CN104248654A - Application of karanjin or Pongamia pinnata extract in anti-influenza virus drugs - Google Patents
Application of karanjin or Pongamia pinnata extract in anti-influenza virus drugs Download PDFInfo
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Abstract
The invention discloses an application of a Pongamia pinnata general flavone extract or karanjin in the preparation of influenza virus treatment and/or prevention drugs. The Pongamia pinnata general flavone extract is extracted from stem branches and/or leaves of Pongamia pinnata, contains 50-70wt% of general flavones, and also contains karanjin; the content of the general flavone is determined through an ultraviolet spectrophotometric method with karanjin as a reference substance; and the content of karanjin is 5-10wt%.
Description
Technical field
The present invention relates to medical art, be specifically related to the application of Indian beech extractive of general flavone or karanjin.
Background technology
Influenza Virus orthomyxoviridae family (Orthomyxoviridae), tunicary 8 fragments of tool, the strand of 11 kinds of protein of encoding altogether bears RNA viruses.Influenza virus causes grippal cause of disease, is divided into first, second, the third three types, wherein threatens the mankind maximum with influenza A.Human influenza virus is H1, H2 and H3 hypotype mainly, wherein the highest with H1N1 hypotype case fatality rate, therefore the present invention mainly with H1N1 type influenza virus for the antiviral activity of object of study to Indian beech extractive of general flavone and karanjin is studied.
At present, influenza still belongs to the acute upper respiratory tract infectious disease failing effectively to control, have infectiousness high, propagate rapidly, the feature such as happening and prevelence.Because the antigenic variation ability of influenza virus is strong, and vaccine only has preventive effect to known influenza virus sub-strain, and invalid for the novel influenza produced by antigenic drift or antigenicity conversion, therefore, every several years influenza virus just once in a big way popular.According to statistics, the whole world has 50-100 ten thousand people to die from influenza every year on average.As can be seen here, influenza as a kind of viral infectious not only serious threat public health, and give country and society bring heavy financial burden.Although known influenza virus is controlled, the antigenic variation due to influenza virus is very competent and quite frequent; the research and production of vaccine relatively lags behind; protective rate for Susceptible population is not high, and therefore, new flu outbreak all likely breaks out at any time.In this case, find the anti-influenza virus medicament with preventive and therapeutic action and just seem particularly important and urgent.
Indian beech Pongamia pinnata (L.) Merr. is the mangrove associates of pulse family (Leguminosae) Pongamia, another name Semen Pongamiae Pinnatae.The Pongamia whole world only has a kind, is distributed widely in India, Malaysia and south China coastal.In China's oil treatment mange, abscess and rheumatism of squeezing out with its seed among the people.In India, its seed and seed oil are used for treating leukoderma, leprosy, rheumatism, and leaf is used for the treatment of hemorrhoid, tumor, diminishing inflammation of wound etc.Modern pharmacological research shows, Indian beech extractive of general flavone has antibacterial, antiinflammatory, analgesia, anti-herpesvirus, antiulcer isoreactivity, but there is not yet the report (Huang Xinbi of resisiting influenza virus, dragon Shengjing city. the chemical composition of mangrove associates Indian beech and Advance on Pharmacological Activities [J]. Chinese herbal medicine, 2004,35 (9): 1073-1076.).The present invention proves through test, and Indian beech extractive of general flavone and the mdck cell of karanjin to influenza a virus infection have good resisiting influenza virus effect; In addition, in mouse influenza animal model, good anti-influenza virus activity is shown equally.This prompting Indian beech extractive of general flavone and karanjin have good exploitation prospect at treatment influenza a virus infection disease areas.
Summary of the invention
An object of the present invention is to provide Indian beech extractive of general flavone or karanjin is treating and/or preventing the application in influenza virus medicine, for the treatment of influenza virus property disease clinically provides one natural drug safely and effectively.
Technical scheme of the present invention is:
Indian beech extractive of general flavone or karanjin are for the preparation of the application in treatment and/or anti-influenza virus medicament.
