CN1256343C - Chickweed total flavones , their production method , use and preparation containing the same - Google Patents
Chickweed total flavones , their production method , use and preparation containing the same Download PDFInfo
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- CN1256343C CN1256343C CN 03103609 CN03103609A CN1256343C CN 1256343 C CN1256343 C CN 1256343C CN 03103609 CN03103609 CN 03103609 CN 03103609 A CN03103609 A CN 03103609A CN 1256343 C CN1256343 C CN 1256343C
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Abstract
The present invention discloses total chickweed flavonoid containing New Zealand vitexin-1, an extraction method for the total chickweed flavonoid, and the application of the total chickweed flavonoid to preparing a medicine for resisting AIDS viruses and herpes viruses. The present invention also discloses various medicine preparations containing the total chickweed flavonoid, particularly a spray preparation containing the total chickweed flavonoid.
Description
Technical field
The preparation that the present invention relates to chickweed total flavones, preparation method, purposes and contain the chickweed total flavones.
Background technology
Chickweed is the dry herb of pinkwort Stellaria media (L.) Cyr..Successive dynasties book on Chinese herbal medicine and the record of books many places it have treatment to dislike sore, dermopathic effect." U.S. chemical abstract " till the 134th volume, the chemical ingredients of the Stellaria plant of being reported mainly is flavones and the former class of flavonoid glycosides, saponin and saponin, ring peptide class, forulic acid and ester class, chlorogenic acid, neochlorogenic acid, coffic acid, quininic acid, xitix class, oxalic acid and caoxalate, monoacyl semi-lactosi lipoid, alkaloids or the like from the nineteen twenty to the calendar year 2001.CN1377680A discloses a kind of preparation method and its usage of broad-spectrum antiviral medicament, it is that the total organic phenolic acid that contains sulphur soluble in water, nitrogen, calcium that extracts from plant chickweed or its congener is or/and total organic phenolate and glucosides class high polarity compound compositions thereof can suppress hiv virus, hepatitis virus, influenza virus, parainfluenza virus, adenovirus, rhinovirus, simplexvirus etc.Also reported relevant chickweed and other medicines combination in the existing document and made the paste of treatment eczema, the makeup of skin regeneration also have chickweed to prevent alopecia, dentoalveolitis, intestinal adhesion disease and are used for the purposes of aspects such as oral cavity cleaning.
Summary of the invention
The inventor carries out extraction separation according to phytochemical extraction separation program to the Stellaria plant, pharmacodynamics with anti-herpesvirus and hiv virus is that index is screened, find that the chickweed total flavones contains structure and is confirmed as 6-C-wood sugar-8-C-glucose-celery flavine glycosides, be puriri glucoside-1 (vicenin-1), its structure such as figure below.Above-mentioned physicochemical constant and spectral data, with Yasukawa K, et al Yakugaku Zasshi1986,106 (6): 517-519 and Su Yalun, Wang Yulan, Yang Jun mountain herbal medicine 1993,24 (7): 343-344, the basically identical of report in 378.
6-C-wood sugar-8-C-glucose-celery flavine glycosides
Research finds that also the chickweed total flavones not only has the effect of anti-herpesvirus and hiv virus, and toxic side effect is little, and this is the existing not available great advantage of antiviral Western medicine; Meanwhile it also has good analgesic effect, can alleviate the patient suffering very soon, has avoided Western medicine easily to produce the shortcoming of resistance strain again.Thereby finished the present invention.
Purpose of the present invention just provides the chickweed total flavones that contains puriri glucoside-1.
Another purpose of the present invention provides the method that preparation contains the chickweed total flavones of puriri glucoside-1.
Another object of the present invention provides the chickweed total flavones that contains puriri glucoside-1 is treated antiviral in preparation purposes.
A further object of the present invention provides the chickweed total flavones that contains puriri glucoside-1 medicine as activeconstituents.
Another purpose of the present invention provides the sprays that the chickweed total flavones that contains puriri glucoside-1 is made activeconstituents.
The chickweed total flavones that contains puriri glucoside-1 of the present invention can extract acquisition according to the common method of extracting flavones from Stellaria plant or other platymiscium.They comprise Stellaria plant slender lobule chickweed Stellaria leptohylla Hance, long lobe chickweed Stellaria hungeana Fenzl., brown lobe Stellaria alsine Grim. Stellaria alsine var.Phaeuspetala Hand.-Mazz., Anhui chickweed Stellariaanhwiensis Migo., apicule chickweed Stellaria apiculata Wils.4987Stellaria (L) Scop., slender lobule Alishan chickweed Stellaria arisanensis var.Leptophylla Hayata., north chickweed Stellariaborealis Bigel., little fine hair chickweed Stellaria tomentalla Ohwi., David's chickweed Stellariadavidii Hemsl., the husky chickweed Stellaria arenaria Maxim. that gives birth to, short lobe chickweed Stellariabrachypetala Bge., China chickweed Stellaria chinensis Regel., Alishan chickweed Stellariaarisanensis Hayata., northeast chickweed Stellaria cherleriae (Fisch.) Will., thick leaf chickweed Stellaria crassifolia, wrinkle leaf chickweed Stellaria crispate Wall., Stellaria alsine Grim. Stellaria alsineGrimm., the chickweed Stellaria decumbens Edgew. that crouches lays down, forked cyme chickweed Stellariadichasioides Williams., narrow leaf bifid chickweed Stellaria dichotoma var.LanceolataBge., two southern chickweed Stellaria delavayi Franch., Stellaria dianthifolia Williams Stellaria dianthifoliaWilliams., the needle-like chickweed Stellaria decunbens var.Acicularia Edgew.EtHook.f. that crouches that lays down, bifid chickweed Stellaria dichotoma L., line leaf bifid chickweed Stellaria dichotomavar.Stephenjiana Willd., Stellaria diversiflora Maxim. Stellaria diversiflora Maxim., concave veins chickweed Stellaria depressa Schnid., turn over white chickweed Stellaria discolor Turcz., standing grain leaf chickweed Stellaria graminea L., chickweed Stellaria diffusa Wills. looses in the shop, Du Shi chickweed Stellariaduthiei Gandoger., line stem chickweed Stellaria filicaulis Mak., the different colored chickweed Stellariadiversiflora var.Gymnandra Franch. of naked stamen, spend more chickweed Stellaria florida Fisch., chickweed Stellaria pilosa Franch. becomes mildewed, line handle chickweed Stellaria filipes Komar., blunt calyx chickweed Stellaria amblyosepala Schrenk., dredge pubescence standing grain leaf chickweed Stellaria graminea var.Pilosula Maxim., turn green standing grain leaf chickweed Stellaria graminea, Stellaria maximowixziana Franch. Stellariamaximowixziana Franch var.Viridescens Maxim., Jiangzi's chickweed Stellariagyantsensis Williams., Stellaria henryi williams Stellaria henryi Williams., the chickweed Stellariahsinganensis Kitagawa. of Xingan, rosy clouds grass chickweed Stellaria gypsophiloides Fenzl., chickweed Stellaria media (L.) Cyr., Stellaria micrantha Hayata Stellaria micrantha Hayata., introversion chickweed Stellaria infracta Maxim., gentle chickweed Stellaria mitans Williams., goose intestines chickweed Stellarianeglecta Weihe., chickweed Stellaria neo-alustris Kitagawa. is given birth in new natural pond, eight stamen chickweed Stellaria octandra Fobedim., red certain kind of berries chickweed Stellaria oxycoccoides Komar., stone is given birth to chickweed Stellaria saxatilis Buch-Ham., handle flower chickweed Stellaria peduncularis Bge., Taiwan chickweed Stellaria cicrantha Hayata., chickweed Stellaria palustria L. is given birth in the natural pond, Turkestan chickweed Stellaria turkestanica Schischk., exhibition leaf chickweed Stellaria patentifoliaKitagawa., imitation stone is given birth to chickweed Stellaria pseudosaxatilis Hand.-Mass., five chickweed Stellaria wutaica Hand.-Mazz., net arteries and veins chickweed Stellaria reticulivena Hayata., rock chickweed Stellaria rupestris Hemsl., embrace stem stone and give birth to chickweed Stellaria saxatilis var.Amplexicaulis Hand.-mazz., three type chickweed Stellaria trimorpha Nakai., xiaofanlu Stellaria pusilla Schmid., flint lobe chickweed Stellaria radians L., accurate Ge Er chickweed Stellariasoongorica Roshev., Su Shi chickweed Stellaria souliei Williams., star hair chickweed Stellariastellato-pilosa Hayata., garden calyx chickweed Stellaria stronglosepala Hand.-Mazz., intend umbrella flower chickweed Stellaria subumbellata Edgew., wetland chickweed Stellaria uda Williams., green colored chickweed Stellaria virdiflora Pax et O.Hoffm., Stellaria wushanensis Williams Stellaria wushanensisWilliams., umbrella flower chickweed Stellaria umbellate Turcz., Yunnan chickweed Stellariayunnanensis franch.j, and ox Stellaria plant ox chickweed Malachiumaquaticum (L.) Fries.
