CN101474247A - Application of spatholobus stem extract in preparing medicament for resisting influenza virus and enterovirus - Google Patents
Application of spatholobus stem extract in preparing medicament for resisting influenza virus and enterovirus Download PDFInfo
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- CN101474247A CN101474247A CNA2008101546008A CN200810154600A CN101474247A CN 101474247 A CN101474247 A CN 101474247A CN A2008101546008 A CNA2008101546008 A CN A2008101546008A CN 200810154600 A CN200810154600 A CN 200810154600A CN 101474247 A CN101474247 A CN 101474247A
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Abstract
The invention discloses the application of a spatholobus stem extract in preparing medicines for preventing influenza virus and enterovirus. Proved by experiments, the spatholobus stem extract has obvious function of preventing the influenza virus and the enterovirus.
Description
Technical field
The present invention relates to the purposes of Caulis Spatholobi extract, particularly relate to the application of Caulis Spatholobi extract in the preparation antiviral drugs.
Background technology
Human life security in many viral disease serious threats, as enterovirus (EV) numerous types, the infection that causes is varied especially, and morbidity is many again to be occurred with syndrome, state of an illness weight great disparity, the infant patient can be broken out severe cardiac myositis or severe pneumonia, and especially neonatal eruption and prevalence can cause death and die.A kind of syndrome can be caused, can be caused different syndromes with a kind of (type) virus by different virus, is the universal phenomenon in the enterovirus genus viral infection.Majority is the performance of subclinical type behind the patient infection enterovirus, is difficult for being found, and it is bigger therefore effectively and in time to make the etiological diagnosis difficulty.Enterovirus be again by excrement-mouth and (or) approach that easily scatters such as respiratory tract infects, should belong to virus acidproof (pH3.0), ether-resistant simultaneously, still can survive during stomach by the people, survive the long period in a humid environment, can from sewage and mud, be separated to, can detect virus water, soil, vegetable, shellfish, contact contaminated water source, food, marine product, tableware and may cause the propagation of community, often cause sporadic popular or large tracts of land is popular.Enterovirus is except that poliovirus at present, and still not having vaccine can be for prevention.Therefore, enterovirus is that current serious influences one of important cause of disease of human health.In addition, human health in the same serious threat of influenza that causes of influenza virus (IV).It belongs to orthomyxoviridae family, is the RNA viruses of peplos, comprises the influenza A virus of domestic animals such as human influenza virus and pig, horse and birdss such as chicken, bird.The human influenza virus can be divided into first, second, the third three types according to the nucleoprotein antigen difference, and its antigenicity very easily morphs.Human influenza is exactly the acute upper respiratory tract infectious disease that is caused by influenza virus, has the infectiousness height, propagates the characteristics such as popular that rapidly, easily take place.This is because virus can only be very fast in the hypotype speed of mutation of the particularity of living cells growth and influenza virus, makes chemicals and influenza virus vaccine be difficult to bring into play its optimal treatment and preventive effect.Especially influenza A virus just breaks out once worldwide being very popular every about 10 years.At present, the threat of influenza still exists, and according to statistics, the whole world has 50-100 ten thousand people to die from influenza every year on average.This shows, influenza as a kind of viral infectious not only serious threat public health, and brought heavy financial burden for country and society.Influenza is accompanied by people's life all the time as the viral infectious of serious harm public health.Though known influenza virus is controlled, because the antigenic variation of influenza virus is very competent and quite frequent; the development and the production of vaccine relatively lag behind; protective rate for Susceptible population is not high, and therefore, new flu outbreak all might break out at any time.In this case, seek Tamiflu and just seem particularly important and urgent with prevention and therapeutical effect.
At present, the disease that enterovirus, influenza virus etc. is caused still lacks narrow spectrum specific treatment medicine, the main means that adopt symptomatic treatment, mostly the medicine that is adopted is broad spectrum activity antiviral drugs such as ribavirin, amantadine etc., needs badly and seeks effective medicine.
