CN103585225B - The application of a kind of Herba of Artemisiae admsii extract in preparation antiviral drugs - Google Patents
The application of a kind of Herba of Artemisiae admsii extract in preparation antiviral drugs Download PDFInfo
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Abstract
The present invention relates to a kind of novelty teabag of Herba of Artemisiae admsii extract, this novelty teabag is the application of Herba of Artemisiae admsii extract in preparation antiviral drugs.Described Herba of Artemisiae admsii extract is prepared as follows: take Herba of Artemisiae admsii as raw material, first extract volatile oil, then uses 50%-95% ethanol extraction, is obtained by purification with macroreticular resin.Herba of Artemisiae admsii extract provided by the present invention is significant to preparation antiviral drugs.
Description
Technical field
The invention belongs to biomedicine field, relate to the application of a kind of Chinese medicinal material extract in preparation antiviral drugs, specifically relate to the application of a kind of Herba of Artemisiae admsii extract in preparation antiviral drugs.
Background technology
At present a large amount of bacterial drug resistance using antibiotic to cause constantly rises has become one of difficult problem of jointly paying close attention in the whole world.Especially the resistant rate of staphylococcus aureus reaches more than 90%.Herpes simplex virus type II mainly causes genital area mucocutaneous infections.Virus enters body through respiratory tract, oral cavity, genital mucosa and damaged skin, dives and occupy in human normal mucosa, blood, saliva and sensory nerve ganglion cell.When Abwehrkraft des Koepers declines, as heating, gastrointestinal dysfunction, menstruation, gestation, focal infection and mood change time, the HSV-II hidden in body is activated and falls ill.The antiviral drugs that synthetic drug is comparatively affirmed in the treatment of herpes simplex is acyclovir (ACV), but the persister of clinical continuous discovery ACV in recent years, and drug resistance has growth trend with the prolongation of courses of pharmaceuticals.
Therefore, antibacterial, the antiviral drugs of developing new high-efficiency low-toxicity have broad prospects, the multiple target effect mechanism of Chinese medicine, low toxic and side effects, be easy to metabolism, the advantage such as to have no drug resistance makes it become the focus of antibacterial, the antiviral drugs of screening high-efficiency low-toxicity.
Herba of Artemisiae admsii is the dry aerial parts of feverfew Herba of Artemisiae admsii ArtemisiaadamsiiBesser.Florescence in summer gathers, and removing impurity, cleans, dry aqueous vapor, cutting, rub out fragrance, dry in the shade, dry.Record in " Drug Standard of Ministry of Public Health of the Peoples Republic of China Tibetan medicine (first) ", standard number: WS3-BC-0050-95.Cold in nature, bitter in the mouth.There is effect of heat-clearing and toxic substances removing.For pneumonia, gastritis, laryngitis, tonsillitis, conjunctivitis etc.The common drug of the minority areas such as illiteracy is hidden for China.But about the basic research of Herba of Artemisiae admsii is still very limited, the follow-up promotion and application of this medical material are restricted.Modern study rarely has report about the chemical composition of Herba of Artemisiae admsii and pharmacological action.
Above-mentioned document and patent etc., there is not yet Herba of Artemisiae admsii or Herba of Artemisiae admsii extract is antibacterial, the report of antivirus action research.
Summary of the invention
The object of the present invention is to provide a kind of application in preparation antiviral drugs of Herba of Artemisiae admsii extract.
The present invention is achieved through the following technical solutions:
The present invention's Herba of Artemisiae admsii used is the dry aerial parts of feverfew Herba of Artemisiae admsii ArtemisiaadamsiiBesser.