The preferred chickweed total flavones that obtains to contain puriri glucoside-1 through the following steps: get chickweed crude drug or bright grass (converting into hay) lower alcohol with 8 times of amounts 30～40%, being warmed to 40～50 ℃ of reduced-pressure backflows in extractor extracts three times, each 1～3 hour, united extraction liquid; Measuring density under decompression is concentrated into 40 ℃ with extract is the extracting solution of 1.05-1.1; Adding 95% ethanol makes alcohol concn reach 60-70% in above-mentioned concentrated solution, and alcohol precipitation (pure hypostasis is used in addition) filters and obtains filtrate, and filtrate is concentrated, and vacuum-drying gets the dried feed medicine.
Another preparation method of chickweed total flavones of the present invention comprises and gets chickweed crude drug or bright grass (converting into hay) with the lower alcohol of 8 times of amounts 30～40%, is warmed to 40～50 ℃ of extractions three times in extractor, each 1～3 hour, and united extraction liquid; Measuring density under with decompression extract being concentrated into 40 ℃ again behind the reclaim under reduced pressure solvent is the extracting solution of 1.05-1.1; Filtrate after concentrating is gone up D101 macroporous resin bed separates, with the distilled water wash-out and discard this part elutriant, again with ethanol or the methanol-eluted fractions of 40-95%, during wash-out, adopting thin layer chromatography to compare with reference substance puriri glucoside-I to elutriant monitors, collects the elutriant that contains corresponding color spot.This part elutriant is through concentrating under reduced pressure, and vacuum-drying gets chickweed general flavone material medicine, and wherein general flavone content is 50-90%.
Another preparation method of chickweed total flavones of the present invention comprises: get chickweed crude drug or bright grass (converting into the hay) lower alcohol with 8 times of amounts 30～40%, being warmed to 40～50 ℃ of reduced-pressure backflows in extractor extracts three times, each 1～3 hour, united extraction liquid; Measuring density under the reclaim under reduced pressure solvent is concentrated into 40 ℃ with extract is the extracting solution of 1.05-1.1; Spissated filtrate is gone up polyamide column separates, earlier with the distilled water wash-out, again with ethanol or the methanol-eluted fractions of 40-95%, during wash-out, adopting thin layer chromatography to compare with reference substance puriri glucoside-I to elutriant monitors, collects the elutriant that contains corresponding color spot.This part elutriant is through concentrating under reduced pressure, and vacuum-drying gets chickweed general flavone material medicine, and wherein general flavone content is 70-90%.
Lower alcohol wherein is selected from ethanol, methyl alcohol, isopropylcarbinol or propyl carbinol, preferred alcohol.
Chickweed total flavones of the present invention has antiviral activity, and virus of the present invention comprises virus of AIDS, simplexvirus, Respirovirus, enterovirus, papovavirus, common cold virus and adenovirus.Chickweed total flavones of the present invention especially has curative effect preferably to hiv virus and simplexvirus.So chickweed total flavones of the present invention can be prepared into the caused disease of the above-mentioned virus of pharmacological agent as activeconstituents, simplexvirus wherein also comprises hsv-1, hsv-2 and varicella zoster virus.The disease that herpes simplex and varicella zoster virus cause comprises skin bleb, property bleb, cornea bleb or varicella and zoster.
Stellaria total flavones of the present invention can be used as activeconstituents and pharmaceutically receivable carrier or excipient are prepared into various formulations, preferred exterior-applied formulation.These formulations comprise sprays, emulsion, creme, paste, patch, eye drops, nasal drop, suppository, film etc., preferred sprays.
Sprays of the present invention is made up of as activeconstituents and sprays auxiliary material the chickweed total flavones.The preferred propylene glycol of sprays auxiliary material, mentha camphor and ethanol.
The concentration of chickweed total flavones can be 0.25-4% in the sprays of the present invention, preferred 0.25% concentration.Get the chickweed total flavones and add water and be made into 0.25%, 0.5%, 1%, 2%, 4% finished product respectively, wherein ethyl p-hydroxybenzoate concentration is 0.1%, and content of propylene glycol is 10%, and screening determines that the concentration of chickweed total flavones is 0.25% through pharmacodynamics.
Used dosing solvent can be the ethanol of water or 20-75% concentration in the sprays of the present invention.Preferred 40% ethanol.Getting total flavones, to be made into content respectively be 4%, 20%, 40%, 60%, 75% ethanolic soln, wherein add 0.1% ethyl p-hydroxybenzoate in the water liquid, observe stability, sprayability, the solution evaporation of each solution, after placing for some time, water liquid, 20% ethanolic soln, 40% ethanolic soln are all more stable, and the solution that 60% and 70% ethanol is mixed with then all has obvious sediment to produce.Water liquid and 20% ethanolic soln be because pure content is lower, should not volatilize, absorb after being sprayed on skin as the sprays soup, so select final dosing concentration 40% ethanol.
Used solubility promoter can be propylene glycol, glycerol in the sprays of the present invention, preferred propylene glycol, more preferably 20% propylene glycol.Add 20% propylene glycol and 20% glycerol respectively in 40% ethanolic soln, evaluation method according to sprays is selected suitable solubility promoter with intuitive analysis methods analyst test-results, the result shows, the sprays clarity of not adding solubility promoter is better, solution spray is better, solution is sprayed on the skin and is evenly distributed, but draws moist relatively poorly, and skin feel is inviscid; With the glycerol is the spray solution muddiness that solubility promoter is made into, and solution spray is better, and solution is sprayed on the skin and is evenly distributed, draw moist better, skin feel viscosity is bigger; The spray solution clarity that adds propylene glycol and be solubility promoter is good, and solution spray is better, and solution is sprayed on the skin and is evenly distributed, draw moist better, skin feel has slight viscosity.As seen propylene glycol is good as the spray solution clarity that solubility promoter is made into, and it is moist good to draw, and propylene glycol viscosity is less, toxicity and pungency are less, and solubility property is good, increase the product stability of preparation, and certain Transdermal absorption effect is arranged, can strengthen the absorption of effective constituent, improve curative effect.
Solubility promoter concentration can be selected propylene glycol 10-30%, and preferred 10%.
Sprays of the present invention is optional can to add perfume compound.The borneol of the aromatic odour of preferentially selecting for use and mentha camphor.
Embodiment
Embodiment 1
Get 200g chickweed 60% extraction using alcohol, fling to ethanol after, add the 500ml water dissolution, with equivalent n-butanol extraction seven times, combining extraction liquid, the reclaim under reduced pressure propyl carbinol must contain the chickweed total flavones.
Embodiment 2
Get 200g chickweed 60% extraction using alcohol, fling to ethanol after, add the 200ml water dissolution, on pretreated macroporous resin, elder generation's water wash-out, water elution liquid discards, and uses 40% ethanol elution again, collect 40% ethanol eluate, behind the recovery ethanol, obtain containing the chickweed total flavones.