Caulis Spatholobi is to continue to use activating blood herbs in thousand simply, all put down in writing its effect of " removing congestion; tissue regeneration promoting blood " ancient times in many pieces of herbal treatises, and be referred to as " panacea of blood system " (the new medical college in Jiangsu. Caulis Spatholobi [M]. Chinese medicine dictionary (first volume). Shanghai: Science and Technology of Shanghai publishing house, 1997.120).The traditional Chinese medical science think its have enrich blood invigorate blood circulation, the function of relaxing muscles and tendons and activating QI and blood in the collateral, be used for the treatment of menoxenia, blood deficiency and yellow complexion, numbness complete diseases such as paralysis, rheumatic arthralgia (Chinese medicine modern study and application. Caulis Spatholobi [M]. Beijing: Xueyuan Press, the 3rd volume: 2539).Whole bloods such as the leukocyte that modern clinical Caulis Spatholobi commonly used (single with or be main prescription with Caulis Spatholobi) treatment a variety of causes (as chemicotherapy, disease in the blood system) causes, platelet, erythrocyte resemble the minimizing disease.In recent years Caulis Spatholobi chemical constituent and pharmaceutical research are proved to have better action at aspects such as antitumor, antioxidation, antiviral.The research of Caulis Spatholobi antivirus action only is confined to herpesvirus and HIV (human immunodeficiency virus) etc. at present, does not also have the report about other virus.
Summary of the invention
The purpose of this invention is to provide the application of a kind of Caulis Spatholobi extract in preparation resisiting influenza virus and anti-enterovirus medicine.
Technical scheme of the present invention is summarized as follows:
The application of Caulis Spatholobi extract in preparation resisiting influenza virus and anti-enterovirus medicine.
Advantage of the present invention:
Experimental results show that: Caulis Spatholobi extract has the effect of significant resisiting influenza virus and anti-enterovirus.
The specific embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
Embodiment 1
Caulis Spatholobi extract adopts following method preparation:
(1) Caulis Spatholobi of the 1000g after will pulverizing added 2000ml water logging bubble 2 hours, was heated to 80 ℃, soaked extraction in 2 hours, and filtration is extracted 3 times, and filtrate is concentrated into 1000ml.
Embodiment 2
Caulis Spatholobi extract adopts following method preparation:
(1) Caulis Spatholobi of the 1000g after will pulverizing added 1000ml water logging bubble 3 hours, was heated to 60 ℃, soaked extraction in 3 hours, and filtration is extracted 4 times, and filtrate is concentrated into 1000ml.
Embodiment 3
Caulis Spatholobi extract adopts following method preparation:
(1) Caulis Spatholobi of the 1000g after will pulverizing added 3000ml water logging bubble 1 hour, was heated to 100 ℃, soaked extraction in 1 hour, and filtration is extracted 2 times, and filtrate is concentrated into 1000ml.
The Chinese crude drug Caulis Spatholobi that embodiment 1-3 is adopted selects homemade high-quality Chinese crude drug Caulis Spatholobi, the place of production: major production areas, China south.
With reference to State Standard of the People's Republic of China GB15193.3-94, GB15193.5-94, GB15193.8-94, GB15193.7-94, GB15193.4-94, GB15193.14-94 and GB15193.13-94, carry out rat acute toxicity test, acute toxicity test in mice, mouse bone marrow cells micronucleus test (male), mouse bone marrow cells micronucleus test (female), mouse sperm deformity test, mouse testis chromosomal aberration test, Salmonella reversion test, the tertogenicity test of rat system and rat and fed experiment in 30 days, the result shows large and small Mus LD50〉30.0g/kg; The Caulis Spatholobi extract (1000mg/kg) of the oral embodiment of the invention of mice 1 preparation, one day 1 time, serve on 15 days, under waking state, observe its spiritual nervous system, cardiovascular system respiratory system of unifying, all no abnormal performance of result.
