CN102579523A - Acute turpinia leaf total flavone ethanol percolation extract as well as preparation method and application thereof - Google Patents
Acute turpinia leaf total flavone ethanol percolation extract as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an acute turpinia leaf total flavone ethanol percolation extract, which is prepared from acute turpinia through ethanol percolation extraction. The ethanol percolation extraction comprises the following steps: taking acute turpinia leaf coarse powder, using 30 to 90 percent of ethanol as the extracting agent, soaking for 12 to 72 hours, carrying out percolation at the speed of 1 to 8 ml/kg/min, collecting percolation liquid accounting for 4 to 15 times the amount of medicinal materials, recovering the ethanol, absorbing aqueous solution with a non-polar macroreticular resin, eluting with ethanol of below 20 percent for removing impurities, eluting with 25 to 90 percent of ethanol, collecting eluate, recovering the ethanol, and then carrying out concentration and drying, so as to obtain the acute turpinia leaf total flavonoid ethanol percolation extract. The total flavone content is above 50 percent, and the main chemical components of flavonoids are ligustroflavone, rhoifolin, hyperin, quercetin, luteolin and apigenin and one or more of glycosides or aglycones of flavonoids. The acute turpinia leaf total flavone ethanol percolation extract can be used as the neuraminidase inhibitor for preventing and treating flu, and can be prepared into pharmaceutically accepted dosage forms.
Description
Technical field
The present invention relates to a kind of acute turpinia leaf general flavone ethanol reflux extract.
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 acute turpinia leaf general flavone ethanol reflux extract; 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 acute turpinia leaf general flavone ethanol reflux extract.
Another object of the present invention has provided the purposes of the medicine of acute turpinia leaf general flavone ethanol reflux extract preparation prevention and treatment influenza and complication disease thereof;
Another object of the present invention has provided the pharmaceutical composition that contains acute turpinia leaf general flavone ethanol reflux extract.
Acute turpinia leaf general flavone ethanol reflux extract of the present invention obtains through the Turpinia pomifera(Roxb) D O. alcohol percolation is extracted, and the alcohol percolation method for distilling is: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, with 30～90% ethanol as extractant; Flood after 12～72 hours, carry out percolation, collect the percolate of 4～15 times of amounts of medical material with 1～8ml/kg/ minute speed; Reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 25～90% ethanol elutions are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferably, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 24～48 hours with 40～80% ethanol; Carry out percolation with 1～7ml/kg/ minute speed, collect the percolate of 4～13 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with below 20% is removed impurity, and reuse 30～85% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 24～36 hours with 45～75% ethanol; Carry out percolation with 1～6m/kg/ minute speed, collect the percolate of 4～11 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with below 20% is removed impurity, and reuse 35～80% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 36 hours with 50～70% ethanol; Carry out percolation with 1～5ml/kg/ minute speed, collect the percolate of 4～10 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with below 20% is removed impurity, and reuse 40～80% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferably, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 24 hours with 55～70% ethanol; Carry out percolation with 2～5ml/kg/ minute speed, collect the percolate of 5～8 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with below 20% is removed impurity, and reuse 45～80% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 12 hours with 60～70% ethanol; Carry out percolation with 3～5ml/kg/ minute speed, collect the percolate of 4～6 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with below 20% is removed impurity, and reuse 50～75% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 36 hours with 65% ethanol; Carry out percolation with 4ml/ minute speed, collect the percolate of 7～10 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with below 20% is removed impurity, and reuse 50～70% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 72 hours with 70% ethanol; Carry out percolation with 4～5ml/kg/ minute speed, collect the percolate of 4 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with below 20% is removed impurity, and reuse 50% ethanol is collected eluent, reclaims ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferably, described acute turpinia leaf general flavone ethanol reflux extract, wherein general flavone content is more than 50%.