Embodiment 3
With 40% alcohol reflux of 8 times of amounts 3 times, each 1.5 hours, united extraction liquid, decompression recycling ethanol to 40 a ℃ survey relative density is 1.10-1.15, go up pretreated macroporous resin column, sample 0.8 gram on per 10 gram macroporous resins, first water wash-out, it is closely colourless to be eluted to water liquid, ultraviolet detection does not have obvious absorption peaks, uses 40% ethanol elution again, collects elutriant, decompression recycling ethanol to 40 a ℃ survey relative density is 1.25, is drying to obtain the chickweed total flavones.
Embodiment 4
Get chickweed crude drug or bright grass (converting into hay) methyl alcohol, in extractor, be warmed to 40～50 ℃ and extract three times with 8 times of amounts 40%, each 1～3 hour, united extraction liquid; Measuring density under with decompression extract being concentrated into 40 ℃ again behind the reclaim under reduced pressure solvent is the extracting solution of 1.05-1.1; Spissated filtrate is gone up polyamide column separate, earlier with the distilled water wash-out, with 80% ethanol or methanol-eluted fractions, during wash-out, adopt thin layer chromatography to compare with reference substance puriri glucoside-I to elutriant and monitor again, collection contains the elutriant of corresponding color spot.This part elutriant is through concentrating under reduced pressure, and vacuum-drying gets chickweed general flavone material medicine, and yield is 1.73%, and wherein general flavone content is 70-90%.
Embodiment 5
According to embodiment 4 described methods, difference is to replace chickweed with Stellaria micrantha Hayata, and yield is 1.37%.
Embodiment 6
According to embodiment 4 described methods, difference is to replace chickweed with the husky chickweed of giving birth to, and yield is 1.18%.
Embodiment 7
According to embodiment 4 described methods, difference is to replace chickweed with the Anhui chickweed, and yield is 1.69%.
Embodiment 8
According to embodiment 4 described methods, difference is to replace chickweed with leafy chickweed, and yield is 1.52%.
Embodiment 9
According to embodiment 4 described methods, difference is to replace chickweed with Stellaria henryi williams, and yield is 1.39%.
Embodiment 10
According to embodiment 4 described methods, difference is to replace chickweed with the slender lobule chickweed, and yield is 1.70%.
Embodiment 11
According to embodiment 4 described methods, difference is to replace chickweed with magnificent chickweed, and yield is 1.37%.
Embodiment 12
According to embodiment 4 described methods, difference is to replace chickweed with Stellaria dianthifolia Williams, and yield is 1.59%.
Embodiment 13
Get long lobe chickweed 60% extraction using alcohol of 200g, fling to ethanol after, add the 200ml water dissolution, on pretreated macroporous resin, elder generation's water wash-out, water elution liquid discards, and uses 40% ethanol elution again, collect 40% ethanol eluate, behind the recovery ethanol, obtain containing the chickweed total flavones.
Embodiment 14
With the chickweed that becomes mildewed with 40% alcohol reflux of 8 times of amounts 3 times, each 1.5 hours, united extraction liquid, decompression recycling ethanol to 40 a ℃ survey relative density is 1.10-1.15, go up pretreated macroporous resin column, sample 0.8 gram on per 10 gram macroporous resins, first water wash-out, it is closely colourless to be eluted to water liquid, ultraviolet detection does not have obvious absorption peaks, uses 40% ethanol elution again, collects elutriant, decompression recycling ethanol to 40 a ℃ survey relative density is 1.25, is drying to obtain the chickweed total flavones.
Embodiment 15
Get wrinkle leaf chickweed crude drug or bright grass (converting into hay) methyl alcohol, in extractor, be warmed to 40～50 ℃ of reduced-pressure backflows and extract three times with 8 times of amounts 40%, each 1～3 hour, united extraction liquid; Measuring density under decompression is concentrated into 40 ℃ with extract is the extracting solution of 1.05-1.1; Spissated filtrate is gone up polyamide column separate, earlier with the distilled water wash-out, with 80% ethanol or methanol-eluted fractions, during wash-out, adopt thin layer chromatography to compare with reference substance puriri glucoside-I to elutriant and monitor again, collection contains the elutriant of corresponding color spot.This part elutriant is through concentrating under reduced pressure, and vacuum-drying gets chickweed general flavone material medicine, and wherein general flavone content is 70-95%.
Embodiment 16
Get 200g exhibition leaf chickweed 60% extraction using alcohol, fling to ethanol after, add the 500ml water dissolution, with equivalent n-butanol extraction seven times, combining extraction liquid, the reclaim under reduced pressure propyl carbinol must contain the chickweed total flavones.
Embodiment 17
Get 200g Taiwan chickweed 60% extraction using alcohol, fling to ethanol after, add the 200ml water dissolution, on pretreated macroporous resin, elder generation's water wash-out, water elution liquid discards, and uses 40% ethanol elution again, collect 40% ethanol eluate, behind the recovery ethanol, obtain containing the chickweed total flavones.
Embodiment 18
The Yunnan chickweed of getting 200g is with 40% alcohol reflux of 8 times of amounts 3 times, each 1.5 hours, united extraction liquid, decompression recycling ethanol to 40 a ℃ survey relative density is 1.10-1.15, go up pretreated macroporous resin column, sample 0.8 gram on per 10 gram macroporous resins, first water wash-out, it is closely colourless to be eluted to water liquid, ultraviolet detection does not have obvious absorption peaks, uses 40% ethanol elution again, collects elutriant, decompression recycling ethanol to 40 a ℃ survey relative density is 1.25, is drying to obtain the chickweed total flavones.
Embodiment 19
Get umbrella leaf chickweed crude drug or bright grass (converting into hay) methyl alcohol, in extractor, be warmed to 40～50 ℃ of reduced-pressure backflows and extract three times with 8 times of amounts 40%, each 1～3 hour, united extraction liquid; Measuring density under the reclaim under reduced pressure solvent is concentrated into 40 ℃ with extract is the extracting solution of 1.05-1.1; Spissated filtrate is gone up polyamide column separate, earlier with the distilled water wash-out, with 80% ethanol or methanol-eluted fractions, during wash-out, adopt thin layer chromatography to compare with reference substance puriri glucoside-I to elutriant and monitor again, collection contains the elutriant of corresponding color spot.This part elutriant is through concentrating under reduced pressure, and vacuum-drying gets chickweed general flavone material medicine, and wherein general flavone content is 70-90%.
The preparation of embodiment 20 sprayss
Chickweed total flavones with preparation among 40% the dissolve with ethanol embodiment 4 obtains clear and bright uniform solution, add again that 10% propylene glycol is made solubility promoter and 0.5% mentha camphor is made perfume compound, above-mentioned solution is dissolved in the propellent of 20-65% then in the wiring solution-forming system, and the container of packing into is made.
Embodiment 21: newborn creme
Chickweed total flavones 5g adds the wetting ability emulsion matrix to 1000g, is distributed into 15g/ and props up.It is antiviral to be used for strong skin.
Embodiment 22: gelifying agent
Get chickweed total flavones 2.5g, borneol 10g and ethyl p-hydroxybenzoate 1g, adding water soluble matrix is distributed into the 15g/ bottle to 1000g.Be used for the treatment of herpes simplex and virus diseases such as varicella-zoster, pointed condyloma.
Embodiment 23: eye drops
Get chickweed total flavones 2.5g, ethyl p-hydroxybenzoate 0.3g and sodium-chlor 5.6g, solubilizing agent is an amount of, adds injection water 1000ml, is distributed into 5ml/ and props up.Be used for the treatment of the herpetic keratitis that causes by virus.
Embodiment 24: nasal drop
Get chickweed total flavones 3.0g and propylene glycol 200g, other right amount of auxiliary materials is made into 1000ml, is distributed into the 10ml/ bottle, is used for the treatment of the nasal mucosa herpes simplex.
Embodiment 25: suppository
Get chickweed total flavones 5.0g, vitamin-E 10g and semi-synthetic fatty acid ester 1477g, make 1000 pieces.Be used for the treatment of virus diseases such as herpes simplex-2 type, pointed condyloma.
