CN104248654B - The application of karanjin or Indian beech extract in anti-influenza virus medicament - Google Patents
The application of karanjin or Indian beech extract in anti-influenza virus medicament Download PDFInfo
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Abstract
The invention discloses Indian beech extractive of general flavone or karanjin to prepare for the application in treatment and/or anti-influenza virus medicament.The Indian beech extractive of general flavone is obtained from Indian beech stem branch and/or leaf extraction, and the content of general flavone is 50%~70% weight；Wherein, containing karanjin；The content of the general flavone is using karanjin as reference substance, using determined by ultraviolet spectrophotometry；The content of karanjin is 5%~10% weight.
Description
Technical field
The present invention relates to pharmaceutical technology field, and in particular to the application of Indian beech extractive of general flavone or karanjin.
Background technology
Influenza Virus orthomyxoviridae family (Orthomyxoviridae), have tunicary 8 fragments, encode 11 hatching eggs altogether
The single-stranded negative RNA virus of white matter.Influenza virus is the cause of disease for causing influenza, is divided into first, second, the third three types, wherein with first
Type influenza threatens the mankind maximum.Human influenza virus is mainly H1, H2 and H3 hypotype, wherein with H1N1 hypotype case fatality rate highests,
Therefore the present invention is mainly research object to the disease-resistant of Indian beech extractive of general flavone and karanjin using H1N1 types influenza virus
Cytotoxic activity is studied.
At present, influenza still belongs to the acute upper respiratory tract infectious disease for failing effectively to control, have infectiousness is high, it is rapid to propagate,
The features such as happening and prevelence.Because the antigenic variation ability of influenza virus is strong, and vaccine only has to known influenza virus sub-strain
Prevention effect, and it is invalid for the novel influenza as caused by antigenic drift or antigenicity conversion, therefore, every few years
Influenza virus just once large range of prevalence.According to statistics, the whole world has the people of 50-100 ten thousand to die from influenza every year on average.Thus
It can be seen that influenza is as a kind of viral infectious, not only serious threat public health, and it is heavy to give country and society to bring
The financial burden of weight.Although known influenza virus has been controlled, due to the antigenic variation ability pole of influenza virus
Strong and quite frequent, the development and production of vaccine relatively lag behind, not high for the protective rate of Susceptible population, therefore, new influenza
It is very popular and is likely to break out at any time.In this case, the anti-influenza virus medicament with preventive and therapeutic action is found just
Be particularly important with it is urgent.
Indian beech Pongamia pinnata (L.) Merr. is that the semi-ma ngrove of pulse family (Leguminosae) Pongamia is planted
Thing, alias poongaoil pongamia seed.The Pongamia whole world only has a kind, and it is coastal to be distributed widely in India, Malaysia and south China.
China's oil treatment scabies leprosy, running sore and rheumatism among the people squeezed out with its seed.In India, its seed and seed oil are used for treating hickie
Disease, leprosy, rheumatism, leaf are used to treat hemorrhoid, tumour, diminishing inflammation of wound etc..Modern pharmacological research shows that Indian beech is always yellow
Ketone extract has antibacterial, anti-inflammatory, analgesia, anti-herpesvirus, antiulcer isoreactivity, but there is not yet the report of resisiting influenza virus
(Huang Xinbi, the chemical composition and Advance on Pharmacological Activities [J] Chinese herbal medicines of imperial Shengjing city mangrove associates Indian beech, 2004,35
(9):1073-1076.).The present invention tests prove that, Indian beech extractive of general flavone and karanjin are to influenza A virus sense
There is the mdck cell of dye good resisiting influenza virus to act on；In addition, equally shown very in mouse influenza animal model
Good anti-influenza virus activity.This prompting Indian beech extractive of general flavone and karanjin are in treatment influenza a virus infection
Disease areas has good exploitation prospect.
The content of the invention
It is an object of the present invention to provide Indian beech extractive of general flavone or karanjin in treatment and/or flu-prevention
Application in virus drugs, a kind of safely and effectively natural drug is provided for the treatment of clinically influenza virus property disease.
The technical scheme is that：
Indian beech extractive of general flavone or karanjin are being prepared for answering in treatment and/or anti-influenza virus medicament
With.
Further, the Indian beech extractive of general flavone is obtained from Indian beech stem branch and/or leaf extraction, general flavone
Content is 50%~70% weight；
Wherein, containing karanjin；
The content of the general flavone is using karanjin as reference substance, using determined by ultraviolet spectrophotometry；
The content of karanjin is 5%~10% weight.
