CN102633850A - Rhoifolin extraction method and usage of drug prepared by rhoifolin - Google Patents
Rhoifolin extraction method and usage of drug prepared by rhoifolin Download PDFInfo
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Abstract
The invention relates to rhoifolin and usage of a drug prepared by the rhoifolin. The rhoifolin extraction method includes the steps: taking folium turpiniae or pummelo peel, adding 5-15 times of amount of ethanol with the concentration of 30%-90%, performing reflux extraction for 1-3 times, each reflux extraction takes 1-3 hours and filtering; and combining filtrate, recovering the ethanol, eluting water solution by macroporous resin, collecting eluted portions, depressurizing, concentrating and drying to obtain mixed total glycoside of the rhoifolin prior to separation, combining rhoifolin fluid parts, crystallizing and obtaining a rhoifolin pure product. The rhoifolin can serve as neuraminidase inhibitor to prevent and treat influenza, and can be made into dosage forms acceptable in pharmacy.
Description
Technical field
The present invention relates to a kind of process for extracting and preparation pharmaceutical use thereof of Rhoifoloside.
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 life style also help 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 transformation reactions 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 symptom, 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 Ro 64-0796/002 (Tamiflu)-neuraminidase inhibitor.Yet influenza virus vaccine vaccination drawback is that the inoculation crowd is selective, is not that everybody can inoculate.In addition, the influenza virus vaccine protection ratio 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.Catch cold in our many kind treatments of 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 " Ro 64-0796/002 (Tamiflu; (3R; 4R, 5S)-amino-3 (1-third the 2-ethoxyethyl acetate)-1-tetrahydrobenzene-1 carboxylic acid, ethyl ester phosphoric acid salt of 4-ethanamide-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.Tamiflu is that the esterase that is positioned at liver and enteron aisle 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 enteron aisle organ dysfunction are undesired.In addition, the renal insufficiency patient also wants careful usefulness.
It is clearing heat and detoxicating that traditional traditional Chinese medicine thinks that leaf of Turpinia pomifera (Roxb) D O. has, relieving sore throat and diminishing swelling, promoting blood circulation and stopping pain.Be used for acute tonsillitis larynx numbness, swelling and pain in the throat, sore swollen toxin falls and pounces on the pain of injury.Pummelo Peel has to loose trembles with fear eliminating dampness, sharp gas, dissolving phlegm.Be used for the cough due to wind and cold, the larynx phlegm of itching is many, and dyspepsia is got sick from drinking too much wine, and the jailbird that vomits is vexed.Rhoifoloside is one of leaf of Turpinia pomifera (Roxb) D O. and Pummelo Peel staple, does not find that leaf of Turpinia pomifera (Roxb) D O. and Pummelo Peel and contained Rhoifoloside thereof have the effect that suppresses influenza infection, duplicates, and does not also find to have the effect that suppresses neuraminidase.
Summary of the invention
The purpose of this invention is to provide Rhoifoloside; It can suppress influenza neuraminidase hydrolysis cell surface sialyl; Cause influenza virus not combine with cell surface receptor and to get in the cell; And reduce influenza virus in intracellular generation, and duplicate thereby suppress influenza virus effectively pointedly, more importantly be that the present invention provides a kind of Rhoifoloside can overcome the side reaction drawback of existing medicine.
Said Rhoifoloside provided by the invention, molecular formula: C
27H
30O
14, molecular weight: 578.52, the structural formula of said Rhoifoloside is following:
Said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O. or Pummelo Peel, add 30%～90% alcohol reflux 1～3 time of 5～15 times of amounts, each refluxing extraction 1～3 hour filters; Merging filtrate reclaims ethanol, and the aqueous solution is collected the wash-out part through the macroporous resin column wash-out, concentrating under reduced pressure, drying, the total glycosides of mixing of Rhoifoloside, through separating, merge Rhoifoloside stream part, crystallization obtains the pure article of Rhoifoloside.
Preferably, wherein the aqueous solution is through macroporous resin column, and water, 5%～10% ethanol, 30%～55% ethanol, 0.7～1.3% sodium hydroxide solution wash-out are collected 30%～55% ethanol elution part, concentrating under reduced pressure, drying respectively.