Embodiment 4
The anti-enterovirus effect of Caulis Spatholobi extract
One, the external anti-enterovirus effect research of Caulis Spatholobi extract
1, Caulis Spatholobi extract in Vero E6 cell culture to the inhibitory action of enterovirus
(1) cell culture: the culture bottle that covers with Vero E6 cell adds 0.25% pancreatin (preparation of Hanks liquid), and 37 ℃ digested 5 minutes, and added the RPMI-1640 culture fluid and (contain hyclone 10%, 3% glutamine 1%) piping and druming, 1:3 goes down to posterity, and 2-3d covers with, add the cell counting count board counting, be configured to 10
5Cells/ml, inoculating cell culture plate, the every hole 0.2ml of 96 orifice plates, the every hole 1ml of 24 orifice plates, 37 ℃, 5% CO
2Cultivate 24h, cell experimentizes after growing up to monolayer.
(2) Caulis Spatholobi extract pair cell toxicity test: after will adding Versene Digestive system 4ml digestion with 0.25% pancreatin 1ml respectively to the Vero E6 cell of enterovirus susceptible, with growth-promoting media blow off, mixing, 100 μ l/ holes are inoculated in 96 porocyte culture plates, 37 ℃, 5% CO
2Grow into cell monolayer behind middle cultivation 24h～48h, discard culture fluid, stand-by.Dilute by variable concentrations keeping liquid with RPMI-1640 behind the given the test agent autoclaving, do 6 concentration of 2 times of serial dilutions and carry out scalping.Each concentration inoculation preprepared cell 3 hole, every hole 100 μ l.With positive control drug ribavirin injection (not filtering), by different concentration dilutions, every concentration 4 holes, every hole 100 μ l.Establish normal cell Vero E6 simultaneously, 37 ℃, 5% CO
2The middle cultivation.Rose in 1st, observation of cell form day by day under inverted microscope, with every hole less than 25%, pathological changes is+; 25%～50% is ++; 50%～75% is +++; 75%～100% is ++ ++ (with pathological changes ++ ++ be terminal point, be 4d observing time).Calculate maximal non-toxic concentration C C according to Reed Muench method
0With the poisonous concentration C C of half
50
(3) Caulis Spatholobi extract inhibitory action that enterovirus is duplicated: inoculating cell Vero E6 inhales and removes culture fluid, with 100 * TCID after 96 orifice plates grow up to 70%～80% monolayer
50Virus 100 μ l absorption Vero E6 cell is hatched 2h for 37 ℃, and the flush away free virus adds with the Caulis Spatholobi sample liquid 100 μ l that keep the liquid doubling dilution, 37 ℃, 5% CO
2Continue in the incubator to cultivate, the observation of cell pathological changes, record pathological changes hole count, calculation sample calculates medium effective concentration (IC to the suppression ratio of virus
50) and therapeutic index (TI).
(4) Caulis Spatholobi extract is to the inhibitory action of enterovirus adherent cell: inoculating cell is inhaled and is removed culture fluid, 100 * TCID after 96 orifice plates grow up to 70%～80% monolayer
50Virus 100 μ l and 100 μ l Caulis Spatholobi sample diluting liquids add (volume ratio is 1:1) simultaneously, hatch 2h for 37 ℃, remove liquid, add 100 μ l and keep liquid, 37 ℃, 5% CO
2Continue to cultivate 96h in the incubator, the observation of cell pathological changes, record pathological changes hole count, calculation sample calculates medium effective concentration (IC to the suppression ratio of virus
50) and therapeutic index (TI).
(5) the Caulis Spatholobi extract pretreatment cell is to the retardation (protective effect) of enterovirus infection: inoculating cell is after 96 orifice plates grow up to 70%～80% monolayer; culture fluid is removed in suction, adds 100 μ l sample diluting liquids, and cell and sample are hatched 2h for 37 ℃; discard sample liquid, add 100 * TCID
50Virus liquid 100 μ l are hatched 2h for 37 ℃, and the flush away free virus adds and keeps liquid and hatch 200 μ l, 37 ℃, 5% CO
2Continue to cultivate 96h in the incubator, the observation of cell pathological changes, record pathological changes hole count, calculation sample is to the suppression ratio of virus, calculation of half inhibitory concentration (IC
50) and therapeutic index (TI).