Indian beech extractive of general flavone of the present invention is prepared by following methods：
(1) water intaking Calusena lansium stem branch and/or leaf, extracted 1~3 time with 50%~90% alcohol reflux, extracted 1~3 hour every time,
Merge extract solution；
(2) step 1 gained extract solution is concentrated under reduced pressure into no alcohol taste, centrifuges, take supernatant；
(3) by polyamide chromatography post separation 2~5 column volumes are eluted with water, then use in the supernatant obtained by step 2
20~70% column volumes of ethanol elution 2~10, collect ethanol eluate；
(4) concentration, drying ethanol eluent obtain Indian beech extractive of general flavone.
Further, the preparation method of the Indian beech extractive of general flavone, comprises the following steps：
(1) water intaking Calusena lansium stem branch and/or leaf, extracted 2 times with 80% alcohol reflux, every time extraction 2 hours, merge extract solution；
(2) step 1 gained extract solution is concentrated under reduced pressure into no alcohol taste, centrifuges, take supernatant；
(3) 3 column volumes are eluted with water by the supernatant obtained by step 2 by polyamide chromatography post separation, then with 50%
5 column volumes of ethanol elution, collect ethanol eluate；
(4) concentration, drying ethanol eluent obtain Indian beech extractive of general flavone.
Karanjin of the present invention has HPLC chromatogram as shown in Figure 4, wherein, the characteristic peak of chromatogram is：Water
Calusena lansium element retention time：10.8min；
Chromatographic condition is：Using octadecylsilane chemically bonded silica as filler；Mobile phase：Methanol -0.2%HAc (43:57)；
Flow velocity：1ml/min；Detection wavelength：260nm.
Karanjin of the present invention is to purify and obtain from Indian beech extractive of general flavone, is comprised the following steps：
(1) by reversed-phase silica gel chromatography post separation, with 30%~60% ethanol elution, karanjin crude product is obtained；
(2) with the karanjin crude product obtained by absolute ethyl alcohol dissolving step (1), it is 5%~30% to add water to concentration of alcohol, is put
Recrystallization is put, is produced.
The preparation method of preferable karanjin, comprises the following steps：
(1) by reversed-phase silica gel chromatography post separation, with 40% ethanol elution, karanjin crude product is obtained；
(2) with the karanjin crude product obtained by absolute ethyl alcohol dissolving step (1), it is 10% to add water to concentration of alcohol, places weight
Crystallization, is produced.
A further object of the present invention is to provide a kind of pharmaceutical composition of resisiting influenza virus, and described pharmaceutical composition contains
Karanjin of the present invention and/or Indian beech extractive of general flavone.
Further, described pharmaceutical composition includes pharmaceutically acceptable carrier and/or excipient.
Brief description of the drawings
Fig. 1 is the structural formula of Indian beech table.
Fig. 2 is the cytotoxicity of Ribavirin and the inhibitory action figure of infected by influenza.
Fig. 3 is mdck cell toxicity and the infected by influenza H1N1 suppression of Indian beech extractive of general flavone and karanjin
Effect；
Wherein, left figure NEP009 represents Indian beech extractive of general flavone, and right figure NEP014 represents karanjin.
Fig. 4 is the HPLC chromatogram of karanjin.
Embodiment
By following examples it will be further appreciated that advantages of the present invention and feature, it is to the present invention that should not be construed
The limitation of scope.
Instrument and reagent in following examples, it is common commercially available in addition to specified otherwise.
【Embodiment 1】The preparation and analysis of Indian beech extractive of general flavone
Fetch water Calusena lansium branches and leaves 1Kg, crushes, adds 80% ethanol 10L heating and refluxing extractions twice, extracts 2h every time, merges extraction
Liquid, no alcohol taste (being concentrated into v/w ≈ 3~4) is concentrated under reduced pressure into Rotary Evaporators, centrifuges, take supernatant, be loaded into and first use ethanol
Wash away after residual solvent using purified water as (column volume 1L) on the polyamide chromatography post of initial solvent, loading flow velocity is per hour 1
Individual column volume；Then, first with purifying 3 column volumes of water elution, then with 50% ethanol elution, 5 column volumes, elution flow rate is per small
When 2 column volumes, collect 50% ethanol eluate, be concentrated under reduced pressure, dry, Indian beech extractive of general flavone 13.6g is obtained, with water
Calusena lansium element is reference substance (described in preparation method following article embodiment 2, purity 99.5%), under 260nm wavelength, through ultraviolet point
General flavone content is calculated as 53.2% with karanjin in light photometry measure extract；With high performance liquid chromatography (chromatographic column：With
Octadecylsilane chemically bonded silica is filler；Mobile phase：Methanol -0.2%HAc (43:57)；Flow velocity：1ml/min；Detect ripple
It is long：It is 13.1% 260nm) to determine Indian beech cellulose content in extractive of general flavone.