Preferably, the acute turpinia leaf general flavone ethanol reflux extract of stating wherein also comprises flavonoid chemical constituent and acceptable liposoluble ingredient medically thereof.
Preferably, described acute turpinia leaf general flavone ethanol reflux extract, wherein the flavonoid chemical constituent contains one or more in nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin, Quercetin, luteolin, apigenin and glycosides thereof or the aglycon.
Preferably, described acute turpinia leaf general flavone ethanol reflux extract, wherein contain nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin reach 4.5%, 1.2% respectively, more than 2.5%.
Preferably, described acute turpinia leaf general flavone ethanol reflux extract wherein contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin total amount and reaches more than 23%.
The purposes that the present invention also provides acute turpinia leaf general flavone ethanol reflux extract to be used to prepare prevention and to treat the medicine of influenza and complication disease thereof; Be used to suppress influenza virus, suppress influenza virus FM1, suppress neuraminidase, its complication is meant renal failure or refers to splenic trauma or/and injury of lung.
The present invention also provides a kind of acute turpinia leaf general flavone ethanol reflux extract preparation, and said preparation is a main active with described acute turpinia leaf general flavone ethanol reflux extract.
Acute turpinia leaf general flavone ethanol reflux extract of the present invention can extract effective inhibition neuraminic acid enzyme component; Acute turpinia leaf general flavone ethanol reflux extract 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, acute turpinia leaf general flavone ethanol reflux extract 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.
The specific embodiment
Acute turpinia leaf general flavone ethanol reflux extract of the present invention, it is obtain through the Turpinia pomifera(Roxb) D O. alcohol percolation is extracted, the alcohol percolation method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, with 30～90% ethanol as extractant; Flood after 12～72 hours, carry out percolation, collect the percolate of 4～15 times of amounts of medical material with 1～8ml/kg/ minute speed; Reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 25～90% ethanol elutions are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferably, described acute turpinia leaf general flavone ethanol reflux extract, wherein general flavone content is more than 50%.
Preferably; Described acute turpinia leaf general flavone ethanol reflux extract; Wherein also comprise flavonoid chemical constituent and acceptable liposoluble ingredient medically thereof, wherein the flavonoid chemical constituent contains one or more in nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin, Quercetin, luteolin, apigenin and glycosides thereof or the aglycon.Wherein contain nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin reach 4.5%, 1.2% respectively, more than 2.5%.
Preferred, wherein contain nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin total amount and reach more than 23%.
Embodiment 1
Method for preparing: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 24 hours, carry out percolation with 7ml/kg/ minute speed with 50% ethanol; Collect the percolate of 11 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 15% is removed impurity, and 50% ethanol elution of 6 times of amounts of reuse column volume is collected eluent; Reclaim ethanol, concentrate drying; Get acute turpinia leaf general flavone ethanol reflux extract, general flavone content 53.6% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 15%, 5%, 3% respectively.
Embodiment 2
Method for preparing: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 48 hours, carry out percolation with 6ml/kg/ minute speed with 60% ethanol; Collect the percolate of 9 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 18% is removed impurity, and 55% ethanol elution of 5 times of amounts of reuse column volume is collected eluent; Reclaim ethanol, concentrate drying; Get acute turpinia leaf general flavone ethanol reflux extract, general flavone content 57.3% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 13%, 6%, 5% respectively.
Embodiment 3
Method for preparing: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 40 hours, carry out percolation with 5ml/kg/ minute speed with 65% ethanol; Collect the percolate of 8 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 20% is removed impurity, and 60% ethanol elution of 5 times of amounts of reuse column volume is collected eluent; Reclaim ethanol, concentrate drying; Get acute turpinia leaf general flavone ethanol reflux extract, general flavone content 63.7% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 10%, 8%, 7% respectively.
