CN102935098A - Acute turpinia leaf general flavone ethanol reflux extract, and preparation method and application thereof - Google Patents
Acute turpinia leaf general flavone ethanol reflux extract, and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an application of an acute turpinia leaf general flavone ethanol reflux extract in preparing drugs for preventing and treating diseases of flu complications which refer to splenic injuries or/and lung injuries. The acute turpinia leaf general flavone ethanol reflux extract is prepared from acute turpinia leaves through ethanol reflux extraction, wherein the extraction method comprises the steps of taking acute turpinia leaves; adding 30%-90% ethanol with a volume 5-10 times that of the acute turpinia leaves to carry out reflux extraction for 1 to 3 times, wherein each time of reflux extraction lasts for 1 to 3 hours; filtering; merging filter liquid; recovering the ethanol from the filter liquid; absorbing water solution by using a nonpolar macroporous absorbent resin; eluting to remove impurities by using the ethanol with a concentration of lower than 20 percent; then, eluting by using 25%-90% ethanol; collecting elution liquid; recovering the ethanol; and concentrating and drying to obtain the acute turpinia leaf general flavone ethanol reflux extract.
Description
Technical field
The present invention relates to a kind of acute turpinia leaf general flavone ethanol reflux extract and its production and use.
Background technology
Influenza virus is day by day serious 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, not only populous, and living habit also is conducive to 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, especially new kind influenza virus, 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 effectively do not treat at present method and the treatment means of influenza virus and disease (influenza) thereof, as carry out the inflammatory reaction treatment for clinical symptoms, cure 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).Although the non-specific anti virus herb of most lays claim to antivirus action, is not for influenza virus, and antivirus action mechanism is 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 has the effect of clear and definite inhibition influenza virus.Western medicine " oseltamivir phosphate (oseltamivir phosphate capsule; (3R; 4R; 5S)-amino-3 (1-the third 2-ethoxyethyl acetate)-1-cyclohexene-1 carboxylic acid, ethyl ester phosphate of 4-acetamide-5-) " be neuraminidase inhibitor, infected by influenza enters cell specific inhibitory effect, but not only expensive, and have that some patients take rear appearance vomiting, feel sick, the 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 in vivo liver and intestinal is converted into active metabolite and plays the neuraminidase effect that suppresses, and also will affect 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 copies, suppresses influenza neuraminidase hydrolysis cell surface sialic acid, thereby cause influenza virus not to be combined with cell surface receptor and to enter 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 to be combined with cell surface receptor and to enter in the cell, copy thereby can effectively suppress influenza virus, more importantly overcome the side reaction drawback of medicine.
Another object of the present invention has provided a kind of preparation method 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, by leaf of Turpinia pomifera (Roxb) D O. is obtained with alcohol reflux, wherein extracting method 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%, use again 25～90% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferably, concrete extracting method is as follows: 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%, use again 30～85% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete extracting method is as follows: concrete extracting method is as follows: 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%, use again 35～80% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete extracting method is as follows: 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%, use again 40～75% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete extracting method is as follows: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 60～75% ethanol of 6～9 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid,, filter, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, remove impurity with the ethanol elution below 20%, use again 45～70% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferably, concrete extracting method is as follows: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 65% ethanol of 7 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid,, filter, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, remove impurity with the ethanol elution below 20%, use again 50～65% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete extracting method is as follows: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 70% ethanol of 8 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid,, filter, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, remove impurity with the ethanol elution below 20%, use again 55% ethanol elution, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
Preferred, concrete extracting method is as follows: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 70% ethanol of 8 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid,, filter, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 15% is removed impurity, uses 55% ethanol elution again, collects eluent, Recycled ethanol, concentrated, 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 flavonoids and acceptable liposoluble ingredient medically thereof, flavonoids 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 respectively 4.5%, 1.2%, 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 present invention also provides the purposes of acute turpinia leaf general flavone ethanol reflux extract for the preparation of the medicine of prevention and treatment influenza and complication disease thereof, be used for suppressing influenza virus, influenza virus FM1, suppress neuraminidase, complication refers to renal failure, and its complication 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 take described acute turpinia leaf general flavone ethanol reflux extract as main active.