Further, described Indian beech extractive of general flavone extracts from Indian beech stem branch and/or leaf and obtain, and the content of total flavones is 50% ~ 70% weight;
Wherein, containing karanjin;
The content of described total flavones take karanjin as reference substance, adopts 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 method:
(1) to fetch water Clausena lansium (Lour.) Skeels stem branch and/or leaf, with 50% ~ 90% alcohol reflux 1 ~ 3 time, extract 1 ~ 3 hour, merge extractive liquid, at every turn;
(2) step 1 gained extracting solution is evaporated to without alcohol taste, centrifugal, get supernatant;
(3) supernatant of step 2 gained is separated by polyamide chromatography post, washes 2 ~ 5 column volumes with water, then use 20 ~ 70% ethanol elution, 2 ~ 10 column volumes, collect ethanol elution;
(4) concentrated, drying ethanol eluent obtains Indian beech extractive of general flavone.
Further, the preparation method of described Indian beech extractive of general flavone, comprises the following steps:
(1) to fetch water Clausena lansium (Lour.) Skeels stem branch and/or leaf, with 80% alcohol reflux 2 times, to extract 2 hours, merge extractive liquid, at every turn;
(2) step 1 gained extracting solution is evaporated to without alcohol taste, centrifugal, get supernatant;
(3) supernatant of step 2 gained is separated by polyamide chromatography post, washes 3 column volumes with water, then use 50% ethanol elution, 5 column volumes, collect ethanol elution;
(4) concentrated, drying ethanol eluent obtains Indian beech extractive of general flavone.
Karanjin of the present invention has HPLC chromatogram as shown in Figure 4, and wherein, the characteristic peak of chromatogram is: karanjin retention time: 10.8min;
Chromatographic condition is: take octadecylsilane chemically bonded silica as filler; Mobile phase: methanol-0.2%HAc (43:57); Flow velocity: 1ml/min; Determined wavelength: 260nm.
Karanjin of the present invention is purified from Indian beech extractive of general flavone and obtains, and comprises the following steps:
(1) be separated by reversed-phase silica gel chromatography post, with 30% ~ 60% ethanol elution, obtain karanjin crude product;
(2) with the karanjin crude product of anhydrous alcohol solution step (1) gained, adding water to concentration of alcohol is 5% ~ 30%, places recrystallization, to obtain final product.
The preparation method of preferred karanjin, comprises the following steps:
(1) be separated by reversed-phase silica gel chromatography post, with 40% ethanol elution, obtain karanjin crude product;
(2) with the karanjin crude product of anhydrous alcohol solution step (1) gained, adding water to concentration of alcohol is 10%, places recrystallization, to obtain final product.
Another 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 comprises pharmaceutically acceptable carrier and/or excipient.
Accompanying drawing explanation
Fig. 1 is the structural formula of Rheum alexandrae Batal. epidermis.
Fig. 2 is the cytotoxicity of ribavirin and the inhibitory action figure of infected by influenza.
Fig. 3 is the mdck cell toxicity of Indian beech extractive of general flavone and karanjin and the inhibitory action of infected by influenza H1N1;
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.
Detailed description of the invention
Advantage and disadvantage of the present invention can be understood further by following examples, should not be construed and limit the scope of the present invention.
Instrument and reagent in following examples, be common commercially available except specified otherwise.
The preparation of [embodiment 1] Indian beech extractive of general flavone and analysis
Water intaking Clausena lansium (Lour.) Skeels branch and leaf 1Kg, pulverize, add 80% ethanol 10L heating and refluxing extraction twice, each extraction 2h, merge extractive liquid, is evaporated to without alcohol taste (being concentrated into v/w ≈ 3 ~ 4) with Rotary Evaporators, centrifugal, get supernatant, be loaded into after first washing away residual solvent with ethanol with purified water be initial solvent polyamide chromatography post on (column volume 1L), loading flow velocity is 1 column volume per hour; Then, first use purified water eluting 3 column volumes, then use 50% ethanol elution, 5 column volumes, elution flow rate is 2 column volumes per hour, collect 50% ethanol elution, concentrating under reduced pressure, dry, obtain Indian beech extractive of general flavone 13.6g, with karanjin, for reference substance, (preparation method is as described in Examples below 2, purity is 99.5%), under 260nm wavelength, in determined by ultraviolet spectrophotometry extract, general flavone content counts 53.2% with karanjin; With high performance liquid chromatography (chromatographic column: with octadecylsilane chemically bonded silica for filler; Mobile phase: methanol-0.2%HAc (43:57); Flow velocity: 1ml/min; Determined wavelength: 260nm) to measure karanjin content in extractive of general flavone be 13.1%.
