallus domesticus, its growth promoter and normal control group basically identical.Embryo Gallus domesticus was all survived in 96 hours.Embryo Gallus domesticus gives turpinia Montana leaf ethanol reflux extractive stock solution and does not see chicken embryo death, so can think that TD0 is 9.0g/100ml.(3). turpinia Montana leaf ethanol reflux extractive inhibitory action to influenza virus in Embryo Gallus domesticus is seen table 5.
Table 5. turpinia Montana leaf ethanol reflux extractive infects the inhibitory action of Embryo Gallus domesticus to influenza virus
Compare with the virus control group: * P＜0.05
Can be known that by table 5 turpinia Montana leaf ethanol reflux extractive has significant inhibitory effect (P＜0.05) at 22.5～90.0mg/ml to influenza virus, ED50 is 22.5mg ± 1.92mg/ml, and TI is 16.34 ± 1.35.
Experimental data 4: turpinia Montana leaf ethanol reflux extractive influences the FM1 influenza virus
Get turpinia Montana leaf ethanol reflux extractive, add suitable quantity of water and make dissolving, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify that turpinia Montana leaf ethanol reflux extractive suppresses 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). turpinia Montana leaf ethanol reflux extractive adopts the DMEM of serum-free that turpinia Montana leaf ethanol reflux extractive is 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) was observed 3 days continuously, with " +～++ ++ " the record result, press the Reed-Muench method and calculate medicine median toxic concentration (TD50) and maximal non-toxic concentration (TD0).(3). turpinia Montana leaf ethanol reflux extractive 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 ℃, 5%CO
2Cultivate in the incubator, inhale next day and remove culture fluid in the hole, add 100TCID50 influenza virus liquid, every hole 100 μ l inhale behind 37 ℃ of absorption 1h and remove supernatant.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 turpinia Montana leaf ethanol reflux extractive, 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., for three days on end, calculates 50% of medicine and suppresses pathological changes concentration (IC50) and therapeutic index (TI).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
-481(2). the TD0 of turpinia Montana leaf ethanol reflux extractive mdck cell is respectively 1026.4mg ± 30.27mg/100ml.(3). after turpinia Montana leaf ethanol reflux extractive 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 6.
Table 6. turpinia Montana leaf ethanol reflux extractive is to the IC50 (mg/100ml) of FM1 influenza virus and TI (x ± s)
Can know that by table 6 turpinia Montana leaf ethanol reflux extractive 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 suppression ratio of CPE carries out the dosage of turpinia Montana leaf ethanol reflux extractive and medicine are to being tangible positive correlation between the CPE suppression ratio.
Experimental data 5: turpinia Montana leaf ethanol reflux extractive is to the death protection result of influenza virus infection FM1 strain in the mice body
Get turpinia Montana leaf ethanol reflux extractive, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify the dead protective effect of turpinia Montana leaf ethanol reflux extractive 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 10 days dead mouse situation, calculating LD50 by the Reed-Muench method is 10-
1.36So confirm that the used modeling concentration of experiment is 10LD50.(2). turpinia Montana leaf ethanol reflux extractive is 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, turpinia Montana leaf ethanol reflux extractive 11.25mg/ml, 22.5mg/ml, 45.0mg/ml, 90.0mg/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 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 7.(3). turpinia Montana leaf ethanol reflux extractive is 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, turpinia Montana leaf ethanol reflux extractive 11.25mg/ml, 22.5mg/ml, 45.0mg/ml, 90.0mg/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 7.
Table 7. turpinia Montana leaf ethanol reflux extractive is to the death protection result of influenza virus infection FM1 strain in the mice body
Annotate: ※ ※ P＜0.01VS influenza virus model group
Table 8. turpinia Montana leaf ethanol reflux extractive is to the spleen index and the exponential influence of lung of influenza virus infection FM1 strain in the mice body
Annotate: ##P＜0.01VS normal control group ※ p＜0.05VS influenza virus model group
(1). can know that by table 7 turpinia Montana leaf ethanol reflux extractive has significant protective effect (p＜0.01) at 45.0～90.0mg/ml to the influenza virus infecting mouse.
(2). can know that by table 8 turpinia Montana leaf ethanol reflux extractive has significant effect (p＜0.05) at 90.0mg/ml to the lung index suppression ratio of influenza virus infecting mouse.