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 ability of neuraminic acid enzymatic activity, 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 by suppressing influenza virus surface neuraminidase, and then the inhibition influenza virus enters the cell the inside, inhibition has entered the influenza virus of cell the inside and has copied, propagation, thereby reduced the infection of influenza virus to cell, growth, 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 by leaf of Turpinia pomifera (Roxb) D O. is added ethanol extraction, and concrete extracting method is as follows: 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%, use again 25～90% ethanol elutions, collect eluent, Recycled ethanol, concentrated, 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 flavonoids and acceptable liposoluble ingredient medically thereof, flavonoids 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 respectively 4.5%, 1.2%, 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
Preparation method: 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, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 10% is removed impurity, with 70% ethanol elution of 7 times of amount column volumes, collects eluent again, Recycled ethanol, concentrated, drying gets 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 respectively 15%, 5%, 4%.
Embodiment 2
Preparation method: get leaf of Turpinia pomifera (Roxb) D O., each 45% ethanol with 14 times of volumes is as the extraction solvent, reflux, extract, 1 time, 2 hours, merge extractive liquid, filters filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 15% is removed impurity, with 50% ethanol elution of 6 times of amount column volumes, collects eluent again, Recycled ethanol, concentrated, drying gets 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 respectively 8%, 12%, 5%.
Embodiment 3
Preparation method: get leaf of Turpinia pomifera (Roxb) D O., each 50% ethanol with 12 times of volumes is as the extraction solvent, reflux, extract, 2 times, each 1.5 hours, merge extractive liquid, filters filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 18% is removed impurity, with 55% ethanol elution of 5 times of amount column volumes, collects eluent again, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract, general flavone content 59.6% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches respectively 11%, 7%, 9%.
Embodiment 4
Preparation method: get leaf of Turpinia pomifera (Roxb) D O., each 60% ethanol with 11 times of volumes is as the extraction solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid, filters filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 20% is removed impurity, with 60% ethanol elution of 5.5 times of amount column volumes, collects eluent again, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract, general flavone content 63.5% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches respectively 10%, 8%, 5%.
Embodiment 5
Preparation method: get leaf of Turpinia pomifera (Roxb) D O., each 65% ethanol with 10 times of volumes is as the extraction solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid, filters filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 20% is removed impurity, with 60% ethanol elution of 6 times of amount column volumes, collects eluent again, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract, general flavone content 66.8% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches respectively 7%, 10%, 7%.
Embodiment 6
Preparation method: get leaf of Turpinia pomifera (Roxb) D O., each 70% ethanol with 9 times of volumes is as the extraction solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid, filters filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 20% is removed impurity, with 60% ethanol elution of 6 times of amount column volumes, collects eluent again, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract, general flavone content 74.9% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches respectively 17%, 3%, 4%.
Embodiment 7
Preparation method: get leaf of Turpinia pomifera (Roxb) D O., each 70% ethanol with 8 times of volumes is as the extraction solvent, reflux, extract, 3 times, each 1 hour, merge extractive liquid, filters filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 18% is removed impurity, with 45% ethanol elution of 7 times of amount column volumes, collects eluent again, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract, general flavone content 80.3% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches respectively 12%, 2%, 3%.
Embodiment 8
Preparation method: get leaf of Turpinia pomifera (Roxb) D O., each 75% ethanol with 7 times of volumes is as the extraction solvent, reflux, extract, 3 times, each 1.5 hours, merge extractive liquid, filters filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 19% is removed impurity, with 60% ethanol elution of 5 times of amount column volumes, collects eluent again, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract, general flavone content 66.6% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches respectively 6%, 5%, 10%.
Embodiment 9
Preparation method: get leaf of Turpinia pomifera (Roxb) D O., each 80% ethanol with 6 times of volumes is as the extraction solvent, reflux, extract, 3 times, each 2 hours, merge extractive liquid, filters filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 17% is removed impurity, with 65% ethanol elution of 6 times of amount column volumes, collects eluent again, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract, general flavone content 58.1% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches respectively 10%, 7%, 11%.
