CN102526153B - Vernonia anthelmintica flavone components, preparation method and application thereof - Google Patents
Vernonia anthelmintica flavone components, preparation method and application thereof Download PDFInfo
- Publication number
- CN102526153B CN102526153B CN 201210045334 CN201210045334A CN102526153B CN 102526153 B CN102526153 B CN 102526153B CN 201210045334 CN201210045334 CN 201210045334 CN 201210045334 A CN201210045334 A CN 201210045334A CN 102526153 B CN102526153 B CN 102526153B
- Authority
- CN
- China
- Prior art keywords
- content
- group
- caulis vernoniae
- vernoniae andersonii
- flavone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention relates to vernonia anthelmintica flavone components, a preparation method and application thereof. The flavone components are fisetin, butein, 7,8,3',4'-tetrahydroxy flavone, 5,7,8,3',4'-pentahydroxy chalcone, 6,8,3',5'-tetrahydroxy-dihydroflavone, liquiritigenin and isoliquiritigenin, which are prepared from plant vernonia anthelmintica through extraction, separation and purification. Application of each flavone component in preparation of a medicament for treating leucoderma provides a new medicament choice for treating leucoderma.
Description
Technical field:
The present invention relates to a kind of dimension medicine Caulis Vernoniae andersonii flavonoid component and its production and use, particularly in the purposes for preparing anti-vitiligo medicine.
Background technology:
Caulis Vernoniae andersonii is Compositae Compositae Vernonia annual herb plant Caulis Vernoniae andersonii Vernonia anthelmintica (Linn.) Willd, medicinal part is its mature fruit, three grades xeothermic inducing diuresis and reducing edema, another name India's mountain Fructus Foeniculi (" Chinese Plants will "), Ai Te pulling force (" Uighur ").Beginning is stated from " Chinese Plants will ", " Uygur's medical material standard " and " Uigurs medicine will " (first volume) is also on the books.Hotan, Aksu, From Western Yunnan has cultivation, and also there is plantation in the states such as India, Pakistan.For Xinjiang Uygur medicine medication, its Uigurs medicine is called the OK a karaoke club Fructus Cumini Cymini, is to be most commonly used to increase one of pigment medicine in Uygur medicine.Record in Drug Standard of Ministry of Public Health of the Peoples Republic of China-Uigurs medicine fascicle, Uygur medicine thinks that Caulis Vernoniae andersonii removes abnormal phlegmatic temperament, anthelmintic, detumescence, dispersing cold for relieving pain.For the stomachache of raw property and hepatopathy, vitiligo etc.The herbal Uygur of China powder stick record Caulis Vernoniae andersonii promotes pigmentation, recovers skin color, cures mainly vitiligo.Caulis Vernoniae andersonii is mainly containing flavones ingredient and volatile oil; Its main active on the treatment vitiligo is flavone compound, has the functions such as activity of tyrosinase, increase skin photosensitization, the vitiligo focus position skin microcirculation of improving, adjusting immunity, melanophore propagation and trace element supplement; Vernonia anthelmintica willd extract has been made into several formulations at present, is applied to the clinical treatment vitiligo, has obtained significant effect.
Less for the research report of Caulis Vernoniae andersonii chemical composition at present, the present invention be take Natural Medicine Chemistry as means, Caulis Vernoniae andersonii has been carried out to extraction, separation, purification, separate first the leukodermic active component that obtains medical treatment from Caulis Vernoniae andersonii, by its drug effect of vivo and vitro experimental evaluation, and be prepared into the leukodermic medicine for the treatment of.
Summary of the invention:
The object of the invention is to, flavonoid component of a kind of Caulis Vernoniae andersonii and its production and use is provided, this flavonoid component is butin, butein, 7, 8, 3 ', 4 '-kaempferol, 5, 7, 8, 3 ', 4 '-the penta hydroxy group chalcone derivative, 6, 8, 3 ', 5 '-the tetrahydroxy flavanone, seven kinds of flavonoid components of glycyrrhizin and isoliquiritigenin, to adopt and extract from plant Caulis Vernoniae andersonii seed, separation and purification obtain flavonoid component, again using each flavonoid component as the purposes at the leukodermic medicine of preparation treatment, for the new selection of having treated leukodermic drug provision.