The application of a kind of Herba of Artemisiae admsii extract of the present invention in preparation antiviral drugs, the preparation method of described Herba of Artemisiae admsii extract is:
(1) Herba of Artemisiae admsii, soaks 4-12 hour, adopts extraction by steam distillation volatile oil, extraction time 1-4 hour, extraction time 1-3 time, and water consumption is 8-15 times of Herba of Artemisiae admsii weight, collects volatile oil, obtains Herba of Artemisiae admsii extract A; Herba of Artemisiae admsii aqueous extract is filtered, obtains extracting solution A and medicinal residues A respectively;
(2) step (1) being obtained medicinal residues A 50%-95% ethanol is solvent, heating and refluxing extraction, and extraction time is 1-3 time, and each extraction time is 1-3 hour, each solvent load is 8-15 times of Herba of Artemisiae admsii weight, filters, and extracting solution reclaims ethanol, concentrated, dry, obtain Herba of Artemisiae admsii extract B;
(3) by the extracting solution A that step (1) obtains, be concentrated into relative density d=1.10-1.20, pass through macroporous adsorptive resins, first wash with water, then use the alcoholic solution eluting of concentration 50%-90%, collect different concentration ethanol eluent, concentrate drying, obtains Herba of Artemisiae admsii extract C;
(4) above-mentioned Herba of Artemisiae admsii extract A, Herba of Artemisiae admsii extract B, Herba of Artemisiae admsii extract C, wherein one or both or three kinds mix by a certain percentage, obtain Herba of Artemisiae admsii extract of the present invention.
The preparation method of Herba of Artemisiae admsii extract is:
(1) Herba of Artemisiae admsii, soaks 4-12 hour, adopts extraction by steam distillation volatile oil, extraction time 1-4 hour, extraction time 1-3 time, and water consumption is 8-15 times of Herba of Artemisiae admsii weight, collects volatile oil, obtains Herba of Artemisiae admsii extract A; Herba of Artemisiae admsii aqueous extract is filtered, obtains extracting solution A and medicinal residues A respectively;
(2) step (1) being obtained medicinal residues A 50%-95% ethanol is solvent, heating and refluxing extraction, and extraction time is 1-3 time, and each extraction time is 1-3 hour, each solvent load is 8-15 times of Herba of Artemisiae admsii weight, filters, and extracting solution reclaims ethanol, concentrated, dry, obtain Herba of Artemisiae admsii extract B;
(3) by the extracting solution A that step (1) obtains, be concentrated into relative density d=1.10-1.20, pass through macroporous adsorptive resins, first wash with water, then use the alcoholic solution eluting of concentration 50%-90%, collect different concentration ethanol eluent, concentrate drying, obtains Herba of Artemisiae admsii extract C;
(4) above-mentioned Herba of Artemisiae admsii extract A, Herba of Artemisiae admsii extract B, the mixing of Herba of Artemisiae admsii extract C, obtain Herba of Artemisiae admsii extract of the present invention.
The preparation method of preferred Herba of Artemisiae admsii extract of the present invention is:
(1) Herba of Artemisiae admsii, soaks 8 hours, and adopt extraction by steam distillation volatile oil, 2 hours extraction times, extraction time 2 times, water consumption is 13 times of Herba of Artemisiae admsii weight, collects volatile oil, obtains Herba of Artemisiae admsii extract A; Herba of Artemisiae admsii aqueous extract is filtered, obtains extracting solution A and medicinal residues A respectively;
(2) step (1) being obtained medicinal residues A 70% ethanol is solvent, heating and refluxing extraction, and extraction time is 2 times, and each extraction time is 2 hours, each solvent load is 12 times of Herba of Artemisiae admsii weight, filters, and extracting solution reclaims ethanol, concentrated, dry, obtain Herba of Artemisiae admsii extract B;
(3) by the extracting solution A that step (1) obtains, relative density d=1.13 is concentrated into, by ADS-3 macroporous adsorptive resins, first wash with water, then use the alcoholic solution eluting of concentration 70%, collect 70% concentration ethanol eluent, concentrate drying, obtains Herba of Artemisiae admsii extract C;
(4) by above-mentioned Herba of Artemisiae admsii extract A, Herba of Artemisiae admsii extract B, the mixing of Herba of Artemisiae admsii extract C, Herba of Artemisiae admsii extract of the present invention is obtained.