Embodiment 10
Preparation method: get leaf of Turpinia pomifera (Roxb) D O., each 85% ethanol with 5 times of volumes is as the extraction solvent, reflux, extract, 3 times, each 2.5 hours, merge extractive liquid, filters filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 15% is removed impurity, with 70% ethanol elution of 4 times of amount column volumes, collects eluent again, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract, general flavone content 53.0% contains nuezhenoside, Radix seu Folium Tosicodendri Delavayi glucoside, hyperin and reaches respectively 13%, 8%, 15%.Experimental data 1, extraction process optimization research
Leaf of Turpinia pomifera (Roxb) D O. mainly contains the chemical compounds such as flavonoid, organic acid, flavone compound is better 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 (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 results are shown in Table 2.
Table 2L9 (3
4) orthogonal test table
Variance analysis
Orthogonal experiments is carried out variance analysis, the results are shown in Table 3.
The variance analysis of table 3 take the total flavones extraction ratio as index
＊F
1-0.05(2，2)＝19.0
＊＊F
1-0.01(2，2)＝99.0
Conclusion: intuitive analysis as can be known, extraction time (C) and to add amount of alcohol (B) larger on extraction effect impact, concentration of alcohol (A) and extraction time (D) are less on the extraction effect impact.Table 3 the results of analysis of variance has further been verified the intuitive analysis conclusion, and namely factor C, B have considerable influence to extraction effect successively.According to producing practical situation, analyze in conjunction with the K value, determine that extraction conditions is: get leaf of Turpinia pomifera (Roxb) D O., add 10 times of amount 70% ethanol and carry 3 times, extract 2 hours at every turn, and get final product.
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 nonflavanoids after with ethanol extraction, must be further purified processing to alcohol extract, to improve the content of flavonoids effective constituent, impurity mostly 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 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, front 500ml collects one bottle, and the every 150ml in back collects one bottle, carries out the flavone qualitative identification, to the 2000ml flavone positive reaction of beginning to have.(quality that is equivalent to crude drug is 50g) not yet detected flavone when applied sample amount was 2000ml, so tentatively determine the extracting solution of every 1000ml macroporous resin loading 100g medical material, 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, the results are shown in Table 4.
The investigation of table 4 eluting concentration
Annotate: an expression HPLC does not detect flavones ingredient.
30%～50%, and effective ingredient can not leak and washes flavonoid glycoside with the optium concentration of ethanol elution as can be seen from Table 4.15% 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, 30%～50% ethanol then can be used as the desorption agent of flavone, can improve like this content of total flavonoid glycoside in effective site, 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, be splined on the 1000ml resin behind the extracting solution Recycled ethanol, washing 4000ml, 15% ethanol is washed 6000ml, and 50% ethanol is washed 6000ml.The results are shown in 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, simultaneously after eluting 3500ml, eluent paler colour (eluting power that is impurity descends) is so 15% ethanol part elution volume is defined as 3500ml (being every 1ml resin 3.5ml 15% ethanol elution).When 5000ml, effective ingredient eluting is complete during 50% ethanol elution, so with 50% ethanol 4000ml eluting (be 1ml resin with 4.5ml 45% ethanol elution).
Experimental data 3: acute turpinia leaf general flavone ethanol reflux extract is to the inhibitory action of neuraminic acid enzymatic activity
Get the acute turpinia leaf general flavone ethanol reflux extract that embodiment 5 preparation methoies obtain, 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 respectively 0,1,2,5,7.5,10 μ l H5N1 neuraminidases again; 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). detecting 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 again the about 1min of mixing;
E.37 carry out fluoremetry after ℃ hatching 20～30min.Excitation wavelength is 360nm, and emission wavelength is 440nm.