Embodiment 26: film
Get PVA
17-8882g, add the distilled water that 5-7 doubly measures, move in the water-bath after immersion is expanded and heat, after making whole dissolvings, other gets titanium dioxide 3g and adds in the supernatant liquor after with colloidal grinding, stir evenly, under agitation add polysorbate-80 5g, glycerine 5g and chickweed total flavones 10g then gradually, make 10% ethanolic soln.The back placement that stirs is spent the night, and removes bubble, makes film.Every contains chickweed total flavones 0.5mg.Be used for the treatment of the treatment of virus diseases such as herpes simplex-2 type, pointed condyloma.
Test example 1: the chickweed total flavones that contains puriri glucoside-1 in cell cultures to the inhibition test of HIV-1, HSV-I, HSV-II and VZV virus
In African green monkey kidney cell (VERO) and human embryonic lung diploid fibroblast (2BS) cultivation, detect the effect of anti-varicella zoster virus of chickweed total flavones (VZV) and herpes simplex virus I-type, II type (HSV-I, HSV-II).Prove that it has the effect of external anti-above-mentioned three kinds of viruses.
Adopt virocyte pathology (CPE) method and tetrazolium bromide (MTT) staining to test calculation of half inhibitory concentration (IC
50) and therapeutic index (TI).Test divides single administration group and three administration groups, and triplicate.
1.1 test
1.1.1 single administration group
1. viral CPE method:
VERO cell and 2BS cell are inoculated 96 well culture plates with 400,000/ml concentration, cultivate 24 hours for 37 ℃, add the 10-100TCID of HSV-I, HSV-II and VZV virus respectively
50Viral liquid, every hole 100 μ l, 37 ℃ absorption 1 hour, discard viral liquid.Add soup, all with 10 concentration of maximal non-toxic concentration doubling dilution of medicine, every concentration 4 holes, every hole 100 μ l.Be that the chickweed total flavones is 5000 μ g/ml, 2500 μ g/ml, 1250 μ g/ml, 625 μ g/ml, 312.5 μ g/ml, 156.2 μ g/ml, 78.1 μ g/ml, 39.1 μ g/ml, 19.5 μ g/ml, 9.8 μ g/ml.Acycloguanosine is 2500 μ g/ml, 1250 μ g/ml, 625 μ g/ml, 312.5 μ g/ml, 156.3 μ g/ml, 78.1 μ g/ml, 39.1 μ g/ml, 19.5 μ g/ml, 9.8 μ g/ml, 4.9 μ g/ml.Establish the contrast of virus control and normal cell simultaneously, 37 ℃, 5%CO
2Cultivated 5-7 days, per 24 hours inverted microscopes are observed viral CPE down, " +++-++ ++ " occur to virus control cytopathy (CPE), calculate IC
50
2. viral MTT staining:
Behind the observation of cell pathology CPE result, add the every hole 10 μ l of MTT staining fluid, put 37 ℃ and cultivated 4 hours, add 10%SDS 100 μ l then and make lysis, measure with microplate reader wavelength 570nm after the cracking, record OD value is calculated IC
50
1.1.2 three administration groups
Three administration group of methods are with an administration group.Per 24 hours of three administration groups discard old soup, add same concentration liquid again, and virus control and cell contrast are changed and kept liquid, continuous three days.
1.2 experimental result
1.2.1 pilot study result
The toxic action of drug cell:
Chickweed total flavones and positive control medicine acycloguanosine, relatively the toxicity of its pair cell the results are shown in subordinate list 2. and attached photograph 1～14 in VERO cell and 2BS cell cultures.
1. the chickweed total flavones is to the toxicity result of VERO cell
Checking medicine: chickweed total flavones CPE method: maximal non-toxic concentration (TD
0) be 5000 μ g/ml, median toxic concentration (TD
50) be 7496.7 μ g/ml.Mtt assay maximal non-toxic concentration (TD
0) be 5000 μ g/ml, median toxic concentration (TD
50) be 7811.4 μ g/ml.
Positive control medicine: acycloguanosine CPE method maximal non-toxic concentration (TD
0) be 2500 μ g/ml, median toxic concentration (TD
50) be 3878.7 μ g/ml.Mtt assay maximal non-toxic concentration (TD
0) be 2500 μ g/ml, median toxic concentration (TD
50) be 3945.1 μ g/ml.
2. the chickweed total flavones is to the toxicity result of 2BS cell
Checking medicine: chickweed total flavones CPE method maximal non-toxic concentration (TD
0) be 5000 μ g/ml, median toxic concentration (TD
50) be 7833.1 μ g/ml.Mtt assay maximal non-toxic concentration (TD
0) be 5000 μ g/ml, median toxic concentration (TD
50) be 7750.6 μ g/ml.
Positive control medicine: acycloguanosine CPE method maximal non-toxic concentration (TD
0) be 2500 μ g/ml, median toxic concentration (TD
50) be 3916.8 μ g/ml.Mtt assay maximal non-toxic concentration (TD
0) be 2500 μ g/ml, median toxic concentration (TD
50) be 3892.8 μ g/ml.
In VERO and 2BS cell cultures, measure HSV-I, HSV-II and VZV virus median infective dose (TCID
50), adopt virocyte pathology CPE method
1. HSV-I virus: median infective dose (TCID
50) be 10
-7
2. HSV-II virus: median infective dose (TCID
50) be 10
-7
3. VZV virus: median infective dose (TCID
50) be 10
-3
1.2.2 official test result (seeing attached list 3)
Chickweed total flavones of the present invention and acycloguanosine in VERO and 2BS cell cultures to the restraining effect of HSV-I, HSV-II type and VZV virus.
1.2.2.1 the chickweed total flavones is to the restraining effect of HSV-I C-type virus C
1. single administration group:
Checking medicine: chickweed total flavones CPE method, medicine medium effective concentration (IC
50) be 10.6 μ g/ml, minimum effective concentration (MIC) is 39.1 μ g/ml, therapeutic index (TI) is 127.8.Mtt assay medicine medium effective concentration (IC
50) be 10.9 μ g/ml, minimum effective concentration (MIC) is 39.1 μ g/ml, therapeutic index (TI) is 127.8.
Positive control medicine: acycloguanosine CPE method, medicine medium effective concentration (IC
50) be＜4.9 μ g/ml, minimum effective concentration (MIC) is＜4.9 μ g/ml, therapeutic index (TI) is＞510.2.Mtt assay medicine medium effective concentration (IC
50) be＜4.9 μ g/ml, minimum effective concentration (MIC) is＜4.9 μ g/ml, therapeutic index (TI) is＞510.2.
2. three administration groups:
Checking medicine: chickweed total flavones CPE method: medicine medium effective concentration (IC
50) be＜9.8 μ g/ml, minimum effective concentration (MIC) is 19.5 μ g/ml, therapeutic index (TI) is 256.4.Mtt assay medicine medium effective concentration (IC
50) be＜9.8 μ g/ml, minimum effective concentration (MIC) is 19.5 μ g/ml, therapeutic index (TI) is 256.4.
Positive control medicine: acycloguanosine CPE method, medicine medium effective concentration (IC
50) be＜4.9 μ g/ml, minimum effective concentration (MIC) is＜4.9 μ g/ml, therapeutic index (TI) is＞510.2.Mtt assay medicine medium effective concentration (IC
50Be＜4.9 μ g/ml, minimum effective concentration (MIC) is＜4.9 μ g/ml, and therapeutic index (TI) is＞510.2.
1.2.2.2 the chickweed total flavones is to the restraining effect of HSV-II C-type virus C
1. single administration group:
Checking medicine: chickweed total flavones CPE method, medicine medium effective concentration (IC
50) be 18.6 μ g/ml, minimum effective concentration (MIC) is 39.1 μ g/ml, therapeutic index (TI) is 127.8.Mtt assay medicine medium effective concentration (IC
50) be 15.7 μ g/ml, minimum effective concentration (MIC) is 39.1 μ g/ml, therapeutic index (TI) is 127.8.