The preparation of [embodiment 2] karanjin and content analysis
The Indian beech extractive of general flavone 10g that Example 1 obtains, after adding 50mL ethanol heating for dissolving, slowly adds water 100mL, centrifugal, gets supernatant and is loaded on the reversed-phase silica gel chromatography post that balanced with 40%; With 40% ethanol elution, thin layer chromatography inspects eluent, collect the stream part containing karanjin, drying under reduced pressure obtains karanjin crude product 2.7g, by karanjin crude product with after anhydrous alcohol solution, adds water to concentration of alcohol and is about 10%, place, recrystallization, obtains karanjin 2.3g, with high performance liquid chromatography (chromatographic column: with octadecylsilane chemically bonded silica for filler; Mobile phase: methanol-0.2%HAc (43:57); Flow velocity: 1ml/min; Determined wavelength: 260nm) to measure karanjin content in extract be 98.2%.
The In Vitro Anti influenza virus effect of [embodiment 3] Indian beech extractive of general flavone or karanjin
Strain: influenza A virus (A/PR/8/34H1N1), purchased from Chinese Typical Representative Organism Depositary.
Cell model: Madin-Darby canine kidney(cell line) (MDCK) system MDCK, purchased from Bo Yan bio tech ltd, Shanghai; Condition of culture: DMEM+10% hyclone, 37 DEG C, 5%CO
2.
The cytotoxicity of medicine detects: have employed
(Invitrogen) test kit detection of drugs is to the toxic action of cell.Experimental principle:
be a kind of oxidation-reduction indicator, absorbance change and fluorescence signal can be produced according to metabolic activity.
soluble in water, its oxidised form enters after cell and produces measurable fluorescence and color change through cyclophorase reduction, for quantitative analysis and the vitro cytotoxicity research of cytoactive and cell proliferation.This mensuration is the ability based on the cell with metabolic activity, reagent being converted to fluorescence and colorimetric indicator, and it is active that impaired and non-activity cell has lower native metabolic, and corresponding signal is lower.Therefore fluorescence signal is strong and weak, can reflect the height of cytoactive.
Method step: mdck cell is inoculated in 96 porocyte culture plates, for subsequent use after cell attachment.With cell maintenance medium (DMEM+5% serum) by medicine from 2 times of continuous 3 times of gradient dilutions of initial concentration, 6 gradients, every Concentraton gradient single hole detects.After 72h is cultivated in dosing, the drug-induced cytopathic effect of light Microscopic observation (CPE), adds
hatch 2h, fluoroscopic examination for 37 DEG C
reduction situation, exciting light 570nm, utilizing emitted light 595nm.
Cytoactive (%)=(sample well-blank)/(cell controls-blank) × 100%
The inhibition test of medicine infected by influenza: detect sensitive not in view of the cytopathic effect caused based on virus and be not easy to quantitative analysis.The present invention adopts and detects the levels of replication that influenza neuraminidase activity represents virus.Experimental principle: influenza neuraminidase (NA), also known as sialidase, is a kind of surface glycoprotein, has enzymatic activity, plays an important role to A, copying of Type B influenza virus.It can the sialic acid residues of cell lysis surface influenza viral receptor end, helps the release of progeny viral particles, and prevents virion aggregation, promote the diffusion of virus.Neuraminidase is the important target spot of anti-influenza virus medicament research and development.It is active that the present invention adopts MUNANA to detect influenza virus NA as substrate.MUNANA (4-methylumbelliferyl-α-N-acetyl-neuraminate) is the specific substrate of influenza virus NA, the catalysate produced under NA effect is under 355nm excitation light irradiation, 460nm fluorescence can be produced, the change of fluorescence intensity, can sensitive reflection neuraminidase activity.After certain hour is cultivated in virus infected cell dosing, get culture fluid supernatant and carry out neuraminidase activity detection, when reaction substrate is excessive, the speed of enzymatic reaction and enzyme concentration are proportional, so can detect the NA enzyme titre reflecting virion in supernatant alive according to this principle.