(6) Caulis Spatholobi extract is to the deactivation of enterovirus: the Caulis Spatholobi extract of getting embodiment 1 preparation of 50 μ g/ml and 30 μ g/ml is hatched 2h with virus stock solution used in 37 ℃ respectively, make 10 times of gradient dilutions with the hybrid virus liquid after keeping liquid and will acting on, be inoculated in 96 well culture plates that cover with cell monolayer and carry out titration of virus, each dilution factor repeats 4 holes, viral liquid with equal extension rate is done contrast, 37 ℃, 5% CO
2Incubator continues to cultivate 96h, and the observation of cell pathological changes is calculated virus titer and the sample inactivation ratio to virus.
2, experimental result
(1) Caulis Spatholobi extract is to the inhibitory action of duplicating of 5 kinds of enterovirus.
(2) Caulis Spatholobi extract can not suppress the adsorption of virus.
(3) Caulis Spatholobi extract has stronger deactivation enterovirus effect, can make the 5 kinds of virus titers of testing reduce by 2～4 orders of magnitude.
Two, Caulis Spatholobi extract resisiting influenza virus effect
1, the inoculation mdck cell is cultured to 70%～80% monolayer in 96 porocyte plates, abandons culture fluid, uses Hank ' s liquid to wash 1 time, adds to be diluted to 100 * TCID
50Viral liquid, every hole 100 μ l, 35 ℃ absorption 2h.Sample liquid is begun to do doubling dilution totally 8 concentration from the half toxic dose.Discard viral liquid after the absorption, use Hank ' s liquid to wash 1 time, add the good medicinal liquid of dilution, each concentration adds 4 holes, establishes the contrast of virus control and cell simultaneously, cultivates 5d for 37 ℃, and observation had or not CPE and write down the result every day.With cell 50% terminal point determining of the drug dilution degree of pathological changes (++) as this medicine minimum effective drug concentration taking place, calculates medium effective concentration (IC
50) and therapeutic index (TI), more than test triplicate at least, all write down the result at every turn.
2, the resisiting influenza virus effect then need be after finishing above CPE observation, with-20 ℃ of culture plate incomes, each experimental group is carried out the experiment of blood clotting titre, judge the minimum effective drug concentration of medicine: get vinyon plate at the bottom of 96 hole circles to this influenza virus, queue every hole from second and add 50 μ l normal saline, the every hole of first row adds medicine 100 μ l to be checked, takes out 50 μ l in first round and does doubling dilution backward; Add 1% guinea-pig red blood cell, 100 μ l, put observed result behind room temperature 30～60min.With erythrocyte 50% coagulation (++) is the final decision result.
3, result of the test
(1) Caulis Spatholobi to get thing better to the influenza virus antiviral effect, best to the inhibition effect of H1N1, EC
50Be 3.19 μ g/ml, TI is 28.4.Antivirus action to other two kinds of influenza virus H3N2 and Influenza B virus is also apparent in view
Caulis Spatholobi is got the drug effect of thing and the external resisiting influenza virus of virazole
(2) with the Caulis Spatholobi extract of 50 μ g/ml and 30 μ g/ml respectively at 35 ℃ with influenza virus Influenza virus A1 (H1N1), Influenza virus A3 (H3N2), Influenza virus B effect 2h after, the mixed liquor blood clotting titre viral with contrast after the difference detection effect, as shown in the table: the Caulis Spatholobi extract of 50 μ g/ml and 30 μ g/ml all can effectively reduce the blood clotting titre of influenza virus.
Caulis Spatholobi extract is to the influence of hirst's hemagglutination titre
(3) the antiviral effect difference of Caulis Spatholobi under the different way of administration.As can be known, Caulis Spatholobi inhibitory action that influenza virus is duplicated〉to the inhibitory action of influenza virus adherent cell〉to the deactivation of influenza virus〉pretreatment cell is to the retardation of influenza infection.The therapy effect that infects adding sample in back influenza virus is best.