Positive control medicine: acycloguanosine CPE method, medicine medium effective concentration (IC
50) be 4.9 μ g/ml, minimum effective concentration (MIC) is 9.8 μ g/ml, therapeutic index (TI) is 255.1.Mtt assay medicine medium effective concentration (IC
50) be＜4.9 μ g/ml, minimum effective concentration (MIC) is 9.8 μ g/ml, therapeutic index (TI) is 255.1.
2. three administration groups:
Checking medicine: chickweed total flavones CPE method, medicine medium effective concentration (IC
50) be 14.0 μ g/ml, minimum effective concentration (MIC) is 39.1 μ g/ml, therapeutic index (TI) is 127.8.Mtt assay medicine medium effective concentration (IC
50) be 13.8 μ g/ml, minimum effective concentration (MIC) is 39.1 μ g/ml, therapeutic index (TI) is 127.8.
Positive control medicine: acycloguanosine CPE method, medicine medium effective concentration (IC
50) be＜4.9 μ g/ml, minimum effective concentration (MIC) is 9.8 μ g/ml, therapeutic index (TI) is 255.1.Mtt assay medicine medium effective concentration (IC
50) be＜4.9 μ g/ml, minimum effective concentration (MIC) is 9.8 μ g/ml, therapeutic index (TI) is 255.1.
1.2.2.3 the chickweed total flavones is to the restraining effect of VZV virus
1. single administration group:
Checking medicine: chickweed total flavones CPE method, medicine medium effective concentration (IC
50) be 13.5 μ g/ml, minimum effective concentration (MIC) is 39.1 μ g/ml, therapeutic index (TI) is 127.8.Mtt assay medicine medium effective concentration (IC
50) be 14.7 μ g/ml, minimum effective concentration (MIC) is 39.1 μ g/ml, therapeutic index (TI) is 127.8.
Positive control medicine: acycloguanosine CPE method, medicine medium effective concentration (IC
50) be＜4.9 μ g/ml, minimum effective concentration (MIC) is 9.8 μ g/ml, therapeutic index (TI) is 255.1.Mtt assay medicine medium effective concentration (IC
50) be＜4.9 μ g/ml, minimum effective concentration (MIC) is 9.8 μ g/ml, therapeutic index (TI) is 255.1.
2. three administration groups:
Checking medicine: chickweed total flavones CPE method, medicine medium effective concentration (IC
50) be 12.6 μ g/ml, minimum effective concentration (MIC) is 19.5 μ g/ml, therapeutic index (TI) is 256.4.Mtt assay medicine medium effective concentration (IC
50) be 12.8 μ g/ml, minimum effective concentration (MIC) is 19.5 μ g/ml, therapeutic index (TI) is 256.4.
Positive control medicine: acycloguanosine CPE method, medicine medium effective concentration (IC
50) be＜4.9 μ g/ml, minimum effective concentration (MIC) is 9.8 μ g/ml, therapeutic index (TI) is 255.1.Mtt assay medicine medium effective concentration (IC
50) be＜4.9 μ g/ml, minimum effective concentration (MIC) is 9.8 μ g/ml, therapeutic index (TI) is 255.1.
: above test-results shows the chickweed total flavones in the extracorporeal antivirus effect test of pesticide effectiveness (in the cell cultures), adopts virocyte pathology CPE method and mtt assay, proves that it has the effect of anti-herpes simplex virus I, II type and varicella zoster virus.
Test example 2: the activity test in vitro that contains the chickweed total flavones anti-HIV-1 of puriri glucoside-1
1 material and method:
Virus: HIV-1018a obtains from the individuality that the preceding HIV-1 of AZT treatment infects.It is provided by Douglas professor Richmen of Univ California-San Diego USA.
Cell: peripheral blood lymphocytes (PBMC) is used the Ficoll-Hypaque density gradient centrifugation method, and the peripheral blood of the healthy of infected by HIV separation never obtains.
Test design:
The mensuration of antiviral activity: with 2 * 10
5The chickweed total flavones that contains puriri glucoside-1 of PBMC/100 microlitre is exposed to the 100TCID with the HIV-1 of inoculation 50 microlitres
50Simultaneously, give puriri glucoside 100 microlitres that add various concentration in each hole.After infecting training in 4 days, get supernatant liquor and measure the generation of p24 antigen.Duplicate quantitative HIV-1p24 antigen according to operation instructions by intact virus antigen capture ELISA (CoulterCorp., Hialeah FL).
Cytotoxic assay: use to comprise
3Possible drug cell toxic action is described in the hyperplasia that the H-thymus pyrimidine is measured.After 96 hole culture dish of the puriri glucoside-1 that contains various concentration were cultivated 4 days, (DuPont NEN, Boston MA) joined 2 * 10 with 1 μ Ci thymus pyrimidine
5Cell/200 microlitre substratum.Add
3Behind the H-thymus pyrimidine, cultivated 6 hours and measured at 37 ℃
3H-TdR.Calculate 50% inhibition concentration (IC with SigmaPlot Scimtific Graphing System
50) and 50% toxic concentration (TC
50), calculate therapeutic index TI value=TC
50/ IC
50
Test-results
HIV-1018a p24 antigen produces (pg/ml) (infected back 4 days, with virus with cell infection 2 hours)
| 800 | 200 | 50 | 12.5 | 3.2 | 0.8 | 0.2 | 0 | IC 50μg/ml | |
| The chickweed total flavones | 131 | 448 | 1798 | 2703 | 3596 | 3489 | 2863 | 3146 | 87.9 |
Conclusion: the chickweed total flavones is 20.8 μ g/ml to the IC50 of the cell of identical time virus inoculation, and TC50 is 475.5 μ g/ml, and therapeutic index is 22.8.The IC50 that the chickweed total flavones infected PBMCs3 hour in advance to HIV-1018a is 22.9 μ g/ml, and TC50 is 475.5 μ g/ml, and therapeutic index is 20.8.
Test example 3: sprays of the present invention infects the result of treatment of II herpes simplex virus type to mouse vagina.
1.1 test
Observation index
(1) after mouse vagina infects HSV-II virus; cause viral vaginitis, local red and swollen, back acroparalysis death; after empirical tests medicine and the treatment of positive control medicine; calculate its sickness rate, mortality ratio, average life day; medicament protection rate and prolongation vital rates and virus control group are relatively; and learn by statistics and handle, calculate t value and P value, judge result of treatment.
(2) will treat preceding and the treatment back vagina cotton swab sample of getting, the suspension of making 1: 5 adopts cytopathy (CPE) method in the VERO cell cultures.The observed and recorded test-results remember that with 26%～50% cytopathy " ++ " remember that with 51%～75% cytopathy " +++" remember that with 76%～100% cytopathy " ++ ++ " calculate 50% infective dose (TCID with the Reed-Muench method with 25% cytopathy note "+"
50).Test-results and virus control group are relatively calculated the inhibiting rate of medicine to HSV-II, judge medication effect.
1.1.1 infective virus
The BaIb/c mouse, female, 18-20 gram, random packet, 10 every group.The 1 milliliter of syringe of will sterilizing is joined homemade hangnail syringe needle, sucks HSV-II virus liquid, and rotation is inserted in the mouse vagina and repeatedly scratched vaginal walls, and injects 10LD simultaneously
50Virus liquid 0.03ml/ only.
2.1.2 medicine grouping
The checking medicine: sprays of the present invention is selected for use with maximal non-toxic dosage (TD
0) 2%, 1%, 0.5%, 0.25%, 0.125%, 5 dosage group of following doubling dilution.
Positive control medicine: 3% acyclovir ointment, 1 dosage group.
Establish the contrast of checking medicine group simultaneously, positive drug group contrast (after infective virus did not scratch vaginal walls, medication was smeared).
Normal mouse contrast (not infective virus, not medication).
(infective virus liquid is 10LD to virus control
50After, take drugless medication).