Method step:
(1) plating cells: mdck cell is inoculated in 96 porocyte culture plates for subsequent use.
(2) virus infected cell: medicine is continuous 3 times of gradient dilutions 6 gradients from 2 times of test concentrations the highest; Culture medium in orifice plate is discarded, adds medicine and virus liquid simultaneously in cell hole, after hatching 1h in 37 DEG C of cell culture incubators, abandon supernatant culture medium, after PBS washing, again add gradient dilution medicine.Be placed in 37 DEG C of cell culture incubators and cultivate 48h, be aided with observation of cell pathological changes (CPE).Get culture fluid supernatant and carry out neuraminidase viability examination.
Blank control wells (normal cell) is established in experiment, enzyme control wells (not adding medicine hole after viral infection) alive, positive drug control hole (adding ribavirin after infection).
(3) neuraminidase activity detects:
Experimental implementation: add 20 μ L substrates (MUNANA) in 96 hole black micro plates, drawing virus-culturing fluid 40 μ L supernatant adds in orifice plate, lucifuge is placed in 37 DEG C and hatches 60min, adds 100 μ L stop buffer/hole cessation reactions, multi-tester measures fluorescent value.Excitation wavelength is: 355nm, emission wavelength 485nm.Calculate the suppressed rate of NA in each detect aperture.
Suppression ratio (%)=100-(sample well-blank)/(enzyme contrast-blank alive) × 100%
Result:
Drug sample stock concentration, most high toxicity test concentrations, most high activity test concentrations, CC
50, EC
50and SI (selection index) is as shown in table 1.
The each drug sample experimental result of table 1.
The cytotoxicity of the positives agents Ribavirin of different orifice plates and the inhibitory action of infected by influenza are as shown in Figure 2.The mdck cell toxicity of Indian beech extractive of general flavone and karanjin and the inhibitory action of infected by influenza H1N1 are as shown in Figure 3.
Conclusion: Indian beech extractive of general flavone or the mdck cell of karanjin to influenza a virus infection have good resisiting influenza virus effect.
Anti-H1N1 virus effect in [embodiment 4] Indian beech extractive of general flavone or karanjin Mice Body
Mice H1N1 virus infects: kunming mice 80, is divided into 8 groups, often organizes 10, male and female half and half.Each group of mice (except the first group of Normal group) H1N1 virus of via intranasal application instillation several times liquid 0.05mL(10
5xTCID
50), carry out first time gastric infusion after instillation 2h.
Drug therapy is tested: via intranasal application instillation H1N1 virus liquid 0.05mL(105EID50), carry out first time gastric infusion after instillation 2h.First group is Normal group, the cell maintenance medium 0.1mL of injection Isodose, gavage normal saline; Second group is virus-infected controls group, gavages normal saline; 3rd group is Indian beech extractive of general flavone low dose group (1mg/mL) treatment group; 4th group is dosage group (10mg/mL) treatment group in Indian beech extractive of general flavone; 5th group is Indian beech extractive of general flavone high dose group (100mg/mL) treatment group; 6th group is karanjin low dose group (1mg/mL) treatment group; 7th group is dosage group (10mg/mL) treatment group in karanjin; 8th group is karanjin high dose group (100mg/mL) treatment group.Once a day, each 0.2mL.Observe the disease symptom after zoogenetic infection.Each group of mice respectively put to death half respectively at after administration the 5th day and the tenth day, and lung is got by sterile working.1/2 of each mouse lung sample is separated for infectious virus, remains the detection of 1/2 lung sample for H1N1 virus nucleic acid.