【Embodiment 2】The preparation of karanjin and content analysis
The Indian beech extractive of general flavone 10g that Example 1 obtains, after adding 50mL ethanol to dissolve by heating, it is slowly added into water
100mL, centrifugation, takes supernatant to be loaded on the reversed-phase silica gel chromatography post balanced with 40%；With 40% ethanol elution, thin layer color
Spectrum inspects eluent, collects the fraction containing karanjin, is dried under reduced pressure to obtain karanjin crude product 2.7g, by karanjin crude product
After being dissolved with absolute ethyl alcohol, it is about 10% to add water to concentration of alcohol, is placed, and recrystallization, karanjin 2.3g is obtained, with efficient liquid
Phase chromatography (chromatographic column：Using octadecylsilane chemically bonded silica as filler；Mobile phase：Methanol -0.2%HAc (43:57)；Stream
Speed：1ml/min；Detection wavelength：It is 98.2% 260nm) to determine Indian beech cellulose content in extract.
【Embodiment 3】The external resisiting influenza virus of Indian beech extractive of general flavone or karanjin acts on
Strain：Influenza A virus (A/PR/8/34H1N1), purchased from Chinese Typical Representative Organism Depositary.
Cell model：MDCK system MDCK, purchased from Shanghai Bo Yan bio tech ltd；Condition of culture:DMEM+
10% hyclone, 37 DEG C, 5%CO2。
The cytotoxicity detection of medicine：Employ(Invitrogen) kit detects medicine to thin
The toxic action of born of the same parents.Experimental principle：It is a kind of oxidation-reduction indicator, suction can be produced according to metabolic activity
Light varience and fluorescence signal.Soluble in water, its oxidised form enters after cell also to be originated in through cyclophorase
Raw measurable fluorescence and color change, the quantitative analysis and vitro cytotoxicity bred for cytoactive and cell are ground
Study carefully.This measure is ability that reagent is converted into fluorescence and colorimetric indicator based on the cell with metabolic activity, be damaged and
Inactive cell has relatively low native metabolic active, and corresponding signal is relatively low.Therefore fluorescence signal is strong and weak, can reflect cell
The height of activity.
Method and step：Mdck cell is inoculated in 96 porocyte culture plates, standby after cell attachment.Use cell maintenance medium
(DMEM+5% serum) from continuous 3 times of gradient dilutions, 6 gradients of 2 times of initial concentrations, detects medicine per concentration gradient single hole.Add
After medicine culture 72h, the drug-induced cytopathic effect of light Microscopic observation (CPE), add37 DEG C of incubations
2h, fluoroscopic examinationReduction situation, exciting light 570nm, transmitting light 595nm.
Cytoactive (%)=(sample well-blank control)/(cell controls-blank control) × 100%
The suppression experiment of medicine infected by influenza：In view of based on virus caused by cytopathic effect detection not enough it is sensitive and
It is not easy to quantitative analysis.The present invention represents the levels of replication of virus using detection influenza virus neuraminidase activity.Experiment
Principle：Influenza neuraminidase (NA), also known as sialidase, it is a kind of surface glycoprotein, there is enzymatic activity, to A, Type B
The duplication of influenza virus plays an important role.It can be helped with the sialic acid residues of cell lysis surface influenza viral receptor end
The release of progeny viral particles, and virion aggregation is prevented, promote the diffusion of virus.Neuraminidase is antiviral drug for influenza
The important target spot of thing research and development.The present invention is using MUNANA as substrate detection influenza virus NA activity.MUNANA(4-
Methylumbelliferyl- α-N-acetyl-neuraminate) be influenza virus NA specific substrate, NA effect under
Caused catalysate can produce 460nm fluorescence, the change of fluorescence intensity can be with sensitive anti-in the case where 355nm excites light irradiation
Reflect neuraminidase activity.After virus infected cell and dosing culture certain time, nutrient solution supernatant is taken to carry out neuraminidase
Activity determination, in reaction substrate excess, the speed of enzymatic reaction and enzyme concentration are proportional, so can be with according to this principle
NA enzyme activity is detected to reflect the titre of virion in supernatant.