2.1.3 pharmacological agent
The viral vaginitis symptom occurs on the 3rd day after the mouse vagina infection HSV-II virus, show as the vagina redness, phenomenons such as misnicturition.Carry out pharmacological agent immediately, medicine spray dosage of the present invention is selected the maximal non-toxic concentration (TD of toxicity test for use
0) 2%, 1%, 0.5%, 025%, 0.125% 5 following dosage group.Positive control medicine 3% acyclovir ointment.The method that test adopts sticking soup of cotton swab and ointment to smear at intravaginal, every group of 10 mouse.Every day, secondary was treated six days continuously.The viral vaginitis symptom occurred on the 3rd day behind another group infective virus, get vagina cotton swab sample before the medication and be placed on (be equipped with 0.5 milliliter the little plastics tubing of physiological saline in) and put-25 ℃ of preservations.Treat with checking medicine and positive control medicine immediately, every day, secondary was treated six days continuously.Got the vagina sample again in 1 day after the drug withdrawal, (treatment back) makes getting sample suspension separation determination virus in the Vero cell cultures of 1: 5.The test triplicate.
1.2 experimental result
1.2.1 sprays of the present invention is to the result of treatment (with protection ratio, prolongation vital rates is indication, judges drug effect) of mouse viral vaginitis
The mouse transvaginal infects HSV-II virus, and the virus infection amount is 10LD
50, 10 every group.The viral vaginitis symptom appearred behind the virus infection on the 3rd day.Carry out pharmacological agent immediately, checking medicine group is selected for use with maximal non-toxic dosage (TD
0) i.e. 1 the dosage group of 2%, 1%, 0.5%, 0.25%, 0.125% and 3% acyclovir ointment of 5 dosage groups of following doubling dilution, every day, secondary was treated 6 days continuously.Three times test-results sees attached list 4.Its medicament protection rate (%) is respectively 63%, 56%, 48%, 44%, 30%, prolongs vital rates (%) and is respectively 59%, 58%, 55%, 52%, 45%, median effective dose (ED
50) be 0.89%.3% its protection ratio of acyclovir ointment (%) is 81%, and prolonging vital rates (%) is 63%.Two kinds of medicines are learned the processing there was no significant difference by statistics, P value＜0.01 (except that 0.125%＜0.05).Illustrate that sprays of the present invention has the effect of anti-II herpes simplex virus type in the body, remarkable with virus control group comparative effectiveness.Therapeutic index according to above-mentioned test-results sprays of the present invention is 32.
1.2.2 sprays of the present invention (is measured TCID with mouse vagina sample suspension isolated viral in cell cultures of taking before and after the test of cure to the result of treatment of mouse viral vaginitis
50The result be index, judge drug effect).
Mouse vagina tangible viral vaginitis symptom occurred, and carried out pharmacological agent after infecting HSV-II virus on the 3rd day.Take the sub-sample of the cotton examination of vagina before the treatment, treat with checking medicine and positive control medicine immediately, every day, secondary was treated six days continuously.Got the vagina sample again in 1 day after the drug withdrawal, and make 1: 5 suspension in the VERO cell cultures, adopt cytopathy (CPE) method, measure virus titer, to judge drug effect.The three batches of test-results, 5. sprayss of the present invention of seeing attached list are respectively 73%, 67%, 63%, 57%, 40% to the inhibiting rate (%) that mouse vagina infects HSV-II virus.Positive control medicine 3% acyclovir is 90% to the inhibiting rate (%) that mouse vagina infects HSV-II virus.Two kinds of medicines are learned the processing there was no significant difference by statistics, and the P value all＜0.01.Illustrate that sprays of the present invention has the effect of anti-II herpes simplex virus type in the body, remarkable with virus control group comparative result.According to the spray therapeutic index of agent of above-mentioned test-results the present invention is 32.
1.3 brief summary
Show that according to test-results sprays dosage of the present invention is in the effect that has anti-II hsv more than 0.25%.
2. the anti-herpes simplex virus test of pesticide effectiveness (tame rabbit cornea test) in the medicament for the eyes water body of the present invention
Detect the result of treatment of the collyrium of the present invention of different concns, the clinical test basis that provides is provided the rabbit viral keratitis.
2.1 test:
2.1.1 infective virus
Rabbit is male, body weight 500～100 grams, random packet.Earlier splash into the rabbit intraocular with 1% aseptic tetracaine before infecting, every eye splashes into 0.04ml, and eyeball anesthesia after about 25 seconds is used disinfectant cornea trepan cut on cornea gently, then with 100ID
50The virus infection rabbit cornea, every cornea 0.2ml, every group of 5 rabbit.The viral keratitis symptom occurred on the 3rd day behind the virus infection, it is muddy red to show as cornea, and pharmacological agent is carried out in the eyelid enlargement immediately, the maximal non-toxic concentration (TD of checking choice of drug externally applied medicine test
0) 2%, 1%, 0.5%, 0.25%, 0.125% 5 following dosage, 5 rabbit of every dosage group external application eye drip, every splashes into about 0.15ml.Positive drug: acyclovir 1mg/ml, every drips 2 about 0.15ml, 5 rabbit of external application eye drip.Every day, secondary was treated six days continuously.Establish checking medicine and positive drug contrast (do not give virus, administration as usual, dosage is with the treatment group) simultaneously.Establish every of normal rabbits cornea contrast (do not give virus, drip physiological saline) simultaneously and splash into 2, about 0.15ml, 5 rabbit of eye drip are established virus control (100ID simultaneously
50Virus quantity) 5 rabbit.Another group is tested behind virus infection and to be got cornea cotton swab sample (sample is placed in the plastics tubing of the physiological saline that 0.5ml is housed) on the 3rd day and put-25 ℃ of preservations.Carry out pharmacological agent immediately, 2%, 1%, 0.5%, 0.25%, 0.125% 5 the following dosage of maximal non-toxic concentration of checking choice of drug externally applied medicine test, 5 rabbit of every dosage group external application eye drip, every splashes into about 0.15ml.Positive drug: acyclovir 1mg/ml, every drips 2 about 0.15ml, 5 rabbit of external application eye drip.Every day, secondary was treated six days continuously.Got the cornea sample after the drug withdrawal in second day again.Make 1: 5 viral suspension, in the Vero cell cultures, adopt cell to become (CPE) method and measure virus titer (TCID
50).The test triplicate.
2.1.2 experimental observation
2.1.2.1 rabbit I herpes simplex virus type eye drip causes viral keratitis, adopts pharmacological agent immediately; every day, secondary was treated six days continuously, and meter record sick damage degree every day and virus control group are relatively; calculate protection ratio, and learn by statistics and handle with X value P value determine effect.
2.1.2.2 rabbit I herpes simplex virus type eye drip, cause viral keratitis, second day (treatment back) takes cornea cotton swab sample respectively after the 3rd day (before the medication) and the drug withdrawal behind virus infection, and make 1: 5 viral suspension, in the Vero cell cultures, adopt cell to become (CPE) method and measure virus titer (TCID
50).(seeing attached list 6) compares with the virus control group, calculates the inhibiting rate of medicine to simplexvirus, and learns by statistics and handle, with X value P value determine effect.
2.2 experimental result
2.2.1 collyrium of the present invention infects hsv test of pesticide effectiveness result to tame rabbit cornea.
Rabbit is through 100ID
50Virus splashes into the damage cornea, cause viral keratitis after three days, carry out pharmacological agent immediately, 2%, 1%, 0.5%, 0.25%, 0.125% 5 the following dosage of maximal non-toxic concentration of checking choice of drug externally applied medicine test, 5 rabbit of every dosage group external application eye drip, every splashes into about 0.15ml.Positive drug: acyclovir 1mg/ml, every drips 2 about 0.15ml, 5 rabbit of external application eye drip.Every day, secondary was treated six days continuously.Meter record sick damage degree every day and virus control group are relatively calculated protection ratio, and learn by statistics and handle with X value P value determine effect.Three times test-results sees Table bright: the collyrium of the present invention of different concns is respectively 100%, 100%, 100%, 93%, 40% to the protection ratio (%) that tame rabbit cornea infects HSV-1 virus.Positive control medicine acyclovir is 100% to the protection ratio (%) that tame rabbit cornea infects HSV-I virus.Two kinds of medicines are learned the processing there was no significant difference by statistics.P value＜0.01.Illustrate that collyrium concentration of the present invention is in the effect that has the anti-I type hsv more than 0.25%.Relatively its result is remarkable with the virus control group.