Detection method: the separation of infectious H1N1 virus in lung, qualification.Get the metainfective 1/2 mouse lung homogenate of above-mentioned H1N1 virus, make suspension with the mdck cell culture fluid containing 100U/mL penicillin, 100 μ g/mL streptomycins, get 0.1mL and infect mdck cell, 37 DEG C, 5%CO
2absorption 2h (every 30min vibration once), abandoning supernatant, adds cell maintenance medium, 37 DEG C, 5%CO
2incubator is cultivated.With Continuous Observation under inverted microscope 6 days, occur that CPE person is for positive, carry out titration of virus after freeze thawing 3 times, and adopt RT-PCR to identify.Do not occur that CPE (cytopathy) person was still considered as being separated feminine gender without CPE person through blind passage 3 generation, discard.H1N1 virus detection of nucleic acids in lung tissue.Get above-mentioned H1N1 metainfective residue 1/2 mouse lung, RNA in extracting tissue, with method extracting H1N1 virus RNA, carry out RT-PCR amplification respectively, product, through agarose gel electrophoresis, EB dyeing, is observed under uviol lamp, contrast with molecular weight standards, person is the positive to occur band at 110bp place.
Experimental result: mdck cell isolated viral result shows, and each group of mouse tissue viral extract being inoculated in culture plate all can not make cell generation pathological changes, by cell blind passage 3 generation, cell growth state is good, occurs without pathological changes.H1N1 detection of nucleic acids result shows, virus control group, low dose group result are positive, blank group, middle dosage group and high dose group result are negative, under middle dose concentration (10mg/mL), Indian beech extractive of general flavone and karanjin can suppress the infection of H1N1 in mouse lung cell and breeding.
The effect of [embodiment 5] Indian beech extractive of general flavone or karanjin anti-H1N1 subtype avian influenza virus in chicken body
SPF chicken is purchased from the emerging large magnificent agriculture SPF chicken Experimental Animal Center in Guangdong; H1N1 virus is purchased from Chinese Typical Representative Organism Depositary.
1, experimental technique
The experiment that Indian beech extractive of general flavone or karanjin control SPF (Specific Pathogen Free, specific pathogen free) chicken in 10 week age infect H1N1 subtype avian influenza virus is divided into 9 groups:
1st group is Normal group, does not infect virus, not administration, amounts to 20 SPF chickens.
2nd group is infect matched group, and H1N1 subtype avian influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL; After infection, not give any medicine, amount to 20 10 week age SPF chicken.
3rd group is SHUANGHUANGLIAN KOUFUYE matched group, amount to 30 10 week age SPF chicken, H1N1 subtype avian influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL; Give simultaneously 15mL/Kg body weight dose SHUANGHUANGLIAN KOUFUYE (Harbin Pharmaceutical Group, Sanjing Pharmaceutical Co., Ltd. produce, lot number: 11041003), every day concentrate drinking-water once, be used in conjunction 7 days.
4th group is Indian beech extractive of general flavone low dose therapy group, amount to 30 10 week age SPF chicken, H1N1 subtype avian influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL; Give the Indian beech extractive of general flavone of 0.5mg/Kg body weight dose, every day concentrates drinking-water once, is used in conjunction 7 days simultaneously.
5th group is dosage treatment group in Indian beech extractive of general flavone, amount to 30 10 week age SPF chicken, H1N1 subtype avian influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL; Give the Indian beech extractive of general flavone of 10mg/Kg body weight dose, every day concentrates drinking-water once, is used in conjunction 7 days simultaneously.
6th group is Indian beech extractive of general flavone high-dose therapy group, amount to 30 10 week age SPF chicken, H1N1 subtype avian influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL; Give the Indian beech extractive of general flavone of 20mg/Kg body weight dose, every day concentrates drinking-water once, is used in conjunction 7 days simultaneously.
7th group is karanjin low dose therapy group, amount to 30 10 week age SPF chicken, H1N1 subtype avian influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL; Give the karanjin of 0.5mg/Kg body weight dose, every day concentrates drinking-water once, is used in conjunction 7 days simultaneously.
8th group is dosage treatment group in karanjin, amount to 30 10 week age SPF chicken, H1N1 subtype avian influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL; Give the karanjin of 10mg/Kg body weight dose, every day concentrates drinking-water once, is used in conjunction 7 days simultaneously.
9th group is karanjin high-dose therapy group, amount to 30 10 week age SPF chicken, H1N1 subtype avian influenza virus dosage is 105EID50/0.1mL, intramuscular injection 0.2mL; Give the karanjin of 20mg/Kg body weight dose, every day concentrates drinking-water once, is used in conjunction 7 days simultaneously.