Three, anti-enterovirus effect in the Caulis Spatholobi extract body
1, mice Coxsackie virusB3 viral infection: 50 of kunming mices, being divided into is 5 groups, 10 every group, male and female half and half.After each organizes injected in mice position routine disinfection, (except that A group normal control group) lumbar injection Coxsackie virus B3 virus liquid 0.1ml (10
5* TCID
50), carry out the gastric infusion first time behind the injection 2h.
2, Drug therapy test: carry out the gastric infusion first time behind the 2h behind the lumbar injection Coxsackie virus B3 virus liquid.The A group is the normal control group, and the cell maintenance medium 0.1ml of injection Isodose gavages normal saline during infective virus when irritating stomach; The B group gavages normal saline for the viral infection matched group; The C group is antiviral Caulis Spatholobi extract low dosage (1mg/ml) treatment group; The D group is dosage (10mg/ml) treatment group in the antiviral Caulis Spatholobi extract; The E group is antiviral Caulis Spatholobi extract high dose (100mg/ml) treatment group.Once a day, each 0.2ml.Disease symptom behind the observation zoogenetic infection.Each treated animal is respectively put to death half respectively at 5d and 10d, get blood system from serum behind the excision eyeball, and animal hearts is got by the sterile working.1/2 of each heart sample is used for infectious virus and separates, the detection that is used for Coxsackie virus B3 viral nucleic acid of residue heart sample 1/2.
3, detection method
(1) separation, the evaluation of infectious Coxsackie virus B3 virus in the heart: get mouse heart 1/2 homogenate that above-mentioned Coxsackie virus B3 infects back 15d and 30d, make suspension with RPMI-1640 cell culture fluid that contains 100U/ml penicillin, 100 μ g/ml streptomycins, get 0.1ml and infect Vero E6 cell, 37 ℃, 5% CO
2Absorption 2h (every the 30min vibration once) abandons supernatant, adds cell maintenance medium, 37 ℃, 5% CO
2Incubator is cultivated.Observe down continuously 6d in inverted microscope, it is positive CPE person to occur, carries out titration of virus after the freeze thawing 3 times, and adopts RT-PCR to identify.CPE person not occurring does not still have CPE person through 3 generations of blind passage and is considered as separating negative, abandons it.
(2) Coxsackie virus B3 viral nucleic acid detects in the heart tissue: after getting mouse heart 1/2 homogenate of above-mentioned Coxsackie virus B3 infection back 5d and 10d, extracting total tissue RNA and viral RNA wherein, with method extracting Coxsackie virus B3, carry out the RT-PCR amplification respectively, product is through agarose gel electrophoresis, EB dyeing, uviol lamp is observed down, and with the contrast of molecular weight standard product, the person is positive band to the occur at the 300bp place.
4, experimental result
Respectively put to death half respectively at 15d and 30d, mouse heart is got by the sterile working, and 1/2 of each heart sample is used for infectious virus and separates, and 1/2 internal organs are used for the detection of Coxsackie virus B3 viral nucleic acid.Vero E6 cell separation virus result shows, is inoculated in respectively organizing mouse tissue virus extracting solution and all can not making cell generation pathological changes of culture plate, and with 3 generations of cell blind passage, cell growth state is good, no pathological changes generation.
And 1/2 internal organs of other sample are extracted total RNA, detect the specific nucleic acid fragment of Coxsackie virus B3 by RT-PCR, the result shows, virus control group, middle dosage group and low dose group result are positive, blank group, high dose group result are negative, under high concentration (100mg/ml), the Caulis Spatholobi extract sample can suppress infection and the breeding of Coxsackie virus B3 in the mouse cardiac muscle cell.
Four, resisiting influenza virus effect in the Caulis Spatholobi extract body
1, mice HIN1 viral infection: 50 of kunming mices, being divided into is 5 groups, 10 every group, male and female half and half.Each organizes mice (except that the A group normal control group) HIN1 of via intranasal application instillation several times virus liquid 0.05ml (10
5* TCID
50), carry out the gastric infusion first time behind the instillation 2h.