The flavonoid component of a kind of Caulis Vernoniae andersonii of the present invention, this flavonoid component is butin, butein, 7,8,3 ', 4 '-kaempferol, 5,7,8,3 ', 4 '-penta hydroxy group chalcone derivative, 6,8,3 ', 5 '-tetrahydroxy flavanone, glycyrrhizin and isoliquiritigenin, wherein the flavonoid component total content is 50-99.9%, each flavonoid component content is 20-99.9%.
The preparation method of the flavonoid component of described Caulis Vernoniae andersonii, this flavonoid component is that concrete operations follow these steps to carry out in dimension medicine Caulis Vernoniae andersonii, carrying out separation and purification:
A, the Caulis Vernoniae andersonii seed meal is broken to the 5-80 order, the solvent of doubly measuring with 6-12 is that the ethanol water that water or concentration are 20-95% extracts 1-4 time, temperature 40-100 ℃, each extraction time 1-3 hour, merge extractive liquid,, reclaim solvent, concentrated, vacuum drying, obtain dry extract, obtains total crude extract;
B, the ethanol water that is 30-90% by the total crude extract water of step a or concentration fully dissolve, then with petroleum ether, chloroform or ethyl acetate extraction 2-7 time, each extractant consumption is 1 with the liquor capacity ratio: 1-4, and extract concentrates and to obtain dry extract;
C, the ethanol water that chloroform-methanol solution or concentration is 40-90% of take are eluent, adopt polyamide, silica gel chromatography or polydextran gel to carry out separation and purification, eluting 6-30 times column volume, collect respectively eluent, by the eluent concentrating under reduced pressure, drying, obtain each component of Caulis Vernoniae andersonii flavone.
Or the total crude extract of step a is separated and obtains the Caulis Vernoniae andersonii flavonoid component with high-speed counter-current chromatograph.
The purposes of the flavonoid component of described Caulis Vernoniae andersonii, the medicine of the flavonoid component of described Caulis Vernoniae andersonii in preparation treatment vitiligo.
Flavonoid component of Caulis Vernoniae andersonii of the present invention and its production and use, wherein the flavonoid component of Caulis Vernoniae andersonii is butin, butein, 7,8,3 ', 4 '-kaempferol, 5,7,8,3 ', 4 '-penta hydroxy group chalcone derivative, 6,8,3 ', 5 '-tetrahydroxy flavanone, glycyrrhizin and isoliquiritigenin, chemical structural formula is respectively formula (1)-Shi (7).
Formula (1) is butin; Formula (2) is butein;
Formula (3) is 6,8,3 ', 5 '-the tetrahydroxy flavanone; Formula (4) is 5,7,8,3 ', 4 '-the penta hydroxy group chalcone derivative;
Formula (5) is 7,8,3 ', 4 '-kaempferol; Formula (6) is isoliquiritigenin;
Formula (7) is glycyrrhizin.
Flavonoid component of Caulis Vernoniae andersonii of the present invention and its production and use, treat leukodermic effective substance in order to inquire into Caulis Vernoniae andersonii, find active component, the present invention adopts relevant pharmacological experiment method to carry out the inside and outside pharmacodynamic experiment, evaluation separates the drug effect of each component of flavone obtained from Caulis Vernoniae andersonii, illustrates that the Caulis Vernoniae andersonii flavonoid component is at the medicinal usage be used for the treatment of aspect vitiligo.
Caulis Vernoniae andersonii flavonoid component of the present invention and the application in preparation treatment vitiligo medicine thereof, after showing that by experiment each flavonoid component of Caulis Vernoniae andersonii is used alone or as a mixture, all can breed by melanophore, raise tyrosinase activity, increase melanin content, promote melanocyte synthetic; Vitiligo animal model skin basal layer melanocyte number, basal layer significantly increase containing the melanin granule cell number, and tyrosinase-related protein is expressed and strengthened.