Preferably, wherein said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 12 times of amount 70% alcohol reflux 2 hours, filter; Filter residue adds 10 times of amount 70% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution wash-out; Collect 45% ethanol elution part, concentrating under reduced pressure, drying.
Preferably, wherein said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 10 times of amount 60% alcohol reflux 2 hours, filter; Filter residue adds 8 times of amount 60% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 10% ethanol, 45% ethanol, 0.8% sodium hydroxide solution wash-out; Collect 45% ethanol elution part, concentrating under reduced pressure, drying.
Preferably, wherein said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 15 times of amount 60% alcohol reflux 2 hours, filter; Filter residue adds 12 times of amount 60% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 8% ethanol, 40% ethanol, 1.2% sodium hydroxide solution wash-out; Collect 40% ethanol elution part, concentrating under reduced pressure, drying.
Preferably, wherein said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 9 times of amount 60% alcohol reflux 2.5 hours, filter; Filter residue adds 8 times of amount 60% alcohol reflux 2 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution is through handle macroporous resin column well, difference water, 5% ethanol, 35% ethanol, 0.7% sodium hydroxide solution wash-out; Collect 35% ethanol elution part, concentrating under reduced pressure, drying.
Preferably, wherein said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 9 times of amount 60% alcohol reflux 2 hours, filter; Filter residue adds 8 times of amount 60% alcohol reflux 2 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 6% ethanol, 30% ethanol, 1% sodium hydroxide solution wash-out; Collect 30% ethanol elution part, concentrating under reduced pressure, drying.
Preferably, wherein said Rhoifoloside adopts following process for extracting to obtain: get Pummelo Peel, add 12 times of amount 70% alcohol reflux 1.5 hours, filter; Filter residue adds 10 times of amount 70% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution wash-out; Collect 45% ethanol elution part, concentrating under reduced pressure, drying.
Preferably, wherein said Rhoifoloside adopts following process for extracting to obtain: get Pummelo Peel, add 10 times of amount 65% alcohol reflux 2 hours, filter; Filter residue adds 8 times of amount 65% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 10% ethanol, 45% ethanol, 0.9% sodium hydroxide solution wash-out; Collect 45% ethanol elution part, concentrating under reduced pressure, drying.
Preferably, wherein said Rhoifoloside adopts following process for extracting to obtain: get Pummelo Peel, add 15 times of amount 60% alcohol reflux 2 hours, filter; Filter residue adds 12 times of amount 60% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 8% ethanol, 40% ethanol, 1.1% sodium hydroxide solution wash-out; Collect 40% ethanol elution part, concentrating under reduced pressure, drying.
Preferably, wherein the D101 macroporous resin column of the aqueous solution through having handled well mixed total glycosides through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merges Rhoifoloside stream part, and crystallization obtains the pure article of Rhoifoloside.
The purposes that the present invention also provides Rhoifoloside to be used to prepare prevention and to treat the medicine of influenza and complication disease thereof.
Preferably, Rhoifoloside is used to suppress influenza virus.
Preferably, Rhoifoloside is used to suppress influenza virus FM1.
Preferably, Rhoifoloside is used to suppress neuraminidase.
Preferably, its complication is meant renal failure.
Preferably, its complication is meant injury of spleen or/and injury of lung.
Rhoifoloside of the present invention can be from the various medicinal materials that contain Rhoifoloside, to obtain Rhoifoloside bullion or Rhoifoloside monomeric compound as extracting in leaf of Turpinia pomifera (Roxb) D O., the Pummelo Peel.
At last, the present invention also provides a kind of Rhoifoloside preparation, and said preparation is a main active ingredient with the above-mentioned Rhoifoloside of the present invention.
Experimental data proves; Rhoifoloside of the present invention is effectively to suppress the neuraminic acid enzyme component, and Rhoifoloside is along with the using dosage size variation, and 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, Rhoifoloside 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 infection, the growth of influenza virus pair cell have been reduced; And prevention and treatment influenza and complication thereof, can also suppress influenza neuraminidase hydrolysis cell surface sialyl, cause influenza virus not combine with cell surface receptor and to get in the cell; And reduce influenza virus in intracellular generation, more importantly be that the present invention provides a kind of Rhoifoloside can overcome the side reaction drawback of existing medicine.