2, Drug therapy test: via intranasal application instillation HIN1 virus liquid 0.05ml (10
5* TCID
50), carry out the gastric infusion first time behind the instillation 2h.The A group is the normal control group, and the cell maintenance medium 0.1ml of injection Isodose gavages normal saline during infective virus when irritating stomach; The B group gavages normal saline for the viral infection matched group; The C group is antiviral Caulis Spatholobi extract low dosage (1mg/ml) treatment group; The D group is dosage (10mg/ml) treatment group in the antiviral Caulis Spatholobi extract; The E group is antiviral Caulis Spatholobi extract high dose (100mg/ml) treatment group.Once a day, each 0.2ml.Disease symptom behind the observation zoogenetic infection.Each treated animal is respectively put to death half respectively at 5d and 10d, and the animal lung is got by the sterile working.1/2 of each lung sample is used for infectious virus and separates, the detection that is used for H1N1 virus nucleic acid of residue lung sample 1/2.
3, detection method
(1) separation of infectious H1N1 virus, evaluation in the lung: get mouse lung 1/2 homogenate that above-mentioned H1N1 infects back 5d and 10d, make suspension with RPMI-1640 cell culture fluid that contains 100U/ml penicillin, 100 μ g/ml streptomycins, get 0.1ml and infect mdck cell, 37 ℃, 5% CO
2Absorption 2h (every the 30min vibration once) abandons supernatant, adds cell maintenance medium, 37 ℃, 5% CO
2Incubator is cultivated.Observe down continuously 6d in inverted microscope, it is positive CPE person to occur, carries out titration of virus after the freeze thawing 3 times, and adopts RT-PCR to identify.CPE person not occurring does not still have CPE person through 3 generations of blind passage and is considered as separating negative, abandons it.
(2) H1N1 virus detection of nucleic acids in the lung tissue: after getting mouse lung 1/2 homogenate of above-mentioned H1N1 infection back 5d and 10d, extracting total tissue RNA and viral RNA wherein, with method extracting H1N1, carry out the RT-PCR amplification respectively, product is through agarose gel electrophoresis, EB dyeing, uviol lamp is observed down, and with the contrast of molecular weight standard product, the person is positive band to the occur at the 110bp place.
4, experimental result
Respectively put to death half respectively at 5d and 10d, mouse lung is got by the sterile working, and 1/2 of each lung sample is used for infectious virus and separates, and 1/2 internal organs are used for the detection of H1N1 virus nucleic acid.Mdck cell isolated viral result shows, is inoculated in respectively organizing mouse tissue virus extracting solution and all can not making cell generation pathological changes of culture plate, and with 3 generations of cell blind passage, cell growth state is good, no pathological changes generation.
And 1/2 internal organs of other sample are extracted total RNA, detect the specific nucleic acid fragment of H1N1 by RT-PCR, the result shows, virus control group and low dose group result are positive, blank group, middle dosage group and high dose group result are negative, (10mg/ml, 100mg/ml), the Caulis Spatholobi extract sample can suppress infection and the breeding of H1N1 in the mouse lung cell under these two concentration.
Carried out antivirus test by the extract to Caulis Spatholobi extract sample, different batches Caulis Spatholobi extract sample and the Different Extraction Method in source, the different places of production, the result shows: source, the different places of production and different extraction are batch to extract antiviral activity no significant difference.
Claims (1)
1. the application of Caulis Spatholobi extract in preparation resisiting influenza virus and anti-enterovirus medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103156096A (en) * | 2013-04-02 | 2013-06-19 | 陈景河 | Pigeon feed additive |
| WO2023174207A1 (en) * | 2022-03-15 | 2023-09-21 | Versitech Limited | A method of obtaining extracts of spatholobus suberectus dunn (ssd), fractions and compostions thereof and using against viral diseases |
-
2008
- 2008-12-26 CN CNA2008101546008A patent/CN101474247A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103156096A (en) * | 2013-04-02 | 2013-06-19 | 陈景河 | Pigeon feed additive |
| WO2023174207A1 (en) * | 2022-03-15 | 2023-09-21 | Versitech Limited | A method of obtaining extracts of spatholobus suberectus dunn (ssd), fractions and compostions thereof and using against viral diseases |
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Open date: 20090708 |