(4). calculate sample for H5N1 neuraminic acid enzymeinhibition percentage ratio according to standard curve, and calculate acute turpinia leaf general flavone ethanol reflux extract for the IC50 of H5N1 neuraminidase after doing concentration curve.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 ability of neuraminic acid enzymatic activity, i.e. also corresponding changing of the height of neuraminic acid enzyme inhibition rate, and become positive correlation;
(3). by above-mentioned experiment as seen, acute turpinia leaf general flavone ethanol reflux extract can be by suppressing influenza virus surface neuraminidase, and then suppress that influenza virus enters the cell the inside, the influenza virus that suppresses to have entered the cell the inside copies, breeds, thereby reduced infection, the growth of influenza virus to cell, and prevention and treatment influenza and complication thereof.
Medical science and study of pharmacy personnel can't not do the inhibition influenza infection, copy 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 on the impact 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 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 simultaneously the normal cell contrast.Culture plate is put 37 ℃, 5%CO
2Cultivate in the incubator, observation of cell pathological changes every day (CPE), Continuous Observation 3 days, 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 suck culture fluid in the hole next day, add 100TCID50 influenza virus liquid, and every hole 100 μ l suck supernatant behind 37 ℃ of absorption 1h.Wash 2 times with phosphate buffer (PBS), take the TD0 of medicine as the 1st hole, with the DMEM liquid of serum-free Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing is made serial dilution again, add respectively in the above-mentioned cell that has infected virus, establish simultaneously virus control and Normal group, 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 larger, shows that the safety range of medicine is larger.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) occurs 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, the results are shown in 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)
As shown in Table 7, the cytopathogenic effect of Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing inhibition FM1 influenza virus all strengthens 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 obvious positive correlation between the CPE suppression ratio.
Experimental data 5: Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing infected by influenza infects the inhibitory action of Embryo Gallus domesticus
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 copied 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, hatch 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). the inhibitory action of Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing infected by influenza 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 simultaneously virus control, physiological saline solution normal control, calculates Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing to the median effective dose (ED50) of 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 was inoculated in Embryo Gallus domesticus, its growth promoter and Normal group were 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 has no 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 of infected by influenza in Embryo Gallus domesticus sees Table 8.
Table 8. Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing infected by influenza infects the inhibitory action of Embryo Gallus domesticus
Compare with the virus control group: * P＜0.05
As shown in Table 8, Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing has significant inhibitory action (P＜0.05) at 11.25～90.0mg/ml infected by influenza, and 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 on the impact 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 that 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.(1). influenza virus FM1 strain virus is inoculated respectively every group of 10 BALB/C mice, 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 determine 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 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 dosage group philosophy gavage, gavage capacity only are 0.4ml/.After 3 days, each group is only used 10LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ under the slight anesthesia of ether except Normal group.Normal group gives the normal saline with volume simultaneously.4 groups of administration are continued administration, Normal 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 results are shown in Table 9.(3). Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing is on the impact of influenza virus infection FM1 strain lung index in the Mice Body: Normal 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 dosage group philosophy gavage, gavage capacity only are 0.4ml/.After 3 days, each group is only used 1.0LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ under the slight anesthesia of ether except Normal group.Normal group gives the normal saline with volume simultaneously.4 groups of administration are continued administration, Normal 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 lung heavy, calculate lung index (lung index=lung quality/weight * 100%); In addition, get spleen and claim spleen heavy, calculate spleen index (spleen index=spleen quality/weight * 100%), the results are shown in 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 on the impact of spleen index and the lung index of influenza virus infection FM1 strain in the Mice Body
Annotate: #P＜0.05VS Normal group is annotated: ##P＜0.01VS Normal group
※ ※ p＜0.001VS influenza virus model group ※ p＜0.05VS influenza virus model group
(1). as shown in 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 influenza virus infected.
(2). as shown in Table 10, Turpinia pomifera(Roxb) D O. total flavones reflux, extract, thing has significant effect (p＜0.01-p＜0.05) at the lung index of 45.0mg/ml-90.0mg/ml influenza virus infected.