The preparation method of Herba of Artemisiae admsii extract of the present invention, is characterized in that: the macroporous adsorbent resin adopted is D101 macroporous adsorbent resin, DM130 macroporous adsorbent resin, ADS-3 macroporous adsorbent resin.
Herba of Artemisiae admsii extract A of the present invention, Herba of Artemisiae admsii extract B, Herba of Artemisiae admsii extract C, the application in preparation antiviral drugs.
The antiviral drugs that Herba of Artemisiae admsii extract of the present invention and chemical drugs or Chinese medicine or natural drug form.
The antiviral drugs that Herba of Artemisiae admsii extract A of the present invention, Herba of Artemisiae admsii extract B, Herba of Artemisiae admsii extract C and chemical drugs or Chinese medicine or natural drug form.
Herba of Artemisiae admsii extract of the present invention, by adding the various adjuvants that pharmaceutics allows, makes the peroral dosage forms such as the tablet on pharmaceutics, granule, capsule.
Herba of Artemisiae admsii extract of the present invention is by various ingredients, and too many levels, multipath ground plays synergism and shows antiviral efficacy, invades to HSV-II the Main Function approach that the blocking effect of cell and direct kill virus are Herba of Artemisiae admsii extracorporeal antivirus effects.HSV-II is tunicary virus, and outermost peplos is made up of lipid and glycoprotein, and wherein any one composition changes, and can make peplos degeneration, thus inactivation of viruses.Experimental result shows that each extract of Herba of Artemisiae admsii is comparatively strong to the direct deactivation of HSV-II, and especially the effect of Herba of Artemisiae admsii extract and Herba of Artemisiae admsii extract A direct deactivation HSV-II is more remarkable than ACV.Therefore infer that these chemical compositions can make peplos degeneration with inactivation of viruses.The blocking effect research that HSV-II invades cell shows, Herba of Artemisiae admsii extract not only has antiviral activity, also has the effect that to a certain degree Cell protection prevents Virus entry.Utilize HSV-II encephalitis model to study Herba of Artemisiae admsii extract to the protective effect of HSV-II infecting mouse, result shows that Herba of Artemisiae admsii extract shows certain antiviral activity.
Detailed description of the invention
Below by specific experiment example and embodiment, the application of Herba of Artemisiae admsii extract in preparation antiviral drugs is described further, but is not limited to the present invention.
Embodiment 1: the preparation of Herba of Artemisiae admsii extract
(1) Herba of Artemisiae admsii 13kg, soaks 8 hours, and adopt extraction by steam distillation volatile oil, 2 hours extraction times, extraction time 2 times, water consumption is 13 times of Herba of Artemisiae admsii weight, collects volatile oil, obtains Herba of Artemisiae admsii extract A; Herba of Artemisiae admsii aqueous extract is filtered, obtains extracting solution A and medicinal residues A respectively;
(2) step (1) being obtained medicinal residues A 70% ethanol is solvent, heating and refluxing extraction, and extraction time is 2 times, and each extraction time is 2 hours, each solvent load is 12 times of Herba of Artemisiae admsii weight, filters, and extracting solution reclaims ethanol, concentrated, dry, obtain Herba of Artemisiae admsii extract B;
(3) by the extracting solution A that step (1) obtains, relative density d=1.13 is concentrated into, by ADS-3 macroporous adsorptive resins, first wash with water, then use the alcoholic solution eluting of concentration 70%, collect 70% concentration ethanol eluent, concentrate drying, obtains Herba of Artemisiae admsii extract C;
(4) by above-mentioned Herba of Artemisiae admsii extract A, Herba of Artemisiae admsii extract B, the mixing of Herba of Artemisiae admsii extract C, Herba of Artemisiae admsii extract of the present invention is obtained.