Embodiment 4
Method for preparing: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 72 hours, carry out percolation with 4ml/kg/ minute speed with 70% ethanol; Collect the percolate of 7 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 20% is removed impurity, and 50% ethanol elution of 5 times of amounts of reuse column volume is collected eluent; Reclaim ethanol, concentrate drying; Get acute turpinia leaf general flavone ethanol reflux extract, general flavone content 82.5% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 7%, 3%, 7% respectively.
Embodiment 5
Method for preparing: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 36 hours, carry out percolation with 2ml/kg/ minute speed with 75% ethanol; Collect the percolate of 6 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 20% is removed impurity, and 50% ethanol elution of 6 times of amounts of reuse column volume is collected eluent; Reclaim ethanol, concentrate drying; Get acute turpinia leaf general flavone ethanol reflux extract, general flavone content 74.6% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 6%, 6%, 11% respectively.
Embodiment 6
Method for preparing: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 24 hours, carry out percolation with 3ml/kg/ minute speed with 80% ethanol; Collect the percolate of 5 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 20% is removed impurity, and 40% ethanol elution of 5 times of amounts of reuse column volume is collected eluent; Reclaim ethanol, concentrate drying; Get acute turpinia leaf general flavone ethanol reflux extract, general flavone content 65.8.6% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 16%, 7%, 4% respectively.
Embodiment 7
Method for preparing: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 72 hours, carry out percolation with 1ml/kg/ minute speed with 85% ethanol; Collect the percolate of 10 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 17% is removed impurity, and 30% ethanol elution of 4 times of amounts of reuse column volume is collected eluent; Reclaim ethanol, concentrate drying; Get acute turpinia leaf general flavone ethanol reflux extract, general flavone content 58.7% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 18%, 6%, 8% respectively.
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), collect four of amount of alcohol (B), percolation speed (C), dip 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 rate of transform
＊F
1-0.05(2，2)＝19.0
＊＊F
1-0.01(2，2)＝99.0
Conclusion: intuitive analysis can know that collection amount of alcohol (B) is bigger to the extraction effect influence, and dip time (A), percolation speed (C) and concentration of alcohol (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 B has appreciable impact to extraction effect.Analyze in conjunction with the K value, confirm that extraction conditions is: get leaf of Turpinia pomifera (Roxb) D O., with 70% alcohol dipping after 72 hours; With 3～6ml/kg/ minute percolation; Collect 10 times of amounts of medical material percolate, reclaim ethanol, be evaporated to thick paste or the dry acute turpinia leaf general flavone ethanol reflux extract that gets.
Experimental data 2, leaf of Turpinia pomifera (Roxb) D O. total flavones purification research
The leaf of Turpinia pomifera (Roxb) D O. medical material proposes a large amount of non-flavone compounds after with ethanol extraction; Must be further purified processing to alcohol extract; Improving the content of flavonoids effective constituent, mostly impurity is inorganic salt, saccharide, pectin and protein-based, esters, pigment etc. in the extracting solution, and the active component in the leaf of Turpinia pomifera (Roxb) D O. is a flavone compound; Therefore, we adopt macroporous adsorbent resin and carry out purification research.
1) applied sample amount is investigated
Get the leaf of Turpinia pomifera (Roxb) D O. medical material and extract in right amount, D-101 macroporous resin (500ml) on the extracting solution, preceding 500ml collects one bottle, and the every 150ml in back collects one bottle, carries out the flavone qualitative identification, the flavone positive reaction is arranged to the 2000ml beginning.(quality that is equivalent to crude drug is 50g) do not detect flavone as yet when applied sample amount was 2000ml, so the preliminary extracting solution of confirming appearance 100g medical material on every 1000ml macroporous resin, i.e. resin volume: the crude drug quality is 10: 1.
2) investigation of eluting concentration
Get Turpinia pomifera(Roxb) D O. herbal extract 100ml (being equivalent to raw medicinal herbs 100g), be splined on 1000ml D101 resin.Water, 15%, 25%, 35%, 45%, 55% ethanol elution carry out assay to 15%, 25%, 35%, 45%, 55% ethanol elution successively, and part eluent evaporate to dryness calculates the rate of extract, and the result sees table 4.