Experimental data 7: acute turpinia leaf general flavone ethanol reflux extract is on the impact 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 preparation methoies obtain;
3) test method: male SD rat, about body weight 220g, feed 10 days normal growths with normal diet first after, be divided at random Normal group, administration experimental group and modeling matched group by body weight, 12 every group.Administration experimental group and modeling matched group are made chronic renal failure (CRF) model with the adenine gavage, with adenine 320mg/ (kgd) gavage, only make the about 2ml/ of suspension with distilled water, totally 20 days; Administration after 20 days, the administration experimental group is with acute turpinia leaf general flavone ethanol reflux extract 0.36g/ (kgd) gavage, acute turpinia leaf general flavone ethanol reflux extract is made suspension solution (0.06g/ml) with distilled water, each about 2ml/ only, the gradation gavage, Normal group and modeling matched group be with equal-volume water gavage, administration after 35 days with the rat etherization. indices is observed in the blood sampling of Mus arteria caudalis.
4) result:
Table 11. acute turpinia leaf general flavone ethanol reflux extract is on the impact of CRF Renal Function in Rats
Annotate: through the T check, administration experimental group and modeling matched group be " * " expression P＜0.05 relatively, and " * * " represents P＜0.01
This experiment is looked sidelong at after the purine gavage sets up rat CRF model with gland, modeling control rats lethargy, body weight obviously reduces, serum Bun, Ser obviously raises, Hb obviously descends, Ret obviously raises, show that Renal Function in Rats is impaired, after giving the acute turpinia leaf general flavone ethanol reflux extract treatment. rat spirit is obviously bestirred oneself, and body weight is recovered gradually, improves 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 effect while of suppressing the neuraminic acid enzymatic activity, 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, its complication refers to splenic trauma or/and injury of lung, described acute turpinia leaf general flavone ethanol reflux extract is by leaf of Turpinia pomifera (Roxb) D O. is obtained with alcohol reflux, wherein extracting method is: get leaf of Turpinia pomifera (Roxb) D O., 30%～90% alcohol reflux 1～3 time that adds 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%, use again 25～90% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
2. purposes as claimed in claim 1, concrete extracting method is as follows: 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%, use again 30～85% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
3. purposes as claimed in claim 2, concrete extracting method is as follows: 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%, use again 35～80% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
4. purposes as claimed in claim 3, concrete extracting method is as follows: 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%, use again 40～75% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
5. purposes as claimed in claim 4, concrete extracting method is as follows: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 60～75% ethanol of 6～9 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid,, filter, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, remove impurity with the ethanol elution below 20%, use again 45～70% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
6. purposes as claimed in claim 5, concrete extracting method is as follows: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 65% ethanol of 7 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid,, filter, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, remove impurity with the ethanol elution below 20%, use again 50～65% ethanol elutions, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
7. purposes as claimed in claim 5, concrete extracting method is as follows: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 70% ethanol of 8 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid,, filter, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, remove impurity with the ethanol elution below 20%, use again 55% ethanol elution, collect eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
8. purposes as claimed in claim 7, concrete extracting method is as follows: get leaf of Turpinia pomifera (Roxb) D O., at every turn with 70% ethanol of 8 times of volumes as extracting solvent, reflux, extract, 2 times, each 2 hours, merge extractive liquid,, filter, filtrate recycling ethanol, aqueous solution adsorbs with nonpolar macroporous adsorption resin, ethanol elution with 15% is removed impurity, uses 55% ethanol elution again, collects eluent, Recycled ethanol, concentrated, drying gets acute turpinia leaf general flavone ethanol reflux extract.
9. such as 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 flavonoids and acceptable liposoluble ingredient medically thereof, flavonoids 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 respectively 4.5%, 1.2%, 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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| CN113045614A (en) * | 2019-12-26 | 2021-06-29 | 江西青峰药业有限公司 | Method for simultaneously separating and purifying multiple flavonoids by using industrial chromatographic technology |
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| CN1872106A (en) * | 2006-04-18 | 2006-12-06 | 徐蕾 | Application of wild basil circle leaves in treating disease of virulence cold |
| CN101011435A (en) * | 2007-02-25 | 2007-08-08 | 胡军 | Shanxiangyuan leaf extract, preparation method and uses thereof |
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