Embodiment 2: the preparation of Herba of Artemisiae admsii extract
(1) Herba of Artemisiae admsii 20kg, soaks 12 hours, and adopt extraction by steam distillation volatile oil, 1 hour extraction time, extraction time 3 times, water consumption is 8 times of Herba of Artemisiae admsii weight, collects volatile oil, obtains Herba of Artemisiae admsii extract A; Herba of Artemisiae admsii aqueous extract is filtered, obtains extracting solution A and medicinal residues A respectively;
(2) step (1) being obtained medicinal residues A 95% ethanol is solvent, heating and refluxing extraction, and extraction time is 1 time, and each extraction time is 3 hours, each solvent load is 15 times of Herba of Artemisiae admsii weight, filters, and extracting solution reclaims ethanol, concentrated, dry, obtain Herba of Artemisiae admsii extract B;
(3) by the extracting solution A that step (1) obtains, relative density d=1.10 is concentrated into, by DM130 macroporous adsorptive resins, first wash with water, then use the alcoholic solution eluting of concentration 90%, collect 90% concentration ethanol eluent, concentrate drying, obtains Herba of Artemisiae admsii extract C;
(4) above-mentioned Herba of Artemisiae admsii extract A, Herba of Artemisiae admsii extract B, the mixing of Herba of Artemisiae admsii extract C, obtain Herba of Artemisiae admsii extract of the present invention.
Embodiment 3: the preparation of Herba of Artemisiae admsii extract
(1) Herba of Artemisiae admsii 18kg, soaks 4 hours, and adopt extraction by steam distillation volatile oil, 4 hours extraction times, extraction time 1 time, water consumption is 15 times of Herba of Artemisiae admsii weight, collects volatile oil, obtains Herba of Artemisiae admsii extract A; Herba of Artemisiae admsii aqueous extract is filtered, obtains extracting solution A and medicinal residues A respectively;
(2) step (1) being obtained medicinal residues A 50% ethanol is solvent, heating and refluxing extraction, and extraction time is 3 times, and each extraction time is 1 hour, each solvent load is 8 times of Herba of Artemisiae admsii weight, filters, and extracting solution reclaims ethanol, concentrated, dry, obtain Herba of Artemisiae admsii extract B;
(3) by the extracting solution A that step (1) obtains, relative density d=1.20 is concentrated into, by D101 macroporous adsorptive resins, first wash with water, then use the alcoholic solution eluting of concentration 50%, collect 50% concentration ethanol eluent, concentrate drying, obtains Herba of Artemisiae admsii extract C;
(4) above-mentioned Herba of Artemisiae admsii extract A, Herba of Artemisiae admsii extract B, the mixing of Herba of Artemisiae admsii extract C, obtain Herba of Artemisiae admsii extract of the present invention.
Embodiment 4: the preparation of Herba of Artemisiae admsii extract
(1) Herba of Artemisiae admsii 25kg, soaks 6 hours, and adopt extraction by steam distillation volatile oil, 3 hours extraction times, extraction time 2 times, water consumption is 15 times of Herba of Artemisiae admsii weight, collects volatile oil, obtains Herba of Artemisiae admsii extract A; Herba of Artemisiae admsii aqueous extract is filtered, obtains extracting solution A and medicinal residues A respectively;
(2) step (1) being obtained medicinal residues A 70% ethanol is solvent, heating and refluxing extraction, and extraction time is 2 times, and each extraction time is 2 hours, each solvent load is 12 times of Herba of Artemisiae admsii weight, filters, and extracting solution reclaims ethanol, concentrated, dry, obtain Herba of Artemisiae admsii extract B;
(3) by above-mentioned Herba of Artemisiae admsii extract A, the mixing of Herba of Artemisiae admsii extract B, Herba of Artemisiae admsii extract of the present invention is obtained.
Embodiment 5: the preparation of Herba of Artemisiae admsii extract sheet
Example 1 Herba of Artemisiae admsii extract 325g, adds starch 115g, mixing, granulates, dry, adds microcrystalline Cellulose 30.5g, magnesium stearate 5g, and mixing, is pressed into 1000, obtains Herba of Artemisiae admsii extract sheet.