2, evaluation methodology
Continuous Observation 14d, the death toll of record chicken, and calculate mortality rate, mean survival time.After infection 1,3,5,7,10 and 14d gather chicken cloaca, larynx cotton swab, the cotton swab sterile saline gathered is made into 10-1 ~ 10-8 suspension, allantoic cavity is seeded in 9 ~ 10 age in days SPF Embryo Gallus domesticus, hatch 48h for 37 DEG C, according to micro-red cell agglutination method, check hemagglutination activity in chick embryo allantoic liquid.If any hemagglutination activity, by hemagglutination inhibition test under the prerequisite getting rid of newcastle disease virus infection, illustrate that bird flu virus exists.
3, experimental result
After infection, the Virus Infection testing result of each group chicken is as shown in table 2, and in whole experimentation, the treatment group of Indian beech extractive of general flavone and karanjin various dose, all without dead, catched and killed rear dissection inspection, do not shown any abnormalities; After off-test, the Virus Infection testing result of each treatment group survival chicken is feminine gender.Illustrate that Indian beech extractive of general flavone and the karanjin of variable concentrations all have good inhibitory action to H1N1 subtype avian influenza virus in vivo.
Table 2 infects the Virus Infection that rear different time gathers each experimental group chicken throat and cloacal swab
The formulation preparation method of [embodiment 6] Indian beech extractive of general flavone or karanjin
(1) preparation method of Indian beech extractive of general flavone tablet
Prescription:
Method for making: Indian beech extractive of general flavone and polyvidone are uniformly dispersed, adds microcrystalline Cellulose, cross-linking sodium carboxymethyl cellulose, and mixing, tabletting, with 16% (w/w) Opadry full water coating solution coating, obtains the tablet of Indian beech extractive of general flavone.
(2) preparation method of Indian beech extractive of general flavone granule
Prescription:
Method for making: Indian beech extractive of general flavone, Icing Sugar, dextrin are crossed 100 mesh sieves respectively, mixing, add 50% ethanol, make soft material, granulation of sieving, 60 DEG C of dryings, use compound membrane bag subpackage after granulate.Obtain the granule of Indian beech extractive of general flavone.
(3) preparation method of Indian beech extractive of general flavone capsule
Prescription:
Method for making: Indian beech extractive of general flavone and starch is even, adds calcium carbonate, and 100 mesh sieves are crossed in mixing, encapsulated, obtain Indian beech extractive of general flavone capsule.
(4) preparation method of karanjin tablet
Prescription:
Method for making: karanjin and polyvidone are uniformly dispersed, adds microcrystalline Cellulose, cross-linking sodium carboxymethyl cellulose, mixing, and tabletting, with 16%(w/w) Opadry full water coating solution coating, obtain the tablet of karanjin.
(5) preparation method of karanjin granule
Prescription:
Method for making: karanjin, Icing Sugar, dextrin are crossed 100 mesh sieves respectively, mixing, add 50% ethanol, make soft material, granulation of sieving, 60 DEG C of dryings, use compound membrane bag subpackage after granulate.Obtain the granule of karanjin.
(6) preparation method of karanjin capsule
Prescription:
Method for making: karanjin and starch is even, adds calcium carbonate, and 100 mesh sieves are crossed in mixing, encapsulated, obtain karanjin capsule.
Claims (10)
1. Indian beech extractive of general flavone or karanjin are for the preparation of the application in treatment and/or anti-influenza virus medicament.
2. apply as claimed in claim 1, it is characterized in that, described Indian beech extractive of general flavone extracts from Indian beech stem branch and/or leaf and obtain, and the content of total flavones is 50% ~ 70% weight;
Wherein, containing karanjin;
The content of described total flavones take karanjin as reference substance, adopts determined by ultraviolet spectrophotometry;
The content of karanjin is 5% ~ 10% weight.