And medical science and study of pharmacy personnel can't not do the inhibition influenza infection, duplicate in advance, or under the prerequisite of the experiment of inhibition neuraminidase, learn that in advance Rhoifoloside has prevention and treats the good result that the influenza virus sexuality is emitted.
In sum, Rhoifoloside provided by the invention and uses thereof brings significant technique effect.
Embodiment
Embodiment 1
Get leaf of Turpinia pomifera (Roxb) D O. 1000g, add 12 times of amount 70% alcohol reflux 2 hours, filter, filter residue adds 10 times of amount 70% alcohol reflux 1.5 hours again; Filter, merge extracted twice liquid, reclaim ethanol; The D101 macroporous resin column of the aqueous solution through having handled well, water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution wash-out are collected 45% ethanol elution part respectively; Concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Rhoifoloside; Through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Rhoifoloside stream part, crystallization; Obtain Rhoifoloside (1.5g, purity: pure article 98.5%), through respectively with the UV of Rhoifoloside standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of Rhoifoloside.
Embodiment 2
Get leaf of Turpinia pomifera (Roxb) D O. 1000g, add 10 times of amount 60% alcohol reflux 2 hours, filter, filter residue adds 8 times of amount 60% alcohol reflux 1.5 hours again; Filter, merge extracted twice liquid, reclaim ethanol; The D101 macroporous resin column of the aqueous solution through having handled well, water, 10% ethanol, 45% ethanol, 0.8% sodium hydroxide solution wash-out are collected 45% ethanol elution part respectively; Concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Rhoifoloside; Through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Rhoifoloside stream part, crystallization; Obtain Rhoifoloside (1.6g, purity: pure article 98.7%), through respectively with the UV of Rhoifoloside standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of Rhoifoloside.
Embodiment 3
Get leaf of Turpinia pomifera (Roxb) D O. 1000g, add 15 times of amount 60% alcohol reflux 2 hours, filter, filter residue adds 12 times of amount 60% alcohol reflux 1.5 hours again; Filter, merge extracted twice liquid, reclaim ethanol; The D101 macroporous resin column of the aqueous solution through having handled well, water, 8% ethanol, 40% ethanol, 1.2% sodium hydroxide solution wash-out are collected 40% ethanol elution part respectively; Concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Rhoifoloside; Through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Rhoifoloside stream part, crystallization; Obtain Rhoifoloside (1.7g, purity: pure article 98.9%), through respectively with the UV of Rhoifoloside standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of Rhoifoloside.
Embodiment 4
Get leaf of Turpinia pomifera (Roxb) D O. 1000g, add 9 times of amount 60% alcohol reflux 2.5 hours, filter, filter residue adds 8 times of amount 60% alcohol reflux 2 hours again; Filter, merge extracted twice liquid, reclaim ethanol; The D101 macroporous resin column of the aqueous solution through having handled well, water, 5% ethanol, 35% ethanol, 0.7% sodium hydroxide solution wash-out are collected 35% ethanol elution part respectively; Concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Rhoifoloside; Through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Rhoifoloside stream part, crystallization; Obtain Rhoifoloside (1.5g, purity: pure article 98.5%), through respectively with the UV of Rhoifoloside standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of Rhoifoloside.
Embodiment 5
Get leaf of Turpinia pomifera (Roxb) D O. 1000g, add 9 times of amount 60% alcohol reflux 2 hours, filter, filter residue adds 8 times of amount 60% alcohol reflux 2 hours again; Filter, merge extracted twice liquid, reclaim ethanol, the D101 macroporous resin column of the aqueous solution through having handled well; Water, 6% ethanol, 30% ethanol, 1% sodium hydroxide solution wash-out are collected 30% ethanol elution part, concentrating under reduced pressure, drying respectively; Get the total glycosides of mixing of 30% above Rhoifoloside,, merge Rhoifoloside stream part, crystallization through silica gel column chromatography, Sephadex LH-20 column chromatography for separation; Obtain Rhoifoloside (1.7g, purity: pure article 98.9%), through respectively with the UV of Rhoifoloside standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of Rhoifoloside.