2.2.2 collyrium of the present invention infects hsv test of pesticide effectiveness result to tame rabbit cornea.
Rabbit is through 100ID
50Virus splashes into the damage cornea, behind the virus infection after three days (before the medication) and the drug withdrawal second day (treatment back) take cornea cotton swab sample respectively.And make 1: 5 viral suspension, in the Vero cell cultures, adopt cell to become (CPE) method and measure virus titer (TCID
50).Compare with the virus control group, calculate the inhibiting rate of medicine, and learn by statistics and handle, with X value P value determine effect simplexvirus.Three test-results (seeing attached list 8): the collyrium of the present invention of different concns is respectively 100%, 100%, 100%, 100%, 13% to the protection ratio (%) that tame rabbit cornea infects HSV-I virus.Positive control medicine acyclovir infects HSV-I%, 100%, 13% to tame rabbit cornea.Positive control medicine acyclovir is 100% to the protection ratio (%) that tame rabbit cornea infects HSV-I virus.Two kinds of medicines are learned the processing there was no significant difference by statistics.P value＜0.01.Illustrate that collyrium concentration of the present invention is in the effect that has the anti-I type hsv more than 0.25%.Relatively its result is remarkable with the virus control group.
2.3 brief summary: show the effect that different concns collyrium of the present invention has the anti-I type hsv according to test-results.
The anti-herpes simplex I C-type virus C test of pesticide effectiveness in the 3 sprays bodies of the present invention
Detect sprays of the present invention guinea pig skin is infected the I herpes simplex virus type test of pesticide effectiveness, for clinical application provides test basis.
3.1 pilot study
3.1.1 drug toxicity is measured
Sprays is to animal skin toxicity test result according to the present invention, and its maximal non-toxic concentration is greater than 5g/kg.
3.1.2 I herpes simplex virus type (HSV-I, SM44 strain) virus virulence is measured
Use trichogen (8%Bas) with guinea pig back skin (4cm earlier
2) depilation, wipe away driedly with 37 ℃ of warm water with skin is clean again, use " cutaneous acupuncture " to remise the skin that has lost hair or feathers then, the skin that had stung is divided into 4 parts, and (area of every skin is 1cm
2).With freshly prepd cell virus suspension with 5 concentration of physiological saline dilution, promptly 10
-0, 10
-1, 10
-2, 10
-3, 10
-4, 5 of every concentration.Every skin is smeared skin infection with the viral liquid of 30 μ l.Observed 14 days, and established the normal guinea pig contrast simultaneously.Calculate median infective dose (LD with Reed Muench method
50).The test triplicate.
3.2 official test
Sprays of the present invention is measured the curative effect that guinea pig skin infects the I herpes simplex virus type:
1. cavy grouping: by sex, weight average grouping, 10 every group.Use trichogen (8%Bas) with guinea pig back skin (4cm earlier
2) depilation, wipe away driedly with 37 ℃ of warm water with skin is clean again, use " cutaneous acupuncture " to remise the skin that has lost hair or feathers then, the skin that had stung is divided into 4 parts, and (area of every skin is 1cm
2), every skin is smeared skin infection with the HSV-I virus stock solution used of 30 μ l.
2. behind the infective virus the 4th day, typical bleb focus promptly appearred in guinea pig back skin, and area reaches 3.6-4.0cm
2(seeing attached photograph).With both treating with sprays of the present invention, medicine spray of the present invention is selected the maximal non-toxic concentration (TD to the external skin test of animal for use
0) 4 dosage of following doubling dilution, both 2%, 1%, 0.5%, 0.25% and 3% acyclovir ointment sprayed and smeared the skin bleb place, treated every day four times, treated continuously 6 days.
3. establish virus control group (not administration behind the infective virus) and normal guinea pig control group (not susceptible poison physiological saline) test triplicate simultaneously.
3.3 test-results
The medication effect observation index:
After the medication every day observed and recorded guinea pig back skin bleb focus changing conditions, record standard is as follows:
Skin bleb focus area is 1/4 o'clock, lesion degree (cm
2) note 1.0-1.6
Skin bleb focus area is 2/4 o'clock, lesion degree (cm
2) note 1.8-2.4
Skin bleb focus area is 3/4 o'clock, lesion degree (cm
2) note 2.6-3.2
Skin bleb focus area is 4/4 o'clock, lesion degree (cm
2) note 3.4-4.0
The skin bleb incrustation reaches 1/4 and is note, lesion degree (cm
2) note 0.8-1.0
The skin bleb incrustation reaches 2/4 and is note, lesion degree (cm
2) note 0.6-0.8
The skin bleb incrustation reaches 3/4 and is note, lesion degree (cm
2) note 0.4-0.6
The skin bleb incrustation reaches 4/4 and is note, lesion degree (cm
2) note 0.2-0.4
Skin bleb decrustation recovery from illness note 0
3.3.1 pilot study result
Drug toxicity is measured: medicine spray of the present invention is greater than 5g/kg to the maximal non-toxic concentration of animal skin externally applied agent.
Virus virulence is measured: I herpes simplex virus type (HSV-I), and through the guinea pig skin infection experiment being measured median infective dose (LD
50) be 10
-0.5, use virus stock solution used during the test of pesticide effectiveness.
3.3.2 official test result
3.3.2.1. sprays of the present invention infects the result of treatment of herpes simplex virus I-type to guinea pig skin:
Behind the guinea pig back skin infections HSV-1 simplexvirus, 1cm promptly appearred on the 2nd day
2About the bleb focus, reached 2-3cm on the 3rd day
2About, the focus area has reached 3.8cm in the time of the 4th day
2About, treat with the medicine spray of the present invention of various dose immediately.Medicine spray of the present invention is selected the maximal non-toxic concentration (TD to the external skin test of animal for use
0) 4 dosage of following doubling dilution, both 2%, 1%, 0.5%, 0.25% and 3% acyclovir ointment sprays and smears the skin bleb place, treats every day four times, treats continuously 6 days.Observed again after the drug withdrawal 2 days.Test-results sees attached list 9 and attached photograph 25～33, and sprays treatment group of the present invention and virus control group relatively its pathology inhibiting rate (%) are respectively 100%, 100%, 100%, 81%.Learn processing sprays dosage of the present invention by statistics and the obvious treatment effect is being arranged, P value＜0.01 more than 0.25%.
3.3.2.2. acyclovir ointment infects the result of treatment of herpes simplex virus I-type to guinea pig skin:
Acyclovir ointment is 100% to the pathology inhibiting rate of guinea pig skin bleb.
3.3.2.3. virus control group:
After the guinea pig back skin infections HSV-1 virus, typical bleb focus promptly appearred in skin of back in the 4th day, and its lesion degree reaches 3.8cm
2About, lesion degree reached 4.0cm in 5-7 days
2, and be mixed with big area and fester, and lower extremity paralysis appears in minority (6) cavy, illustrated that indivedual cavy virus infectiones are heavier, virus has been invaded and lower limb nerve.
3.3.3 test result analysis
Medicine spray of the present invention and virus control group compare, medication the 2nd day, and the skin bleb of cavy promptly alleviates to some extent, and the lesion degree (area) of the 3rd day skin bleb of medication is by 3.6cm
2Narrow down to 2.0cm
2About, the 4th day bleb focus of medication begins shrivelled, and incrustation occurs, and the 6th day bleb lesions position of medication lesion degree (area) of all forming a scab has narrowed down to 0.7cm
2About, skin bleb is almost recovered.And the virus control group test 5-7 days its lesion degree (area) developed into 4.0cm
2Though bleb focus area also reduced gradually in the 9th day, the course of disease time is long.The test-results of positive control medicine acyclovir ointment is consistent with the test-results of sprays of the present invention.