The investigation of table 4 eluting concentration
Annotate :-expression HPLC does not detect flavones ingredient.
From table 4 can find out flavonoid glycoside with the optium concentration of ethanol elution 30%～50%, and effective ingredient can not leak and washes.15% ethanol and 45% ethanol elution part total flavones amount are all lower in addition; And the impurity that 15% ethanol is removed is more; Therefore, 15% ethanol just can be as the eluting solvent of impurity, and 30%～50% ethanol then can be used as the desorption agent of flavone; Can improve the content of total flavonoid glycoside in effective site like this, reach the purpose of separation and purification.
3) investigation of elution volume
Get leaf of Turpinia pomifera (Roxb) D O. medical material 100g and extract, extracting solution is splined on the 1000ml resin after reclaiming ethanol, washing 4000ml, and 15% ethanol is washed 6000ml, and 50% ethanol is washed 6000ml.The result sees table 5.
The investigation of table 5 elution volume
"-" expression does not detect flavones ingredient; "+" expression detects flavones ingredient.
Can find out usefulness from the qualitative examination result; 15% ethanol elution has been removed a large amount of non-flavones ingredients; Having a small amount of effective ingredient runs off; After eluting 3500ml, eluent paler colour (eluting power that is impurity descends) is so 15% ethanol part elution volume is confirmed as 3500ml (being that every 1ml resin is with 3.5ml 15% ethanol elution) simultaneously.When 5000ml, effective ingredient eluting is complete during 50% ethanol elution, so with 50% ethanol 4000ml eluting (being that the 1ml resin is with 4.5ml 45% ethanol elution).
Experimental data 3: acute turpinia leaf general flavone ethanol reflux extract is to the active inhibitory action of neuraminidase
Get acute turpinia leaf general flavone ethanol reflux extract, add suitable quantity of water and make dissolving, use tiring of neuraminidase inhibitor identification kit mensuration acute turpinia leaf general flavone ethanol reflux extract inhibition neuraminidase (N1) and see table 6.
(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 acute turpinia leaf general flavone ethanol reflux extract 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, do standard curve
Sample 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 acute turpinia leaf general flavone ethanol reflux extract after doing concentration curve for the H5N1 neuraminidase for the H5N1 neuraminidase.The suppression ratio IC50 that acute turpinia leaf general flavone ethanol reflux extract reaches neuraminidase is 3.72mg/ml.See table 6.
Table 6. acute turpinia leaf general flavone ethanol reflux extract suppresses the activity of neuraminidase
Can be clear that according to above-mentioned experimental result:
(1). acute turpinia leaf general flavone ethanol reflux extract can extract effective inhibition neuraminic acid enzyme component;
(2). acute turpinia leaf general flavone ethanol reflux extract 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; Acute turpinia leaf general flavone ethanol reflux extract 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 acute turpinia leaf general flavone ethanol reflux extract has prevention and treats the good result that the influenza virus sexuality is emitted.
Experimental data 4: acute turpinia leaf general flavone ethanol reflux extract is to the influence of FM1 influenza virus
Get acute turpinia leaf general flavone ethanol reflux extract, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify that acute turpinia leaf general flavone ethanol reflux extract 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). acute turpinia leaf general flavone ethanol reflux extract adopts the DMEM of serum-free that acute turpinia leaf general flavone ethanol reflux extract 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) 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). acute turpinia leaf general flavone ethanol reflux extract 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 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 acute turpinia leaf general flavone ethanol reflux extract, 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
-4.81(2). the TD0 of acute turpinia leaf general flavone ethanol reflux extract mdck cell is respectively 1044.86mg ± 28.11mg/100ml.(3). after acute turpinia leaf general flavone ethanol reflux extract 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 7.