3. apply as claimed in claim 1, it is characterized in that, described Indian beech extractive of general flavone is prepared by following method:
(1) to fetch water Clausena lansium (Lour.) Skeels stem branch and/or leaf, with 50% ~ 90% alcohol reflux 1 ~ 3 time, extract 1 ~ 3 hour, merge extractive liquid, at every turn;
(2) step 1 gained extracting solution is evaporated to without alcohol taste, centrifugal, get supernatant;
(3) supernatant of step 2 gained is separated by polyamide chromatography post, washes 2 ~ 5 column volumes with water, then use 20% ~ 70% ethanol elution, 2 ~ 10 column volumes, collect ethanol elution;
(4) concentrated, drying ethanol eluent obtains Indian beech extractive of general flavone.
4. apply as claimed in claim 3, it is characterized in that described Indian beech extractive of general flavone is prepared by following method:
(1) to fetch water Clausena lansium (Lour.) Skeels stem branch and/or leaf, with 60% ~ 80% alcohol reflux 2 ~ 3 times, extract 2 ~ 3 hours, merge extractive liquid, at every turn;
(2) step 1 gained extracting solution is evaporated to without alcohol taste, centrifugal, get supernatant;
(3) supernatant of step 2 gained is separated by polyamide chromatography post, washes 3 ~ 4 column volumes with water, then use 40% ~ 60% ethanol elution, 4 ~ 7 column volumes, collect ethanol elution;
(4) concentrated, drying ethanol eluent obtains Indian beech extractive of general flavone.
5. apply as claimed in claim 4, it is characterized in that, described Indian beech extractive of general flavone is prepared by following methods:
(1) to fetch water Clausena lansium (Lour.) Skeels stem branch and/or leaf, with 80% alcohol reflux 2 times, to extract 2 hours, merge extractive liquid, at every turn;
(2) step 1 gained extracting solution is evaporated to without alcohol taste, centrifugal, get supernatant;
(3) supernatant of step 2 gained is separated by polyamide chromatography post, washes 3 column volumes with water, then use 50% ethanol elution, 5 column volumes, collect ethanol elution;
(4) concentrated, drying ethanol eluent obtains Indian beech extractive of general flavone.
6. apply as claimed in claim 1, it is characterized in that, described karanjin has HPLC chromatogram as shown in Figure 4, and wherein, the characteristic peak of chromatogram is: karanjin retention time: 10.8min;
Chromatographic condition is: take octadecylsilane chemically bonded silica as filler; Mobile phase: methanol-0.2%HAc (43:57); Flow velocity: 1ml/min; Determined wavelength: 260nm.
7. the application as described in claim 1 or 6, is characterized in that, described karanjin is purified from Indian beech extractive of general flavone and obtains, and comprises the following steps:
(1) be separated by reversed-phase silica gel chromatography post, with 30% ~ 60% ethanol elution, obtain karanjin crude product;
(2) with the karanjin crude product of anhydrous alcohol solution step (1) gained, adding water to concentration of alcohol is 5% ~ 30%, places recrystallization, to obtain final product.
8. apply as claimed in claim 7, it is characterized in that, the purification step of described karanjin comprises:
(1) be separated by reversed-phase silica gel chromatography post, with 40% ethanol elution, obtain karanjin crude product;
(2) with the karanjin crude product of anhydrous alcohol solution step (1) gained, adding water to concentration of alcohol is 10%, places recrystallization, to obtain final product.
9. a pharmaceutical composition for resisiting influenza virus, is characterized in that, containing karanjin or Indian beech extractive of general flavone.
10. pharmaceutical composition according to claim 9, is characterized in that, comprises pharmaceutically acceptable carrier and/or excipient further.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106581023A (en) * | 2016-12-17 | 2017-04-26 | 郑州仁宏医药科技有限公司 | Western medicine for treating viral pneumonia |
| EP3240557A4 (en) * | 2015-11-09 | 2018-08-29 | Shankar Akhawri | A novel synergistic herbal formulation for degenerative disease cardiovascular diseases, autoimmune, inflammatory and metabolism disorder and the process of preparing the same |
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| EP3240557A4 (en) * | 2015-11-09 | 2018-08-29 | Shankar Akhawri | A novel synergistic herbal formulation for degenerative disease cardiovascular diseases, autoimmune, inflammatory and metabolism disorder and the process of preparing the same |
| CN106581023A (en) * | 2016-12-17 | 2017-04-26 | 郑州仁宏医药科技有限公司 | Western medicine for treating viral pneumonia |
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