Method and step：
(1) plating cells：Mdck cell is inoculated in standby in 96 porocyte culture plates.
(2) virus infected cell：Medicine continuous 3 times of gradient dilutions, 6 gradients from 2 times of highest test concentrations；By orifice plate
Middle culture medium discards, while adds medicine and virus liquid in cell hole, after being incubated 1h in 37 DEG C of cell culture incubators, abandons supernatant training
Base is supported, after PBS washings, adds gradient dilution medicine again.37 DEG C of cell culture incubator culture 48h are placed in, are aided with observation cytopathy
Become (CPE).Nutrient solution supernatant is taken to carry out neuraminidase viability examination.
Experiment sets blank control wells (normal cell), enzyme activity control wells (non-dosing thing hole after virus infection), positive drug
Control wells（Add Ribavirin after infection）.
（3）Neuraminidase activity detects：
Experimental implementation：20 μ L substrates are added in 96 hole black micro plates（MUNANA）, draw the μ L of supernatant of virus-culturing fluid 40
Add in orifice plate, lucifuge is placed in 37 DEG C of incubation 60min, adds 100 μ L terminate liquids/hole terminating reaction, is surveyed on multi-tester
Determine fluorescent value.Excitation wavelength is：355nm, launch wavelength 485nm.Calculate NA in each detection hole and be suppressed rate.
Inhibiting rate（%）=100-（Sample well-blank control）/（Enzyme activity control-blank control）×100%
As a result:
Drug sample stock concentration, highest toxotest concentration, most highly active test concentrations, CC50, EC50And SI (choosings
Select index) as shown in table 1.
Each drug sample experimental result of table 1.
The cytotoxicity of the different positives agents Ribavirins of orifice plate and the inhibitory action of infected by influenza are as shown in Figure 2.
Inhibitory action such as Fig. 3 institutes of the mdck cell toxicity and infected by influenza H1N1 of Indian beech extractive of general flavone and karanjin
Show.
Conclusion：Indian beech extractive of general flavone or karanjin have fine to the mdck cell of influenza a virus infection
Resisiting influenza virus effect.
【Embodiment 4】Anti- H1N1 virus functions in Indian beech extractive of general flavone or karanjin Mice Body
Mouse H1N1 virus infection：Kunming mice 80, is divided into 8 groups, every group 10, male and female half and half.Each group mouse（Remove
Outside first group of Normal group）The H1N1 virus liquids of via intranasal application instillation several times 0.05mL（105XTCID50）, is carried out after instillation 2h
Gastric infusion.
Drug therapy is tested：Via intranasal application instillation H1N1 virus liquids 0.05mL（105EID50）, carry out for the first time after instillation 2h
Gastric infusion.First group is Normal group, injects the cell maintenance medium 0.1mL of Isodose, gavage physiological saline；Second group
For virus-infected controls group, physiological saline is gavaged；3rd group is Indian beech extractive of general flavone low dose group（1mg/mL）Treatment
Group；4th group is Indian beech extractive of general flavone middle dose group (10mg/mL) treatment group；5th group is that Indian beech general flavone extracts
Thing high dose group (100mg/mL) treatment group；6th group is karanjin low dose group (1mg/mL) treatment group；7th group is water
Calusena lansium element middle dose group (10mg/mL) treatment group；8th group is karanjin high dose group (100mg/mL) treatment group.Daily one
It is secondary, each 0.2mL.Observe the disease symptom after zoogenetic infection.The 5th day and the tenth day everywhere after administration for each group mouse
Dead half, and sterile working takes lung.The 1/2 of each mouse lung sample is used for infectious virus and separated, and remaining 1/2 lung sample is used for
The detection of H1N1 viral nucleic acids.