Embodiment 6: the preparation of Herba of Artemisiae admsii extract particles
Example 2 Herba of Artemisiae admsii extract 205g, adds dextrin 135g, mixing, and granulate, granulate, obtains Herba of Artemisiae admsii extract particles.
Embodiment 7: the preparation of Herba of Artemisiae admsii extract capsule
Example 3 Herba of Artemisiae admsii extract 305g, adds starch 85g, mixing, granulates, and dry, granulate, obtains Herba of Artemisiae admsii extract capsule by encapsulated 1000.
Experimental example 1: each extract antibacterial action of Herba of Artemisiae admsii
1. strain and culture medium
Experimental strain is provided by Center for Disease Control (CDC) of Shandong Province and Clinical Laboratory center, Shandong Province.Be listed as follows:
Experiment culture medium: antibacterial MH culture medium, fungus PDA culture medium is all purchased from Nat'l Pharmaceutical & Biological Products Control Institute.
2. fungistatic effect detection method:
2.1 fungistatic effects cylinder plate method measures inhibition zone size
Activation bacterium liquid physiological saline solution is diluted to 10
5~ 10
6cfu/ml is for subsequent use.Add 0.2ml bacterium liquid to dry MH culture medium flat plate, smoothen.Place 5min, make culture medium fully absorb bacterium liquid.6, sterilized Oxford cup placed by each flat board, places 5min, makes Oxford cup adsorbed close in media surface.The each medicine 100ul of 0.1g/ml is added in the cup of Oxford.Positive control levofloxacin and fluconazol.Positive control antibacterial concentration is 40 μ g/ml levofloxacin, and Candida albicans concentration is 200 μ g/ml fluconazol, all adds 100ul.Negative control adds sterilized water.Then flat board is placed in 37 DEG C of constant incubators and cultivates 24h (candida albicans 48h), go out and measure antibacterial circle diameter with slide calliper rule, calculating mean value, represent bacteriostatic activity with antibacterial circle diameter D.Fungistatic effect criterion is: D≤8mm is insensitive, and 8mm < D≤13mm is less sensitive, and 13mm < D≤19mm is medium sensitivity, and D > 19mm is extremely sensitive.
The mensuration of 2.2 minimal inhibitory concentration MIC adopts Microdilution plate method
Test bacterial concentration is 10
6cfu/ml.Drawing the bacterium liquid diluted is added in 96 porocyte culture plates, and every hole 100ul, often kind of medicine 100ul contrast dilution 10 hole successively, and last hole does not add medicine (only adding culture medium and antibacterial) and adds dilution bacterium liquid 100ul, is bacterial growth control hole.Stay string hole not add antibacterial (only adding culture medium and medicine) and do drug control hole, positive control levofloxacin and fluconazol, illustratively do doubling dilution.Be placed in 1min that agitator vibrates, make after in hole, solution fully mixes, microwell plate is added a cover and is placed on and is covered with in the square enamel tray of wet gauze, hatches 24h (candida albicans 48h) in 37 DEG C of incubators, observes and is minimal inhibitory concentration without lowest concentration of drug contained by bacterial growth hole.
3. fungistatic effect
Table 1 different Herba of Artemisiae admsii extract inhibition zone (mm)
Note: a, Herba of Artemisiae admsii extract, Herba of Artemisiae admsii extract C, Herba of Artemisiae admsii extract A, Herba of Artemisiae admsii extract B addition are 1mg/ cup, b, positive control antibacterial concentration are 40 μ g/ml levofloxacin, Candida albicans concentration is 200 μ g/ml fluconazol, all adds 100ul.5%DMSO is negative control.
Table 2 different Herba of Artemisiae admsii extract minimal inhibitory concentration (MIC) (unit mg/ml)
Note: positive control antibacterial levofloxacin, Candida albicans fluconazol, all adds 100ul; 5%DMSO is negative control.