Experimental data 6: turpinia Montana leaf ethanol reflux extractive is to the influence of chronic renal failure
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), the turpinia Montana leaf ethanol reflux extractive that embodiment 4 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, 12 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 turpinia Montana leaf ethanol reflux extractive 1.2g/ (kgd); Turpinia Montana leaf ethanol reflux extractive is processed suspension solution (0.2g/ml) with distilled water, and about at every turn 2ml/, 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 9. turpinia Montana leaf ethanol reflux extractive is to the influence of CRF kidney of rats function
Annotate: through the T check, administration 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; Showing the rat impaired renal function, after giving the turpinia Montana leaf ethanol reflux extractive treatment. rat spirit is obviously bestirred oneself, and body weight is recovered gradually; Improve creatinine clearance rate, promote the discharge of metabolite, reduce serum BUN, SCr concentration; Renal function and anemia are obviously improved, and show that turpinia Montana leaf ethanol reflux extractive has preventive and therapeutic effect to kidney of rats damage due to the adenine.
Can be clear that according to above-mentioned experimental result:
(1). turpinia Montana leaf ethanol reflux extractive can extract effectively preventing chronic renal failure composition;
(2). turpinia Montana leaf ethanol reflux extractive suppresses the active effect of neuraminidase simultaneously having, also have the effect of the chronic renal failure controlled, and has overcome oseltamivir phosphate capsule sternly to clear ability of creatinine and renal failure crowd's side effect, on the contrary prevention effect in addition.
Claims (6)
1. the application of turpinia Montana leaf ethanol reflux extractive in the medicine of preparation inhibition influenza virus FM1; Said turpinia Montana leaf ethanol reflux extractive obtains through leaf of Turpinia pomifera (Roxb) D O. is added ethanol extraction; Concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O.; 40%～85% alcohol reflux 2～3 times that adds 5～13 times of volumes, each reflux, extract, 1～2.5 hour filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
2. application as claimed in claim 1, concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., add 50%～80% alcohol reflux 2～3 times of 5～11 times of volumes, each reflux, extract, 1～2 hour filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
3. application as claimed in claim 2, concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., add 55%～75% alcohol reflux 2～3 times of 5～10 times of volumes, each reflux, extract, 1～1.5 hour filters; Merging filtrate is evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
4. the application of turpinia Montana leaf ethanol reflux extractive in the medicine of preparation inhibition influenza virus FM1, said turpinia Montana leaf ethanol reflux extractive obtains through leaf of Turpinia pomifera (Roxb) D O. is added ethanol extraction, and concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O.; Each 60～75% ethanol with 6～9 times of volumes are as the extraction solvent; Reflux, extract, 2 times, each 2 hours, merge extractive liquid; Filter, be evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
5. application as claimed in claim 4, concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 65% ethanol of 7 times of volumes as extracting solvent; Reflux, extract, 2 times, each 2 hours, merge extractive liquid; Filter, be evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
6. application as claimed in claim 4, concrete method for distilling is following: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 70% ethanol of 8 times of volumes as extracting solvent; Reflux, extract, 2 times, each 2 hours, merge extractive liquid; Filter, be evaporated to thick paste or the dry turpinia Montana leaf ethanol reflux extractive that gets.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101448712A CN101879197B (en) | 2010-04-13 | 2010-04-13 | Turpinia Montana leaf ethanol reflux extractive, preparation method and application thereof |
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| CN2010101448712A CN101879197B (en) | 2010-04-13 | 2010-04-13 | Turpinia Montana leaf ethanol reflux extractive, preparation method and application thereof |
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| CN2011103330226A Division CN102772453A (en) | 2010-04-13 | 2010-04-13 | Turpinia formosana Nakai leaf ethanol backflow extract and preparation method and applications thereof |
| CN2011103330230A Division CN102920752A (en) | 2010-04-13 | 2010-04-13 | Turpinia arguta leaf ethanol reflux extract, preparation method and uses thereof |
| CN2011103329924A Division CN102784177A (en) | 2010-04-13 | 2010-04-13 | Turpinia montana leaf ethanol reflux extractive, preparation method and application thereof |
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