Embodiment 6
Get Pummelo Peel 1000g, add 12 times of amount 70% alcohol reflux 1.5 hours, filter, filter residue adds 10 times of amount 70% alcohol reflux 1.5 hours again; Filter, merge extracted twice liquid, reclaim ethanol; The D101 macroporous resin column of the aqueous solution through having handled well, water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution wash-out are collected 45% ethanol elution part respectively; Concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Rhoifoloside; Through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Rhoifoloside stream part, crystallization; Obtain Rhoifoloside (4.5g, purity: pure article 98.6%), through respectively with the UV of Rhoifoloside standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of Rhoifoloside.
Embodiment 7
Get Pummelo Peel 1000g, add 10 times of amount 65% alcohol reflux 2 hours, filter, filter residue adds 8 times of amount 65% alcohol reflux 1.5 hours again; Filter, merge extracted twice liquid, reclaim ethanol; The D101 macroporous resin column of the aqueous solution through having handled well, water, 10% ethanol, 45% ethanol, 0.9% sodium hydroxide solution wash-out are collected 45% ethanol elution part respectively; Concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Rhoifoloside; Through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Rhoifoloside stream part, crystallization; Obtain Rhoifoloside (4.6g, purity: pure article 98.5%), through respectively with the UV of Rhoifoloside standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of Rhoifoloside.
Embodiment 8
Get Pummelo Peel 1000g, add 15 times of amount 60% alcohol reflux 2 hours, filter, filter residue adds 12 times of amount 60% alcohol reflux 1.5 hours again; Filter, merge extracted twice liquid, reclaim ethanol; The D101 macroporous resin column of the aqueous solution through having handled well, water, 8% ethanol, 40% ethanol, 1.1% sodium hydroxide solution wash-out are collected 40% ethanol elution part respectively; Concentrating under reduced pressure, drying, the total glycosides of mixing of 30% above Rhoifoloside; Through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merge Rhoifoloside stream part, crystallization; Obtain Rhoifoloside (4.6g, purity: pure article 98.7%), through respectively with the UV of Rhoifoloside standard substance, IR, ESI-MS,
1H-NMR,
13C-NMR has relatively confirmed the structure of Rhoifoloside.
Experimental data 1: Rhoifoloside is to the active restraining effect of neuraminidase
Get the Rhoifoloside that embodiment 1 preparation method is obtained, add suitable quantity of water and make dissolving, use tiring of neuraminidase inhibitor identification kit mensuration Rhoifoloside inhibition neuraminidase (N1) and see table 1.
(1). typical curve is prepared: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases and detects damping fluid; B. every hole adds 0,1,2,5,7.5,10 μ l H5N1 neuraminidases more respectively; C. every hole adds 0～20 μ l Milli-Q water again.
(2). the preparation of sample detection: a. every hole in 96 hole luciferase targets adds 70 μ l neuraminidases and detects damping fluid; B. every hole adds 10 μ l H5N1 neuraminidases again; C. every hole adds 0～10 μ l Rhoifoloside 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 suppressor factor and H5N1 neuraminidase are fully interacted, the sample of doing typical curve is also hatched together;
C. every hole adds 10 μ l neuraminidase fluorogenic substrates;
D. vibrate the about 1min of mixing again;
E.37 carry out fluorometric assay after ℃ hatching 20～30min.Excitation wavelength is 360nm, and emission wavelength is 440nm.
(4). calculate the inhibition per-cent of sample according to typical curve, and calculate the IC50 of Rhoifoloside after doing concentration curve for the H5N1 neuraminidase for the H5N1 neuraminidase.The inhibiting rate IC50 that Rhoifoloside reaches neuraminidase is 0.03g/L.See table 1.
Table 1. Rhoifoloside suppresses the activity of neuraminidase
Can be clear that according to above-mentioned experimental result:
(1). Rhoifoloside can extract effective inhibition neuraminic acid enzyme component;
(2). Rhoifoloside 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; Rhoifoloside 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 Rhoifoloside has prevention and treats the good result that the influenza virus sexuality is emitted.