Result shows: Herba of Artemisiae admsii extract has broad-spectrum antiseptic and bactericidal action.To gram positive bacteria: staphylococcus aureus, drug resistance staphylococcus epidermidis, bacillus cereus, bacillus subtilis; Gram negative bacteria: escherichia coli, enterococcus faecalis; Deep infection fungus Candida albicans has the effect killed with Developing restraint, has important practice significance and using value, can have in the food of antibacterial and/or bactericidal action, health product, cosmetics and medicine apply in preparation.
Experimental example 2: each extract antivirus action of Herba of Artemisiae admsii.
1, experiment material
Virus: HSV-II type (3 generation) 20110913, Lanzhou Institute of Biological Products's vaccine research room provides (herpessimplexvirus, HSV-II)
Cell: African green monkey kidney cell (Vero cell) Lanzhou Institute of Biological Products provides.Cultivate with DMEM culture medium+7% Ox blood serum.
Animal: cleaning grade kunming mice, male and female half and half, body weight 18-22g, is purchased from Lanzhou Institute of Biological Products's Animal House.
Medicine and reagent: tetrazolium bromide (MTT, FlukaBiochemika company), dimethyl sulfoxide (dimethylsulfoxide, DMSO, Sigma company produces), DMEM (GIBCO company), standard neonatal Ox blood serum (sino-america joint-venture Lanzhou people's marine growth Engineering Co., Ltd), Aciclovir for injection (ACV, 9-(2-hydroxyl ethoxymethyl-) guanine, Wuhan Pusheng Pharmaceutical Co., Ltd., the accurate word H42020129 of product batch number 110811 traditional Chinese medicines).(embodiment 1 method prepares, and lot number is respectively: 20110908,20110909,20110910,20110911) for Herba of Artemisiae admsii extract, Herba of Artemisiae admsii extract C, Herba of Artemisiae admsii extract A, Herba of Artemisiae admsii extract B
2, experimental technique
2.1 experiment in vitro.
2.1.1 drug cytotoxicity measures (mtt assay)
Be inoculated in 96 porocyte culture plates by cell suspension with 8 × 105/ml density, every hole 200 μ l, 37 DEG C, 5%CO2,100% relative humidity cultivates 24h.Herba of Artemisiae admsii extract, Herba of Artemisiae admsii extract C, Herba of Artemisiae admsii extract A, the equal doubling dilution of Herba of Artemisiae admsii extract B are to following 6 concentration: 16,8,4,2,1,0.5mg/ml, and add in cell suspension hole, each drug level repeats 4 holes, separately establishes normal cell controls.Continue under the same terms to cultivate 33h, observation of cell pathological changes (CPE) situation, and detect cell survival rate with mtt assay.
2.1.2 virus virulence measures (plaque method)
By cell suspension with 8 × 10
5/ ml density is inoculated in 24 porocyte culture plates, every hole 1mL, 37 DEG C, 5%CO2, and 100% relative humidity cultivates 24h.Viral dilution is to following 6 concentration: 10
-3, 10
-4, 10
-5, 10
-6, 10
-7, 10
-8, add in cell suspension hole, each drug level repeats 4 holes, separately establishes normal cell controls, and methylcellulose covering covers, and continues to cultivate 72h, violet staining, counting under the same terms.
Infectious virus amount (PFU/ml)=(speckle number in every hole/every hole virus inoculation amount ml) × viral dilution multiple.
2.1.3 medicine invades the blocking effect (plaque method) of cell to HSV-II
Test sample liquid is mixed with HSV-II equal-volume, with this virus drugs mixed liquor vero cells infection.37 DEG C, 5%CO2 adsorbs 90min, and washing, adds cell maintenance medium and put 37 DEG C, and 5%CO2 cultivates.Every day observes CPE, detects viral suppression after 72h with plaque method.Therapeutic index (TI) is adopted to weigh the suppression effect of drug on viral as evaluation index.Therapeutic index (TI)=half toxic concentration (TC
50)/half suppresses dense (IC
50).Each drug level repeats 4 holes, arranges normal cell controls, drug control and virus control simultaneously.