Detection method：Separation, the identification of infectious H1N1 viruses in lung.Take metainfective 1/2 mouse of above-mentioned H1N1 viruses
Lung homogenate, suspension is made with the mdck cell nutrient solution containing 100U/mL penicillin, 100 μ g/mL streptomysins, takes 0.1mL to feel
Contaminate mdck cell, 37 DEG C, 5%CO22h (every 30min vibrations once) is adsorbed, abandoning supernatant adds cell maintenance medium, 37 DEG C,
5%CO2Incubator culture.With Continuous Observation under inverted microscope 6 days, there is CPE person for the positive, viral drop is carried out after freeze thawing 3 times
It is fixed, and identified using RT-PCR.Do not occur CPE (cytopathy) persons and be still considered as separation feminine gender without CPE person through the generation of blind passage 3,
Discard.H1N1 viral nucleic acids detect in lung tissue.Metainfective remaining 1/2 mouse lungs of above-mentioned H1N1 are taken, extract RNA in tissue,
H1N1 viral RNAs are extracted with method, carry out RT-PCR amplifications respectively, product is dyed through agarose gel electrophoresis, EB, seen under uviol lamp
Examine, compareed with molecular weight standards, occurring band at 110bp, person is the positive.
Experimental result：Mdck cell isolated viral result is shown, is inoculated in each group mouse tissue Viral extraction of culture plate
Liquid can not make cell that lesion occur, and by the generation of cell blind passage 3, cell growth state is good, and no lesion occurs.H1N1 detection of nucleic acids
As a result show, virus control group, low dose group result are positive, and blank control group, middle dose group and high dose group result are in the moon
Property, under middle dosage concentration (10mg/mL), it is thin in mouse lung that Indian beech extractive of general flavone and karanjin can suppress H1N1
Infection and breeding in born of the same parents.
【Embodiment 5】The work of Indian beech extractive of general flavone or the karanjin anti-H1N1 subtype avian influenza virus in chicken body
With
SPF chickens are purchased from the emerging big magnificent agriculture SPF chicken Experimental Animal Centers in Guangdong；H1N1 viruses are protected purchased from Chinese Typical Representative microorganism
Tibetan center.
1st, experimental method
Indian beech extractive of general flavone or karanjin preventing and treating 10 week old SPF (Specific Pathogen Free, no spy
Determine cause of disease) chicken infection H1N1 subtype avian influenza virus experiment be divided into 9 groups：
1st group is Normal group, and virus infection, is not administered, altogether 20 SPF chickens.
2nd group is infection control group, and H1N1 subtype avian influenza virus dosage is 105EID50/0.1mL, intramuscular injection
0.2mL；After infection, not to any medicine, 20 10 week old SPF chickens altogether.
3rd group is Shuanghuanglian oral liquid control group, altogether 30 10 week old SPF chickens, H1N1 subtype avian influenza virus dosage
For 105EID50/0.1mL, intramuscular injection 0.2mL；Shuanghuanglian oral liquid (the Harbin Pharmaceutical Group of 15mL/Kg body weight doses is given simultaneously
Three smart Pharmacy stock Co., Ltd's productions, lot number：11041003), concentrate drinking-water once daily, be used in conjunction 7 days.
4th group is Indian beech extractive of general flavone low dose therapy group, altogether 30 10 week old SPF chickens, H1N1 hypotype fowl
Influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL；The Indian beech for giving 0.5mg/Kg body weight doses simultaneously is total
Chromocor extract, concentrate drinking-water once daily, be used in conjunction 7 days.
5th group is Indian beech extractive of general flavone middle dosage treatment group, altogether 30 10 week old SPF chickens, H1N1 hypotype fowl
Influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL；The Indian beech for giving 10mg/Kg body weight doses simultaneously is total
Chromocor extract, concentrate drinking-water once daily, be used in conjunction 7 days.
6th group is Indian beech extractive of general flavone high-dose therapy group, altogether 30 10 week old SPF chickens, H1N1 hypotype fowl
Influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL；The Indian beech for giving 20mg/Kg body weight doses simultaneously is total
Chromocor extract, concentrate drinking-water once daily, be used in conjunction 7 days.
7th group is karanjin low dose therapy group, altogether 30 10 week old SPF chickens, H1N1 subtype avian influenza virus agent
Measure as 105EID50/0.1mL, intramuscular injection 0.2mL；The karanjin of 0.5mg/Kg body weight doses is given simultaneously, is concentrated daily
Drinking-water once, is used in conjunction 7 days.
8th group is karanjin middle dosage treatment group, altogether 30 10 week old SPF chickens, H1N1 subtype avian influenza virus agent
Measure as 105EID50/0.1mL, intramuscular injection 0.2mL；The karanjin of 10mg/Kg body weight doses is given simultaneously, concentrates drink daily
Water once, is used in conjunction 7 days.
