CN102784178A - Turpinia montana leaf general flavone ethanol reflux extractive, preparation method and application thereof - Google Patents
Turpinia montana leaf general flavone ethanol reflux extractive, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an application of a Turpinia montana leaf general flavone ethanol reflux extractive used for preparing the medicines for preventing and treating influenza complication disease, the complication is renal failure, the Turpinia montana leaf general flavone ethanol reflux extractive is obtained by extracting Turpinia montana leaf through adding ethanol, a preparation method comprises the following steps: taking Turpinia montana leaf, adding 30%-90% of ethanol with 5-15 times volume for reflux extraction for 2-3 times, wherein the duration of each reflux extraction is 1-2.5 hours, filtrating, merging the filtrates, recovering ethanol from the filtrate, adsorbing an aqueous solution by nonpolar macroporous adsorption resin, washing by ethanol with concentration of less than 20% for removing the impurities, washing by 25-90% of ethanol, collecting an eluate, recovering ethanol, concentrating and drying to obtain the Turpinia montana leaf general flavone ethanol reflux extractive.
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; Through leaf of Turpinia pomifera (Roxb) D O. is obtained with alcohol reflux, wherein method for distilling is: 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, filtrate recycling 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 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, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 30～85% ethanol elutions are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete method for distilling is following: 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, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 35～80% ethanol elutions are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
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, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 40～75% ethanol elutions are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
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, filtered filtrate recycling ethanol; Aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%, and reuse 45～70% 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 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 filtrate recycling ethanol; Aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%, and reuse 50～65% ethanol elutions are collected eluent; Reclaim ethanol, concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
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, filtered filtrate recycling ethanol; Aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%, and reuse 55% ethanol elution is collected eluent; Reclaim ethanol, concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
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, filtered filtrate recycling ethanol; Aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with 15% ethanol elution, and reuse 55% ethanol elution is 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, 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, influenza virus FM1, suppress neuraminidase; Complication is meant renal failure, and its complication is meant 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 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., add 30%～90% alcohol reflux 1～3 time of 5～15 times of volumes; Each reflux, extract, 1～3 hour filters; Merging filtrate, filtrate recycling 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, 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%.
Embodiment 1
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., with 90% ethanol of 5 times of volumes as extracting solvent, reflux, extract, 1 time, 3 hours; Merge extractive liquid, filters, filtrate recycling ethanol, and aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 10% is removed impurity, and 70% ethanol elution of 7 times of amounts of reuse column volume is collected eluent; Reclaim ethanol, concentrate drying; Get acute turpinia leaf general flavone ethanol reflux extract, wherein general flavone content 51.6%, contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 15%, 5%, 4% respectively.
Embodiment 2
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 45% ethanol of 14 times of volumes as extracting solvent, reflux, extract, 1 time, 2 hours; Merge extractive liquid, filters, filtrate recycling ethanol, and 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, wherein general flavone content 55.1%, contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 8%, 12%, 5% respectively.
Embodiment 3
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 50% ethanol of 12 times of volumes as extracting solvent, reflux, extract, 2 times, each 1.5 hours; Merge extractive liquid, filters, filtrate recycling ethanol, and 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 59.6% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 11%, 7%, 9% respectively.
Embodiment 4
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 60% ethanol of 11 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours; Merge extractive liquid, filters, filtrate recycling ethanol, and aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 20% is removed impurity, and 60% ethanol elution of 5.5 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 63.5% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 10%, 8%, 5% respectively.
Embodiment 5
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 65% ethanol of 10 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours; Merge extractive liquid, filters, filtrate recycling ethanol, and aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 20% is removed impurity, and 60% 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 66.8% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 7%, 10%, 7% respectively.
Embodiment 6
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 70% ethanol of 9 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours; Merge extractive liquid, filters, filtrate recycling ethanol, and aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 20% is removed impurity, and 60% 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 74.9% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 17%, 3%, 4% respectively.
Embodiment 7
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 70% ethanol of 8 times of volumes as extracting solvent, reflux, extract, 3 times, each 1 hour; Merge extractive liquid, filters, filtrate recycling ethanol, and aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 18% is removed impurity, and 45% ethanol elution of 7 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 80.3% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 12%, 2%, 3% respectively.
Embodiment 8
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 75% ethanol of 7 times of volumes as extracting solvent, reflux, extract, 3 times, each 1.5 hours; Merge extractive liquid, filters, filtrate recycling ethanol, and aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 19% 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 66.6% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 6%, 5%, 10% respectively.
Embodiment 9
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 80% ethanol of 6 times of volumes as extracting solvent, reflux, extract, 3 times, each 2 hours; Merge extractive liquid, filters, filtrate recycling ethanol, and aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 17% is removed impurity, and 65% 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 58.1% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 10%, 7%, 11% respectively.