2.1.4 medicine is to the direct deactivation (plaque method) of HSV-II
Test sample liquid is mixed with HSV-II equal-volume, after 37 DEG C of effect 90min, with this virus drugs mixed liquor vero cells infection.37 DEG C, 5%CO2 adsorbs 90min, washing, and the same method is cultivated and checked.
2.1.5 the inhibitory action (plaque method) of drug on viral granule propagation
By virus infected cell, wash, add the test sample liquid of variable concentrations after absorption 90min, the same method is cultivated and is checked.Viral suppression (%)=(virus control group on average goes out speckle number-medicine-feeding test group and on average goes out speckle number)/virus control group on average goes out speckle number
2.2 experiment in vivo.
2.2.1 medicine is to the determination of acute toxicity of mice
Cleaning grade Kunming mouse random packet, often organizes 6, male and female half and half.According to Vitro Experimental Results, select to suppress the active total extract preferably of HSV-II biosynthesis, with 400,500,625,800,1000,1250mg/kg dosage gastric infusion, separately establish negative control group, give isopyknic normal saline.Normal feed water inlet, Continuous Observation 10d, record dead mouse situation also calculates LD
50.
2.2.2 viral encephalocoele model toxicity test
Cleaning grade Kunming mouse random packet often organizes 10, male and female half and half.After infection site routine disinfection, through the right side
Intracerebroventricular virus, viral dilution is to following 6 concentration: 10
-1, 10
-2, 10
-3, 10
-4, 10
-6, 10
-7, infective dose is every 0.05mL.Normal feed water inlet, Continuous Observation 10d, record dead mouse situation also calculates LD by Reed-Muench method
50.
2.2.3 medicine infects the protective effect of HSV-II mice to encephalocoele
Cleaning grade Kunming mouse random packet often organizes 10, male and female half and half.After infection site routine disinfection, through right ventricle injecting virus, 24h after viral infection, drug treatment.By medicine LD50 1/2 ~ 1/4 as the high test dose of medicine experiment in vivo, LD
501/12 ~ 1/16 as low test dose, height 2 dosage groups established by every medicine.Without the matched group (virus control group, Normal group) of medicine, continuous gastric infusion 5d, observes animal morbidity and death condition, continuous 30d.
3, experimental result
3.1 experiment in vitro.
3.1.1 drug cytotoxicity measures
Table 3 Herba of Artemisiae admsii extract is to the toxicity (unit: mg/ml) of Vero cell
The toxic action of test sample liquid to Vero cell shows as that cell proliferation is slow, granule is more, refractivity is poor, morphologic change, part cell breakage come off.Because cell metabolic activity reduces or death, the viable count that mtt assay detects reduces.
3.1.2 virus virulence measures (plaque method)
Vero cell is more responsive to HSV-II, and the Vero cell pathological changes effect CPE caused by HSV-II infects is characterized as circle contracting swelling, mutually merges formation thyrsiform or starlike, even part cell detachment, forms focus.It is 10 that plaque counting obtains HSV-II type (3 generation) 20110913 infectious virus amount (PFU/ml)
7.57pFU/ml, this HSV-II virus adopts 10
-5time every hole go out speckle number and be about 40, easily count, therefore adopt 10
-5as the viral dilution of subsequent experimental.
3.1.3 Herba of Artemisiae admsii extract invades the blocking effect of cell to HSV-II
Table 4 Herba of Artemisiae admsii extract invades the blocking effect of Vero cell to HSV-II.
3.1.4 Herba of Artemisiae admsii extract is to the direct killing effect of HSV-II
Table 5 Herba of Artemisiae admsii extract is to the direct deactivation of HSV-II.