Experimental data 2: Rhoifoloside is to the restraining effect of influenza virus infected chicken embryo
Get the Rhoifoloside that embodiment 1 preparation method is obtained, use influenza virus A-prime mouse lung adapted strain (FM1) and (H1N1) identify that Rhoifoloside suppresses the ability that the FM1 influenza virus is duplicated and suppresses in the chicken embryo.(1). FM1 influenza virus liquid is inoculated in 10d no-special pathogen in the age chick embryo allantoic cavity, and every embryo 0.2ml is hatched 72h for 37 ℃, observes and calculate half chicken embryo infective dose (EID50).(2). Rhoifoloside adopts the toxic action of chicken embryo; SPSS is done to be inoculated in 10d no-special pathogen in the age chick embryo allantoic cavity behind the serial dilution to Rhoifoloside; Every embryo 0.2ml, each concentration is inoculated 6 embryos, hatches for 37 ℃; Observe chicken embryonic development developmental state, can survive the peak concentration of 96h as the TD of medicine with the chicken embryo.(3). Rhoifoloside restraining effect to influenza virus in the chicken embryo adopts, and the influenza virus liquid of 0.1ml mixes with different dilution Rhoifolosides, and 37 ℃ of effect 2h are inoculated in 10d no-special pathogen in age chick embryo allantoic cavity, and every winding kind 6 embryos are hatched 72h for 37 ℃.The virus attack amount is 50EID50, establishes virus control, SPSS normal control simultaneously, calculates the median effective dose (ED50) of Rhoifoloside to viral inhibition.
(1) the .FM1 influenza virus is calculated through the Reed-Muench method the virulence of chicken embryo, and its EID50 is 10
-5.07
(2). after Rhoifoloside was inoculated in the chicken embryo, it grew and normal control group basically identical.96h chicken embryo is all survived.The chicken embryo gives Rhoifoloside stoste and does not see chicken embryo death, so can think that TD0 is 1.6g/L.(3). Rhoifoloside restraining effect to influenza virus in the chicken embryo is seen table 2.
Table 2. Rhoifoloside is to the restraining effect of influenza virus infected chicken embryo
Compare with the virus control group: * P＜0.05
Can know that by table 2 Rhoifoloside has significant inhibitory effect (P＜0.05), ED at 0.05～0.4g/L to influenza virus
50Be 0.05g ± 0.001g/L, TI is 84.0 ± 3.18.
Experimental data 3: Rhoifoloside influences the FM1 influenza virus
Get the Rhoifoloside that embodiment 1 preparation method is obtained, use influenza virus A-prime mouse lung adapted strain (FM1) and (H1N1) identify that Rhoifoloside suppresses the ability of FM1 influenza virus virulence.(1) .FM1 adopts cell median infective dose (TCID50) micromethod to the toxicity test of dog kidney passage cell (MDCK).(2). Rhoifoloside adopts the DMEM of serum-free that Rhoifoloside is done to be inoculated in the mdck cell hole that forms individual layer behind the serial dilution to the toxicity test of mdck cell, every hole 100 μ l, each extent of dilution repeats 4 holes, establishes the normal cell contrast simultaneously.Culture plate is put 37 ℃, 5%CO
2Cultivate in the incubator, observation of cell pathology every day (CPE) is observed 3d continuously, with " +～++ ++ " the record result, press the Reed-Muench method and calculate medicine median toxic concentration (TD50) and maximal non-toxic concentration (TD0).(3). Rhoifoloside suppresses the effect of FM1 influenza virus and measures: mdck cell 5 * 10
5/ ml, every hole 100 μ l, in 96 orifice plates, 37 ℃, the interior cultivation of 5%CO2 incubator are inhaled and remove nutrient solution in the hole next day, add 100TCID50 influenza virus liquid, every hole 100 μ l, supernatant is removed in suction behind 37 ℃ of absorption 1h.Washing 2 times with phosphate buffered saline buffer (PBS), is the 1st hole with the TD0 of medicine, with the DMEM liquid of serum-free Rhoifoloside is made serial dilution again, adds respectively in the cell of above-mentioned infective virus, establishes virus control and normal control group simultaneously, 37 ℃, 5%CO
2Cultivate in the incubator, observe the mdck cell characteristics of lesion that influenza virus produces every day, i.e. monolayer sex change becomes circle etc., and 3d calculates 50% of medicine and suppresses pathology concentration (IC50) and therapeutic index (TI) continuously.The calculating of TI: TI=TD50/IC50, the TI value is big more, shows that the safety range of medicine is big more.With Kruskal-Walis and Mann-Whitney method of inspection comparison test group and the cytopathic difference of virus control group; Drug dose and inhibiting rate that virus infected cell is avoided cytopathy (CPE) takes place are carried out correlation analysis, judge whether amount validity response relation.