Table 7. acute turpinia leaf general flavone ethanol reflux extract is to the IC50 (mg/100ml) of FM1 influenza virus and TI (x ± s)
Can know that by table 7 acute turpinia leaf general flavone ethanol reflux extract 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 acute turpinia leaf general flavone ethanol reflux extract and medicine are to being tangible positive correlation between the CPE suppression ratio.
Experimental data 5: acute turpinia leaf general flavone ethanol reflux extract infects the inhibitory action of Embryo Gallus domesticus to influenza virus
Get acute turpinia leaf general flavone ethanol reflux extract, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify that acute turpinia leaf general flavone ethanol reflux extract 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 is hatched 72h for 37 ℃, observes and calculate half Embryo Gallus domesticus infective dose (EID50).(2). acute turpinia leaf general flavone ethanol reflux extract adopts the toxic action of Embryo Gallus domesticus; With physiological saline solution acute turpinia leaf general flavone ethanol reflux extract is done to be inoculated in 10d no-special pathogen in age chick embryo allantois intracavity behind the serial dilution; 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). acute turpinia leaf general flavone ethanol reflux extract inhibitory action to influenza virus in Embryo Gallus domesticus adopts; 0.1ml influenza virus liquid and different dilution acute turpinia leaf general flavone ethanol reflux extracts mix; 37 ℃ of effect 2h; Be inoculated in no-special pathogen chick embryo allantoic cavity in 10 day age, 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 the median effective dose (ED50) of acute turpinia leaf general flavone ethanol reflux extract 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 acute turpinia leaf general flavone ethanol reflux extract is 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 acute turpinia leaf general flavone ethanol reflux extract stock solution and does not see chicken embryo death, so can think that TD0 is 8.4g/100ml.(3). acute turpinia leaf general flavone ethanol reflux extract inhibitory action to influenza virus in Embryo Gallus domesticus is seen table 8.
Table 8. acute turpinia leaf general flavone ethanol reflux extract 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 8 acute turpinia leaf general flavone ethanol reflux extract has significant inhibitory effect (P＜0.05) at 11.25～90.0mg/ml to influenza virus, ED50 is 10.24mg ± 0.49mg/ml, and TI is 41.42 ± 7.72.
Experimental data 6: acute turpinia leaf general flavone ethanol reflux extract is to the influence of influenza virus infection FM1 strain in the mice body
Get acute turpinia leaf general flavone ethanol reflux extract, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify the dead protective effect of acute turpinia leaf general flavone ethanol reflux extract 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). acute turpinia leaf general flavone ethanol reflux extract 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, acute turpinia leaf general flavone ethanol reflux extract 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, observe 14d altogether, calculate mortality rate (mortality rate=every group of death toll/every group of total mice * 100%), the result sees table 9.(3). acute turpinia leaf general flavone ethanol reflux extract 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, acute turpinia leaf general flavone ethanol reflux extract 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 9.
Table 9. acute turpinia leaf general flavone ethanol reflux extract is to the death protection result of influenza virus infection FM1 strain in the mice body
Annotate: ※ ※ P＜0.01VS influenza virus model group ※ P＜0.05VS influenza virus model group
Table 10. acute turpinia leaf general flavone ethanol reflux extract is 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 is annotated: ##P＜0.01VS normal control group
※ ※ p＜0.001VS influenza virus model group ※ p＜0.05VS influenza virus model group
(1). can know that by table 9 acute turpinia leaf general flavone ethanol reflux extract has significant protective effect (p＜0.01-p＜0.05) at 45.0～90.0mg/ml to the influenza virus infecting mouse.
(2). can know that by table 10 acute turpinia leaf general flavone ethanol reflux extract has significant effect (p＜0.01-p＜0.05) at 45.0mg/ml-90.0mg/ml to the lung index suppression ratio of influenza virus infecting mouse.