3.1.5 medicine inhibitory action (plaque method) that HSV-II virion is bred
The inhibitory action that table 6 Herba of Artemisiae admsii extract is bred HSV-II virion.
Herba of Artemisiae admsii extract is by various ingredients, and too many levels, multipath ground plays synergism and shows antiviral efficacy, invades to HSV-II the Main Function approach that the blocking effect of cell and direct kill virus are Herba of Artemisiae admsii extracorporeal antivirus effects.HSV-II is tunicary virus, and outermost peplos is made up of lipid and glycoprotein, and wherein any one composition changes, and can make peplos degeneration, thus inactivation of viruses.Experimental result shows that each extract of Herba of Artemisiae admsii is comparatively strong to the direct deactivation of HSV-II, and especially the effect of Herba of Artemisiae admsii extract and Herba of Artemisiae admsii extract A direct deactivation HSV-II is more remarkable than ACV.Therefore infer that these chemical compositions can make peplos degeneration with inactivation of viruses.The blocking effect research that HSV-II invades cell shows, Herba of Artemisiae admsii extract not only has antiviral activity, also has the effect that to a certain degree Cell protection prevents Virus entry.Utilize HSV-II encephalitis model to study Herba of Artemisiae admsii extract to the protective effect of HSV-II infecting mouse, result shows that Herba of Artemisiae admsii extract shows certain antiviral activity.
Claims (6)
1. the application of Herba of Artemisiae admsii extract in the anti-HSV-II virus drugs of preparation, the preparation method of described Herba of Artemisiae admsii extract is:
(1) Herba of Artemisiae admsii, soaks 4-12 hour, adopts extraction by steam distillation volatile oil, extraction time 1-4 hour, extraction time 1-3 time, and water consumption is 8-15 times of Herba of Artemisiae admsii weight, collects volatile oil, obtains Herba of Artemisiae admsii extract A; Herba of Artemisiae admsii aqueous extract is filtered, obtains extracting solution A and medicinal residues A respectively;
(2) step (1) being obtained medicinal residues A 50%-95% ethanol is solvent, heating and refluxing extraction, and extraction time is 1-3 time, and each extraction time is 1-3 hour, each solvent load is 8-15 times of Herba of Artemisiae admsii weight, filters, and extracting solution reclaims ethanol, concentrated, dry, obtain Herba of Artemisiae admsii extract B;
(3) by the extracting solution A that step (1) obtains, be concentrated into relative density d=1.10-1.20, pass through macroporous adsorptive resins, first wash with water, then use the alcoholic solution eluting of concentration 50%-90%, collect different concentration ethanol eluent, concentrate drying, obtains Herba of Artemisiae admsii extract C;
(4) above-mentioned Herba of Artemisiae admsii extract A, Herba of Artemisiae admsii extract B, the mixing of Herba of Artemisiae admsii extract C, obtain Herba of Artemisiae admsii extract.
2. application according to claim 1, is characterized in that the macroporous adsorbent resin that the preparation method of described Herba of Artemisiae admsii extract adopts is D101 macroporous adsorbent resin, DM130 macroporous adsorbent resin, ADS-3 macroporous adsorbent resin.
3. application according to claim 1, is characterized in that the anti-HSV-II virus drugs that described Herba of Artemisiae admsii extract and chemical drugs or Chinese medicine or natural drug form.
4. Herba of Artemisiae admsii extract A according to claim 1, Herba of Artemisiae admsii extract B, the application in the anti-HSV-II virus drugs of preparation.
5. application according to claim 1, is characterized in that the anti-HSV-II virus drugs that described Herba of Artemisiae admsii extract A, Herba of Artemisiae admsii extract B and chemical drugs or Chinese medicine or natural drug form.
6. application according to claim 1, is characterized in that described Herba of Artemisiae admsii extract makes tablet oral on pharmaceutics, granule, capsule by the various adjuvants adding pharmaceutics permission.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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