(1) the .FM1 influenza virus is calculated through the Reed-Muench method the virulence of mdck cell, and its TCID50 is 10
-4.81(2). the TD0 of Rhoifoloside mdck cell is respectively 0.64g ± 0.031g/L.(3). after Rhoifoloside 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 3.
Table 3. Rhoifoloside is to the IC50 (g/L) of FM1 influenza virus and TI (x ± s)
Can be known that by table 3 IC50 of Rhoifoloside is low, TI is high.Rhoifoloside suppresses the cytopathogenic effect of FM1 influenza virus all to be strengthened along with the increase of drug dose.Drug dose and medicine are shown that to the correlation analysis that the inhibiting rate of CPE carries out the dosage of Rhoifoloside and medicine are to the tangible positive correlation of emperor between the CPE inhibiting rate.
Experimental data 4: Rhoifoloside is to the spleen index and the influence of lung exponential of influenza virus infection FM1 strain in the mouse body
Get the Rhoifoloside that embodiment 1 preparation method is obtained, use influenza virus A-prime mouse lung adapted strain (FM1) and (H1N1) identify the dead provide protection of Rhoifoloside influenza virus infection FM1 strain in the mouse 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 mouse collunarium is inoculated 20 μ l.Observe the dead mouse situation of 10d, calculating LD50 by the Reed-Muench method is 10
-1.36So confirm that the used modeling concentration of experiment is 10LD50.(2). Rhoifoloside is to the dead provide protection of influenza virus infection FM1 strain in the mouse body: normal control group, influenza virus FM1 strain virus control group, Rhoifoloside 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/L dose groups etc. are irritated stomach respectively, irritate gastric capacity and only are 0.4ml/.Behind the 3d, each is organized under the slight anesthesia of ether with 10LD50 influenza virus FM1 strain collunarium infecting mouse 20 μ l/ except that the normal control group.The normal control group gives the saline water with volume simultaneously.4 groups of administration are continued administration, normal control group and influenza virus FM1 strain virus control group administered physiological saline 8 days, and dosage is the same.Day by day observe animal morbidity and record death toll, observed altogether 14 days, calculate mortality ratio (mortality ratio=every group of death toll/every group of total mice * 100%), the result sees table 4.(3). Rhoifoloside influences influenza virus infection FM1 strain lung exponential in the mouse body: normal control group, influenza virus FM1 strain virus control group, Rhoifoloside 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/L dose groups etc. are irritated stomach respectively, irritate gastric capacity and only are 0.4ml/.After 3 days, each is organized under the slight anesthesia of ether with 1.0LD50 influenza virus FMl strain collunarium infecting mouse 20 μ l/ except that the normal control group.The normal control group gives the saline water with volume simultaneously.4 groups of administration are continued administration, normal control group and influenza virus FM1 strain virus control group administered physiological saline 8 days, and dosage is the same.Behind virus infection, put to death mouse on the 8th day, weigh, get lung and claim that lung is heavy, calculates lung index (lung index=lung quality/physique amount * 100%); In addition, get spleen and claim that spleen is heavy, calculate spleen index (spleen index=spleen quality/physique amount * 100%), the result sees table 5.