9th group is karanjin high-dose therapy group, altogether 30 10 week old SPF chickens, H1N1 subtype avian influenza virus agent
Measure as 105EID50/0.1mL, intramuscular injection 0.2mL；The karanjin of 20mg/Kg body weight doses is given simultaneously, concentrates drink daily
Water once, is used in conjunction 7 days.
2nd, evaluation method
Continuous Observation 14d, records the death toll of chicken, and calculates the death rate, mean survival time.After infection 1,3,5,7,
10 and 14d collection chickens cloaca, larynx cotton swab, the cotton swab of collection are made into 10-1~10-8 suspensions with sterile saline,
Allantoic cavity is seeded in 9~10 age in days SPF chicken embryos, 37 DEG C of incubation 48h, according to micro erythrocyte agglutination method, checks chick embryo allantois
Hemagglutination activity in liquid.If any hemagglutination activity, by hemagglutination-inhibition test on the premise of newcastle disease virus infection is excluded, explanation has
Avian influenza virus is present.
3rd, experimental result
After infection, the Virus Infection testing result of each group chicken is as shown in table 2, in whole experiment process, Indian beech
The treatment group of extractive of general flavone and karanjin various dose without death, dissects after catching and killing and examined, do not show any abnormalities；Examination
The Virus Infection testing result for testing end Hou Ge treatment groups survival chicken is feminine gender.Illustrate that the Indian beech of various concentrations is always yellow
Ketone extract and karanjin have good inhibiting effect to H1N1 subtype avian influenza virus in vivo.
Different time gathers the Virus Infection of each experimental group chicken throat and cloacal swab after table 2 infects
【Embodiment 6】The formulation preparation method of Indian beech extractive of general flavone or karanjin
(1) preparation method of Indian beech extractive of general flavone tablet
Prescription：
Preparation method：Indian beech extractive of general flavone is uniformly dispersed with PVP, it is fine to add microcrystalline cellulose, cross-linked carboxymethyl
Plain sodium is tieed up, is mixed, tabletting, is coated with the full water coating solution of 16% (w/w) Opadry, produces the tablet of Indian beech extractive of general flavone.
（2）The preparation method of Indian beech extractive of general flavone granule
Prescription：
Preparation method：Indian beech extractive of general flavone, Icing Sugar, dextrin are crossed into 100 mesh sieves respectively, mixed, adds 50% ethanol, system
Into softwood, sieving is pelletized, 60 DEG C of dryings, is dispensed after whole grain with compound membrane bag.Produce the granule of Indian beech extractive of general flavone.
（3）The preparation method of Indian beech extractive of general flavone capsule
Prescription：
Preparation method：Indian beech extractive of general flavone and starch is uniform, calcium carbonate is added, is mixed, crosses 100 mesh sieves, it is encapsulated,
Produce Indian beech extractive of general flavone capsule.
（4）The preparation method of karanjin tablet
Prescription：
Preparation method：Karanjin and PVP are uniformly dispersed, add microcrystalline cellulose, Ac-Di-Sol, is mixed
It is even, tabletting, with 16%（w/w）The full water coating solution coating of Opadry, produce the tablet of karanjin.
（5）The preparation method of karanjin granule
Prescription：
Preparation method：Karanjin, Icing Sugar, dextrin are crossed into 100 mesh sieves respectively, mixed, 50% ethanol is added, softwood is made, is sieved
Granulation, 60 DEG C of dryings, dispensed with compound membrane bag after whole grain.Produce the granule of karanjin.
（6）The preparation method of karanjin capsule
Prescription：
Preparation method：Karanjin is uniform with starch, calcium carbonate is added, is mixed, crosses 100 mesh sieves, it is encapsulated, produce Indian beech
Cellulose capsule agent.
Claims (8)
1. the application of Indian beech extractive of general flavone or karanjin in preparing for anti-influenza virus medicament.
2. application as claimed in claim 1, it is characterised in that the Indian beech extractive of general flavone is from Indian beech stem branch
And/or leaf is extracted and obtained, the content of general flavone is 50%~70% weight；
Wherein, containing karanjin；
The content of the general flavone is using karanjin as reference substance, using determined by ultraviolet spectrophotometry；
The content of karanjin is 5%~10% weight.