3.4 brief summary
The sprays of the present invention that shows various dose according to test-results has the obvious treatment effect to the guinea pig back skin bleb, and is consistent with the test-results of positive control medicine acyclovir ointment.Prove that this medicine has the effect of tangible anti-I type hsv.Continuously treatment is six days, not only can reduce sickness rate, and can obviously shorten the course of disease of skin infections, with the virus control group curative effect of significance is arranged relatively.
Claims (15)
1. the chickweed total flavones contains puriri glucoside-I, it is characterized in that it can obtain with following method:
Chickweed crude drug or bright grass are converted into hay, extracts at 40-50 ℃ of reduced-pressure backflow with 8-12 ethanol, methyl alcohol, isopropylcarbinol or propyl carbinol doubly, 1-3 hour at every turn, united extraction liquid, reclaim under reduced pressure solvent concentrated extracting solution to 40 ℃, mensuration density is 1.05-1.1.
2. according to the described chickweed total flavones of claim 1, it is characterized in that adding ethanol in the chickweed total flavones concentrated solution of claim 1 gained makes alcohol concn reach 60-70%, alcohol precipitation filters, and filtrate concentrates, and vacuum-drying obtains.
3. according to the described chickweed total flavones of claim 1, it is characterized in that macroporous resin bed on the extraction concentrated solution of claim 1 is separated, with the distilled water wash-out, discard water elution liquid again with ethanol or the methanol-eluted fractions of 40-95%, during wash-out, compare the monitoring of elutriant employing thin layer chromatography with puriri glucoside-I, collection contains the elutriant of corresponding color spot, concentrating under reduced pressure, vacuum-drying below 70 ℃ obtains.
4. according to the described chickweed total flavones of claim 1, it is characterized in that polyamide column on the described extraction concentrated solution of claim 1 is separated, with the distilled water wash-out, with ethanol or the methanol-eluted fractions of 40-95%, during wash-out, compare the monitoring of elutriant employing thin layer chromatography again with puriri glucoside-I, collection contains the elutriant of corresponding color spot, concentrating under reduced pressure, vacuum-drying below 70 ℃ obtains.
5. any one described chickweed total flavones among the claim 1-4 is to extract to obtain from following plants: Stellaria plant slender lobule chickweed Stellaria leptohylla Hance, long lobe chickweed Stellaria hungeanaFenzl., brown lobe Stellaria alsine Grim. Stellaria alsine var.Phaeuspetala Hand.-Mazz., Anhui chickweed Stellaria anhwiensis Migo., apicule chickweed Stellaria apiculata Wils.4987Stellaria (L) Scop., slender lobule Alishan chickweed Stellaria arisanensis var.Leptophylla Hayata., north chickweed Stellaria borealis Bigel., little fine hair chickweed Stellaria tomentalla Ohwi., David's chickweed Stellariadavidii Hemsl., the husky chickweed Stellaria arenaria Maxim. that gives birth to, short lobe chickweed Stellaria brachypetalaBge., China chickweed Stellaria chinensis Regel., Alishan chickweed Stellaria arisanensis Hayata., northeast chickweed Stellaria cherleriae (Fisch.) Will., thick leaf chickweed Stellaria crassidolia, wrinkle leaf chickweed Stellaria crispate Wall., Stellaria alsine Grim. Stellaria alsine Grimm., the chickweed Stellariadecumbens Edgew. that crouches lays down, forked cyme chickweed Stellaria dichasioides Williams., narrow leaf bifid chickweed Stellaria dichotoma var.Lanceolata Bge., southwest chickweed Stellaria delavayi Franch., Stellaria dianthifolia Williams Stellaria dianthifolia Williams., the needle-like chickweed Stellaria decunbens var.Acicularia Edgew.Et Hook.f. that crouches that lays down, bifid chickweed Stellaria dichotoma L., line leaf bifid chickweed Stellaria dichotoma var.Stephenjiana Willd., Stellaria diversiflora Maxim. Stellaria diversiflora Maxim., concave veins chickweed Stellaria depressa Schnid., turn over white chickweed Stellaria discolor Turcz., standing grain leaf chickweed Stellaria graminea L., chickweed Stellaria diffusa Wills. looses in the shop, Du Shi chickweed Stellaria duthieiGandoger., line stem chickweed Stellaria filicaulis Mak., the different colored chickweed Stellaria diversifioravar.Gymnandra Franch. of naked stamen, spend more chickweed Stellaria florida Fisch., chickweed Stellaria pilosaFranch. becomes mildewed, line handle chickweed Stellaria filipes Komar., blunt calyx chickweed Stellaria amblyosepalaSchrenk., dredge pubescence standing grain leaf chickweed Stellaria graminea var.Pilosula Maxim., turn green standing grain leaf chickweed Stellaria graminea, Stellaria maximowixziana Franch. Stellaria maximowixziana Franch var.ViridescensMaxim., Jiangzi's chickweed Stellaria gyantsensis Williams., Stellaria henryi williams Stellaria henryiWilliams., the chickweed Stellaria hsinganensis Kitagawa. of Xingan, rosy clouds grass chickweed Stellariagypsophiloides Fenzl., chickweed Stellaria media (L.) Cyr., Stellaria micrantha Hayata Stellaria micranthaHayata., introversion chickweed Stellaria infracta Maxim., gentle chickweed Stellaria mitans Williams., goose intestines chickweed Stellaria neglecta Weihe., chickweed Stellaria neo-alustris Kitagawa. is given birth in new natural pond, eight stamen chickweed Stellaria octandra Fobedim., red certain kind of berries chickweed Stellaria oxycoccoides Komar., stone is given birth to chickweed Stellaria saxatilis Buch-Ham., handle flower chickweed Stellaria pedunculans Bge., Taiwan chickweed Stellaria cicrantha Hayata., chickweed Stellaria palustria L. is given birth in the natural pond, Turkestan chickweed Stellaria turkestanica Schischk., exhibition leaf chickweed Stellaria patentifolia Kitagawa., imitation stone is given birth to chickweed Stellaria pseudosaxatilis Hand.-Mass., five chickweed Stellaria wutaicaHand.-Mazz., net arteries and veins chickweed Stellaria reticulivena Hayata., rock chickweed Stellaria rupestrisHemsl., embrace stem stone and give birth to chickweed Stellaria saxatilis var.Amplexicaulis Hand.-mazz., three type chickweed Stellaria trimorpha Nakai., xiaofanlu Stellaria pusilla Schmid., flint lobe chickweed Stellariaradians L., accurate Ge Er chickweed Stellaria soongorica Roshev., Su Shi chickweed Stellaria soulieiWilliams., star hair chickweed Stellaria stellato-pilosa Hayata., garden calyx chickweed Stellariastronglosepala Hand.-Mazz., intend umbrella flower chickweed Stellaria subumbellata Edgew., wetland chickweed Stellaria uda Williams., green colored chickweed Stellaria virdiflora Pax et O.Hoffm., Stellaria wushanensis Williams Stellaria wushanensis Williams., umbrella flower chickweed Stellaria umbellate Turcz. or Yunnan chickweed Stellaria yunnanensis franch.j.
6. any one described chickweed total flavones is treated hiv virus and simplexvirus I type, the purposes of the medicine of simplexvirus II type and varicella zoster in preparation among the claim 1-5.
7. treat hiv virus and simplexvirus I type for one kind, the medicine of simplexvirus II type and varicella zoster, it contains the described chickweed total flavones of claim 1-5 as activeconstituents and pharmaceutically acceptable carrier or excipient.
8. according to the described medicine of claim 7, wherein contained chickweed total flavones is 0.25-4%.
9. according to the described medicine of claim 8, wherein contained chickweed total flavones is 0.25%.
10. according to any one described medicine among the claim 7-9, it is a sprays.
11. according to the described sprays of claim 10, wherein it makes dosing solution with the ethanol of 20-75%.
12. according to the described sprays of claim 11, wherein it makes dosing solution with 40% ethanol.
13. according to the described sprays in the claim 12, wherein it contains the solubility promoter propylene glycol.
14. according to the described sprays of claim 13, wherein its contained propylene glycol concentration is 10%.
15. according to the sprays described in the claim 14, wherein it also contains 0.5% mentha camphor.
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