The specific embodiment
Below with specific embodiment, the present invention is elaborated:
Embodiment 1
A, by Caulis Vernoniae andersonii seed 5kg, be crushed to 20 orders, by the extraction with aqueous solution of 6 times of amounts 3 times, 100 ℃ of temperature, each 3 hours extraction times, merge extractive liquid,, reclaim solvent, concentrated, vacuum drying, obtain dry extract, obtains total crude extract;
B, by total crude extract concentration, be that 30% ethanol water fully dissolves, with petroleum ether extraction 2 times, each extractant consumption with liquor capacity than approximately 1: 4, the concentrated dry extract that to obtain of extract;
C, take chloroform-methanol as eluent, adopt polyamide to carry out separation and purification, 6 times of column volumes of eluting, collect respectively eluent, by the eluent concentrating under reduced pressure, dry, obtain each component of Caulis Vernoniae andersonii flavone, with dry product weight, calculate containing each flavone total content 50.8%, butin content is 20%, butein content is 40.5%, 7, 8, 3 ', 4 '-kaempferol content is 68.5%, 5, 7, 8, 3 ', 4 '-penta hydroxy group chalcone derivative content is 55.4%, 6, 8, 3 ', 5 '-tetrahydroxy flavanone 55.6%, glycyrrhizin content is 80.4%, isoliquiritigenin content is 99.9%.
Embodiment 2
A, by Caulis Vernoniae andersonii seed 5kg, be crushed to 60 orders, with 20% ethanol water of 10 times of amounts, extract 1 time, 80 ℃ of temperature, 3 hours extraction times, merge extractive liquid,, reclaim solvent, concentrated, vacuum drying, obtain dry extract, obtains total crude extract;
B, total crude extract is fully dissolved with aqueous solution, then uses chloroform extraction 7 times, each extractant consumption with liquor capacity than approximately 1: 1, the concentrated dry extract that to obtain of extract;
C, take 40% ethanol water as eluent, adopt silica gel chromatography to carry out separation and purification, 10 times of column volumes of eluting, collect respectively eluent, by the eluent concentrating under reduced pressure, dry, obtain each component of Caulis Vernoniae andersonii flavone, with dry product weight, calculate containing each flavone total content 60.2%, butin content is 67.3%, butein content is 62.5%, 7, 8, 3 ', 4 '-kaempferol content is 70.4%, 5, 7, 8, 3 ', 4 '-penta hydroxy group chalcone derivative content is 60.8%, 6, 8, 3 ', 5 '-tetrahydroxy flavanone 70.0%, glycyrrhizin content is 99.4%, isoliquiritigenin content is 80.2%.
Embodiment 3
A, by Caulis Vernoniae andersonii seed 5kg, be crushed to 5 orders, with 40% ethanol water of 6 times of amounts, extract 2 times, temperature 60 C, each 1 hour extraction time, merge extractive liquid,, reclaim solvent, concentrated, vacuum drying, obtain dry extract, obtains total crude extract;
B, total crude extract is fully dissolved with 70% ethanol water, then is extracted with ethyl acetate 5 times, each extractant consumption with liquor capacity than approximately 1: 3, the concentrated dry extract that to obtain of extract;
C, take 90% ethanol water as eluent, adopt polydextran gel to carry out separation and purification, 25 times of column volumes of eluting, collect respectively eluent, by the eluent concentrating under reduced pressure, dry, obtain each component of Caulis Vernoniae andersonii flavone, with dry product weight, calculate containing each flavone total content 84.4%, butin content is 88.8%, butein content is 30.5%, 7, 8, 3 ', 4 '-kaempferol content is 40.4%, 5, 7, 8, 3 ', 4 '-penta hydroxy group chalcone derivative content is 70.7%, 6, 8, 3 ', 5 '-tetrahydroxy flavanone 82.9.0%, glycyrrhizin content is 90.2%, isoliquiritigenin content is 90.4%.