Embodiment 10
Method for preparing: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 85% ethanol of 5 times of volumes as extracting solvent, reflux, extract, 3 times, each 2.5 hours; Merge extractive liquid, filters, filtrate recycling ethanol, and aqueous solution adsorbs with nonpolar macroporous adsorption resin; Ethanol elution with 15% is removed impurity, and 70% 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 53.0% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches 13%, 8%, 15% 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), 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, 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 leaf of 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 the acute turpinia leaf general flavone ethanol reflux extract that embodiment 5 method for preparinies are obtained; 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, 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 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 2.92mg/ml, sees 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: Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing is to the influence of FM1 influenza virus
Get Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify that Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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 pomifera(Roxb) D O. total flavones reflux, extract, thing adopts the DMEM of serum-free that Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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 pomifera(Roxb) D O. total flavones reflux, extract, thing 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, next day, culture fluid in the hole was removed in suction, added 100 TCID50 influenza virus liquid, and 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 pomifera(Roxb) D O. total flavones reflux, extract, thing, 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 Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing mdck cell is respectively 841.1mg ± 25.08mg/100ml.(3). after Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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. Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing is to the IC50 (mg/100ml) of FM1 influenza virus and TI (x ± s)
Can know that by table 7 Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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 pomifera(Roxb) D O. total flavones reflux, extract, thing and medicine are to being tangible positive correlation between the CPE suppression ratio.
Experimental data 5: Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing infects the inhibitory action of Embryo Gallus domesticus to influenza virus
Get Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify that Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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). Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing adopts the toxic action of Embryo Gallus domesticus; With physiological saline solution Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing is done to be inoculated in no-special pathogen chick embryo allantois intracavity in 10 day age 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). Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing inhibitory action to influenza virus in Embryo Gallus domesticus adopts; 0.1ml influenza virus liquid and different dilution Turpinia pomifera(Roxb) D O. total flavones reflux, extract, things 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 Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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 pomifera(Roxb) D O. total flavones reflux, extract, thing 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 Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing stock solution and does not see chicken embryo death, so can think that TD0 is 7.6g/100ml.(3). Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing inhibitory action to influenza virus in Embryo Gallus domesticus is seen table 8.
Table 8. Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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 Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing has significant inhibitory effect (P＜0.05) at 11.25～90.0mg/ml to influenza virus, ED50 is 9.31mg ± 0.46mg/ml, and TI is 47.29 ± 3.48.
Experimental data 6: Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing is to the influence of influenza virus infection FM1 strain in the mice body
Get Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing, use influenza virus A-prime Mus lung adapted strain (FM1) and (H1N1) identify of the dead protective effect of Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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 pomifera(Roxb) D O. total flavones reflux, extract, thing 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 pomifera(Roxb) D O. total flavones reflux, extract, thing 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 10 LD50 influenza virus FM1 strain collunarium infecting mouses, 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 9.(3). Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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 pomifera(Roxb) D O. total flavones reflux, extract, thing 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 1.0 LD50 influenza virus FM1 strain collunarium infecting mouses, 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. Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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. Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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 Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing has significant protective effect (p＜0.01-p＜0.05) at 22.5～90.0mg/ml to the influenza virus infecting mouse.
(2). can know that by table 10 Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing 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 5 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 (10)
1. acute turpinia leaf general flavone ethanol reflux extract is in the purposes of the medicine of preparation prevention and treatment influenza complication disease; Said complication is meant renal failure; Said acute turpinia leaf general flavone ethanol reflux extract is through leaf of Turpinia pomifera (Roxb) D O. is obtained with alcohol reflux, and wherein method for distilling is: 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, filtrate recycling 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 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, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 30～85% ethanol elutions 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 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, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 35～80% ethanol elutions 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 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, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%; Reuse 40～75% ethanol elutions are collected eluent, reclaim ethanol; Concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
5. purposes as claimed in claim 4, 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, filtered filtrate recycling ethanol; Aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%, and reuse 45～70% ethanol elutions are collected eluent; Reclaim ethanol, concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
6. purposes as claimed in claim 5, 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 filtrate recycling ethanol; Aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%, and reuse 50～65% ethanol elutions are collected eluent; Reclaim ethanol, concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
7. purposes as claimed in claim 5, 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, filtered filtrate recycling ethanol; Aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with the ethanol elution below 20%, and reuse 55% ethanol elution is collected eluent; Reclaim ethanol, concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
8. purposes as claimed in claim 7, 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, filtered filtrate recycling ethanol; Aqueous solution adsorbs with nonpolar macroporous adsorption resin, removes impurity with 15% ethanol elution, and reuse 55% ethanol elution is collected eluent; Reclaim ethanol, concentrate, drying gets acute turpinia leaf general flavone ethanol reflux extract.
9. like each described purposes of claim 1～8; Wherein the general flavone content of acute turpinia leaf general flavone ethanol reflux extract is more than 50%; Wherein also comprise flavonoid chemical constituent and acceptable liposoluble ingredient medically thereof; The flavonoid chemical constituent contains one or more in nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and Quercetin, luteolin, apigenin and glycosides thereof or the aglycon, wherein nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin reach 4.5%, 1.2% respectively, more than 2.5%.
10. purposes as claimed in claim 9 wherein contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin total amount and reaches more than 23%.
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