Table 4. Rhoifoloside is to the death protection result of influenza virus infection FM1 strain in the mouse body
Annotate: ※ ※ P＜0.01VS influenza virus model group ※ P＜0.05VS influenza virus model group
Table 5. Rhoifoloside is to the spleen index and the influence of lung exponential of influenza virus infection FM1 strain in the mouse body
Annotate: #P＜0.05VS normal control group is annotated: ※ ※ p＜0.001 VS influenza virus model group ※ p＜0.05VS influenza virus model group
(1). can know that by table 4 Rhoifoloside has significant provide protection (p＜0.01) at 0.2～0.8g/L to the influenza virus infecting mouse.
(2). can know that by table 5 Rhoifoloside has significant effect (p＜0.01) at 0.4～0.8g/L to the lung index inhibiting rate of influenza virus infecting mouse.
Experimental data 5: Rhoifoloside is to the influence of renal dysfunction
1) SD rat, 200～240g, Shanghai west pul-Bi Kai laboratory animal responsibility ltd, animal conformity certification number: SCXK (Shanghai) 2003-0002
2) reagent and medicine VITAMIN B4, VITAMIN B4, (content＞98% is the import packing, Chinese Shanghai, lot number 20010520), the Rhoifoloside that embodiment 1 preparation method is obtained;
3) TP: male SD rat, about body weight 220g, earlier feed 10 days normal growths with normal diet after, be divided into normal control group, administration experimental group and modeling control group at random by body weight, 13～15 every group.Administration experimental group and modeling control group are irritated stomach with VITAMIN B4 and are made chronic renal failure (CRF) model, irritate stomach with VITAMIN B4 320mg/ (kgd), only process the about 2ml/ of suspension with zero(ppm) water, totally 20 days; Administration after 20 days; The administration experimental group is irritated stomach with Rhoifoloside 1.6g/ (kgd), and Rhoifoloside is processed suspension solution (0.2g/ml) with zero(ppm) water, and about at every turn 2ml/ only; Stomach is irritated in gradation; Normal control group and modeling control group are irritated stomach with equal-volume water, and administration was used etherization with rat after 35 days, and each item index is observed in the blood sampling of mouse caudal artery.
4) result:
Table 6. Rhoifoloside is to the influence of CRF kidney of rats function
Annotate: through the T check, administration Rhoifoloside and modeling control group and normal control 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, and the modeling control rats is One's spirits are drooping, and body weight obviously reduces; Serum Bun, Ser obviously raise, and Hb obviously descends, and Ret obviously raises; Show the rat impaired renal function; Rat and normal control group difference are not remarkable after giving the Rhoifoloside treatment, and very remarkable with the model group comparing difference, so Rhoifoloside is to the kidney free of toxic effects.
Claims (16)
1. Rhoifoloside is used to prepare the purposes of preventing and treating the medicine of influenza and complication disease thereof, the molecular formula of wherein said Rhoifoloside: C
27H
30O
14, molecular weight: 578.52, the structural formula of said Rhoifoloside is following:
Said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O. or Pummelo Peel, add 30%～90% alcohol reflux 1～3 time of 5～15 times of amounts, each refluxing extraction 1～3 hour filters; Merging filtrate reclaims ethanol, and the aqueous solution is collected the wash-out part through the macroporous resin column wash-out, concentrating under reduced pressure, drying, the total glycosides of mixing of Rhoifoloside, through separating, merge Rhoifoloside stream part, crystallization obtains the pure article of Rhoifoloside.
2. purposes as claimed in claim 1, wherein the aqueous solution is through macroporous resin column, and water, 5%～10% ethanol, 30%～55% ethanol, 0.7～1.3% sodium hydroxide solution wash-out are collected 30%～55% ethanol elution part, concentrating under reduced pressure, drying respectively.
3. according to claim 1 or claim 2 purposes, wherein said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 12 times of amount 70% alcohol reflux 2 hours, filter; Filter residue adds 10 times of amount 70% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution wash-out; Collect 45% ethanol elution part, concentrating under reduced pressure, drying.
4. according to claim 1 or claim 2 purposes, wherein said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 10 times of amount 60% alcohol reflux 2 hours, filter; Filter residue adds 8 times of amount 60% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 10% ethanol, 45% ethanol, 0.8% sodium hydroxide solution wash-out; Collect 45% ethanol elution part, concentrating under reduced pressure, drying.