3. application as claimed in claim 1, it is characterised in that the Indian beech extractive of general flavone is prepared by following methods：
(1) water intaking Calusena lansium stem branch and/or leaf, extracted 1~3 time with 50%~90% alcohol reflux, extracted 1~3 hour every time, closed
And extract solution；
(2) step 1 gained extract solution is concentrated under reduced pressure into no alcohol taste, centrifuges, take supernatant；
(3) 2~5 column volumes are eluted with water by the supernatant obtained by step 2 by polyamide chromatography post separation, then with 20%
~70% column volume of ethanol elution 2~10, collect ethanol eluate；
(4) concentration, drying ethanol eluent obtain Indian beech extractive of general flavone.
4. application as claimed in claim 3, it is characterised in that the Indian beech extractive of general flavone is prepared by following methods：
(1) water intaking Calusena lansium stem branch and/or leaf, extracted 2~3 times with 60%~80% alcohol reflux, extracted 2~3 hours every time, closed
And extract solution；
(2) step 1 gained extract solution is concentrated under reduced pressure into no alcohol taste, centrifuges, take supernatant；
(3) 3~4 column volumes are eluted with water by the supernatant obtained by step 2 by polyamide chromatography post separation, then with 40%
~60% column volume of ethanol elution 4~7, collect ethanol eluate；
(4) concentration, drying ethanol eluent obtain Indian beech extractive of general flavone.
5. application as claimed in claim 4, it is characterised in that the Indian beech extractive of general flavone is prepared by following methods：
(1) water intaking Calusena lansium stem branch and/or leaf, extracted 2 times with 80% alcohol reflux, every time extraction 2 hours, merge extract solution；
(2) step 1 gained extract solution is concentrated under reduced pressure into no alcohol taste, centrifuges, take supernatant；
(3) 3 column volumes are eluted with water by the supernatant obtained by step 2 by polyamide chromatography post separation, then with 50% ethanol
5 column volumes are eluted, collect ethanol eluate；
(4) concentration, drying ethanol eluent obtain Indian beech extractive of general flavone.
6. application as claimed in claim 1, it is characterised in that the karanjin has HPLC chromatogram as shown in Figure 4,
Wherein, the characteristic peak of chromatogram is：Karanjin retention time：10.8min；
Chromatographic condition is：Using octadecylsilane chemically bonded silica as filler；Mobile phase：Volume ratio is 43:57 methanol and
0.2%HAc；Flow velocity：1ml/min；Detection wavelength：260nm.
7. the application as described in claim 1 or 6, it is characterised in that the karanjin is from Indian beech extractive of general flavone
It is middle purification and obtain, comprise the following steps：
(1) by reversed-phase silica gel chromatography post separation, with 30%~60% ethanol elution, karanjin crude product is obtained；
(2) with the karanjin crude product obtained by absolute ethyl alcohol dissolving step (1), it is 5%~30% to add water to concentration of alcohol, is placed
Recrystallization, is produced.
8. application as claimed in claim 7, it is characterised in that the purification step of the karanjin includes：
(1) by reversed-phase silica gel chromatography post separation, with 40% ethanol elution, karanjin crude product is obtained；
(2) with the karanjin crude product obtained by absolute ethyl alcohol dissolving step (1), it is 10% to add water to concentration of alcohol, and placement is tied again
Crystalline substance, produce.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310259698.4A CN104248654B (en) | 2013-06-26 | 2013-06-26 | The application of karanjin or Indian beech extract in anti-influenza virus medicament |
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| CN102241686A (en) * | 2011-05-06 | 2011-11-16 | 南京泽朗农业发展有限公司 | Preparation method of karanjin |
| CN102462727A (en) * | 2010-11-17 | 2012-05-23 | 中国医学科学院药用植物研究所 | Yulangsan general flavone and action of monomer component thereof in preparation of anti-tumor medicament |
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| CN102462727A (en) * | 2010-11-17 | 2012-05-23 | 中国医学科学院药用植物研究所 | Yulangsan general flavone and action of monomer component thereof in preparation of anti-tumor medicament |
| CN102241686A (en) * | 2011-05-06 | 2011-11-16 | 南京泽朗农业发展有限公司 | Preparation method of karanjin |
Non-Patent Citations (1)
| Title |
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| 水罗伞的化学成分和药理研究进展;李熙灿等;《中药新药与临床药理》;20050531;第16卷(第3期);第231-232页以及封三 * |
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