Experimental data 7: acute turpinia leaf general flavone ethanol reflux extract 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 acute turpinia leaf general flavone ethanol reflux extract 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 acute turpinia leaf general flavone ethanol reflux extract 0.36g/ (kgd); Acute turpinia leaf general flavone ethanol reflux extract is processed suspension solution (0.06g/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 11. acute turpinia leaf general flavone ethanol reflux extract 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 acute turpinia leaf general flavone ethanol reflux extract 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 acute turpinia leaf general flavone ethanol reflux extract 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). acute turpinia leaf general flavone ethanol reflux extract can extract effectively preventing chronic renal failure composition;
(2). acute turpinia leaf general flavone ethanol reflux extract has the active effect of the neuraminidase of inhibition simultaneously; The effect that also has the control chronic renal failure; Overcome oseltamivir phosphate capsule sternly to the clear ability of creatinine and renal failure crowd's side effect, otherwise also had prevention effect.
Claims (13)
1. acute turpinia leaf general flavone ethanol reflux extract suppresses the purposes in the medicine of neuraminidase in preparation, and said extract obtains through the Turpinia pomifera(Roxb) D O. alcohol percolation is extracted, and the alcohol percolation method for distilling is: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, with 30～90% ethanol as extractant; Flood after 12～72 hours, carry out percolation, collect the percolate of 4～9 times of amounts of medical material with 1～8ml/kg/ minute speed; Reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 25～90% ethanol elutions are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
2. purposes as claimed in claim 1, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, with 40～80% ethanol as extractant; Flood after 24～48 hours, carry out percolation, collect the percolate of 4～9 times of amounts of medical material with 1～7ml/kg/ minute speed; Reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 30～85% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
3. purposes as claimed in claim 2, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, with 45～75% ethanol as extractant; Flood after 24～36 hours, carry out percolation, collect the percolate of 4～8 times of amounts of medical material with 1～6m/kg/ minute speed; Reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 35～80% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
4. purposes as claimed in claim 3, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, with 50～70% ethanol as extractant; Flood after 36 hours, carry out percolation, collect the percolate of 4～8 times of amounts of medical material with 1～5ml/kg/ minute speed; Reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 40～80% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
5. purposes as claimed in claim 3, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, with 55～70% ethanol as extractant; Flood after 24 hours, carry out percolation, collect the percolate of 5～8 times of amounts of medical material with 2～5ml/kg/ minute speed; Reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 45～80% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
6. purposes as claimed in claim 1, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, with 60～70% ethanol as extractant; Flood after 12 hours, carry out percolation, collect the percolate of 4～6 times of amounts of medical material with 3～5ml/kg/ minute speed; Reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 50～75% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
7. purposes as claimed in claim 4, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 36 hours with 65% ethanol; Carry out percolation with 4ml/ minute speed, collect the percolate of 7～8 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with below 20% is removed impurity, and reuse 50～70% ethanol are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
8. purposes as claimed in claim 1, concrete method for distilling is following: get the leaf of Turpinia pomifera (Roxb) D O. coarse powder, as extractant, flood after 72 hours with 70% ethanol; Carry out percolation with 4～5ml/kg/ minute speed, collect the percolate of 4 times of amounts of medical material, reclaim ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with below 20% is removed impurity, and reuse 50% ethanol is collected eluent, reclaims ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
9. like each described purposes of claim 1～8, wherein general flavone content is more than 50%.
10. like each described purposes of claim 1～8, wherein also comprise flavonoid chemical constituent and acceptable liposoluble ingredient medically thereof.
11. purposes as claimed in claim 10, wherein the flavonoid chemical constituent contains one or more in nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin, Quercetin, luteolin, apigenin and glycosides thereof or the aglycon.
12. purposes as claimed in claim 11, wherein contain nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin reach 4.5%, 1.2% respectively, more than 2.5%.
13. purposes as claimed in claim 12 wherein contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin total amount and reaches more than 23%.
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