5. according to claim 1 or claim 2 purposes, wherein said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 15 times of amount 60% alcohol reflux 2 hours, filter; Filter residue adds 12 times of amount 60% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 8% ethanol, 40% ethanol, 1.2% sodium hydroxide solution wash-out; Collect 40% ethanol elution part, concentrating under reduced pressure, drying.
6. according to claim 1 or claim 2 purposes, wherein said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 9 times of amount 60% alcohol reflux 2.5 hours, filter; Filter residue adds 8 times of amount 60% alcohol reflux 2 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the macroporous resin column of the aqueous solution through having handled well, water, 5% ethanol, 35% ethanol, 0.7% sodium hydroxide solution wash-out respectively; Collect 35% ethanol elution part, concentrating under reduced pressure, drying.
7. according to claim 1 or claim 2 purposes, wherein said Rhoifoloside adopts following process for extracting to obtain: get leaf of Turpinia pomifera (Roxb) D O., add 9 times of amount 60% alcohol reflux 2 hours, filter; Filter residue adds 8 times of amount 60% alcohol reflux 2 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 6% ethanol, 30% ethanol, 1% sodium hydroxide solution wash-out; Collect 30% ethanol elution part, concentrating under reduced pressure, drying.
8. according to claim 1 or claim 2 purposes, wherein said Rhoifoloside adopts following process for extracting to obtain: get Pummelo Peel, add 12 times of amount 70% alcohol reflux 1.5 hours, filter; Filter residue adds 10 times of amount 70% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 10% ethanol, 45% ethanol, 1% sodium hydroxide solution wash-out; Collect 45% ethanol elution part, concentrating under reduced pressure, drying.
9. according to claim 1 or claim 2 purposes, wherein said Rhoifoloside adopts following process for extracting to obtain: get Pummelo Peel, add 10 times of amount 65% alcohol reflux 2 hours, filter; Filter residue adds 8 times of amount 65% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 10% ethanol, 45% ethanol, 0.9% sodium hydroxide solution wash-out; Collect 45% ethanol elution part, concentrating under reduced pressure, drying.
10. according to claim 1 or claim 2 purposes, wherein said Rhoifoloside adopts following process for extracting to obtain: get Pummelo Peel, add 15 times of amount 60% alcohol reflux 2 hours, filter; Filter residue adds 12 times of amount 60% alcohol reflux 1.5 hours again, filters, and merges extracted twice liquid; Reclaim ethanol, the aqueous solution passes through macroporous resin column, respectively water, 8% ethanol, 40% ethanol, 1.1% sodium hydroxide solution wash-out; Collect 40% ethanol elution part, concentrating under reduced pressure, drying.
11. purposes according to claim 1 or claim 2, wherein the macroporous resin column of the aqueous solution through having handled well mixed total glycosides through silica gel column chromatography, Sephadex LH-20 column chromatography for separation, merges Rhoifoloside stream part, and crystallization obtains the pure article of Rhoifoloside.
12. purposes as claimed in claim 11, wherein said Rhoifoloside is used to suppress influenza virus.
13. purposes as claimed in claim 12, wherein said Rhoifoloside is used to suppress influenza virus FM1.
14. purposes as claimed in claim 11, wherein said Rhoifoloside is used to suppress neuraminidase.
15. purposes as claimed in claim 11, wherein said complication is meant renal failure.
16. purposes as claimed in claim 11, wherein said complication is meant injury of spleen or/and injury of lung.
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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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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105541941A (en) * | 2016-01-07 | 2016-05-04 | 广东石油化工学院 | Method for extracting and separating rhoifolin from exocarpium leaves |
| CN106349308A (en) * | 2016-08-02 | 2017-01-25 | 重庆工商大学 | Method for extracting apigenin-7-O-neohesperidoside from paeonia suffruticosa andrews and application of apigenin-7-O-neohesperidoside |
| CN106349308B (en) * | 2016-08-02 | 2019-04-19 | 重庆工商大学 | The method and application of Rhoifolin are extracted in a kind of peony |
| CN108125975A (en) * | 2018-01-29 | 2018-06-08 | 广东省农业科学院动物卫生研究所 | Application of the Rhoifolin in anti-Cryptosporidum parvum drug is prepared |
| 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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