Embodiment 4
A, by Caulis Vernoniae andersonii seed 5kg, be crushed to 80 orders, with 60% ethanol water of 12 times of amounts, extract 3 times, temperature 60 C, each 2 hours extraction times, merge extractive liquid,, reclaim solvent, concentrated, vacuum drying, obtain dry extract, obtains total crude extract;
B, total crude extract is fully dissolved with 70% ethanol water, then uses petroleum ether extraction 5 times, each extractant consumption with liquor capacity than approximately 1: 3, the concentrated dry extract that to obtain of extract;
C, take 60% ethanol water as eluent, adopt silica gel chromatography to carry out separation and purification, 30 times of column volumes of eluting, collect eluent, by the eluent concentrating under reduced pressure, dry, obtain each component of Caulis Vernoniae andersonii flavone, with dry product weight, calculate containing each flavone total content 90.0%, butin content is 99.9%, butein content is 80.4%, 7, 8, 3 ', 4 '-kaempferol content is 90.4%, 5, 7, 8, 3 ', 4 '-penta hydroxy group chalcone derivative content is 70.7%, 6, 8, 3 ', 5 '-tetrahydroxy flavanone 90.9%, glycyrrhizin content is 89.4%, isoliquiritigenin content is 30.4%.
Embodiment 5
A, by Caulis Vernoniae andersonii seed 5kg, be crushed to 40 orders, by the water extraction of 8 times of amounts 4 times, 80 ℃ of temperature, each 3 hours extraction times, merge extractive liquid,, reclaim solvent, concentrated, vacuum drying, obtain dry extract, obtains total crude extract;
B, total crude extract is separated according to a conventional method with high-speed counter-current chromatograph, can obtain the Caulis Vernoniae andersonii flavonoid component, with dry product weight, calculate containing each flavone total content 99.9%, butin content is 75.4%, butein content is 99.9%, 7,8,3 ', 4 '-kaempferol content is 95.2%, 5,7,8,3 ', 4 '-penta hydroxy group chalcone derivative content is 88.7%, 6,8,3 ', 5 '-tetrahydroxy flavanone 99.9%, glycyrrhizin content are 70.4%, isoliquiritigenin content is 50.2%.
Embodiment 6
A, by Caulis Vernoniae andersonii seed 5kg, be crushed to 80 orders, by the concentration of 8 times of amounts, be that 95% ethanol water extracts 3 times, 80 ℃ of temperature, each 3 hours extraction times, merge extractive liquid,, reclaim solvent, concentrated, vacuum drying, obtain dry extract, obtains total crude extract;
B, be that 60% ethanol water fully dissolves by total crude extract concentration again, then use chloroform extraction 6 times, each extractant consumption with liquor capacity than approximately 1: 2, the concentrated dry extract that to obtain of extract;
C, take 90% ethanol water as eluent, adopt polydextran gel to carry out separation and purification, 15 times of column volumes of eluting, collect respectively eluent, by the eluent concentrating under reduced pressure, dry, obtain each component of Caulis Vernoniae andersonii flavone, with dry product weight, calculate containing each flavone total content 80.0%, butin content is 50.2%, butein content is 80.4%, 7, 8, 3 ', 4 '-kaempferol content is 99.9%, 5, 7, 8, 3 ', 4 '-penta hydroxy group chalcone derivative content is 99.9%, 6, 8, 3 ', 5 '-tetrahydroxy flavanone 70.2%, glycyrrhizin content is 30.4%, isoliquiritigenin content is 66.3%.
Embodiment 7
A, by Caulis Vernoniae andersonii seed 5kg, be crushed to 60 orders, by the concentration of 10 times of amounts, be that 60% ethanol water extracts 3 times, temperature 70 C, each 1 hour extraction time, merge extractive liquid,, reclaim solvent, concentrated, vacuum drying, obtain dry extract, obtains total crude extract;
B, total crude extract is fully dissolved with aqueous solution, then is extracted with ethyl acetate 3 times, each extractant consumption with liquor capacity than approximately 1: 1, the concentrated dry extract that to obtain of extract;
C, take chloroform-methanol solution as eluent, adopt polydextran gel to carry out separation and purification, 20 times of column volumes of eluting, collect respectively eluent, by the eluent concentrating under reduced pressure, dry, obtain each component of Caulis Vernoniae andersonii flavone, with dry product weight, calculate containing each flavone total content 90.4%, butin content is 92.2%, butein content is 92.3%, 7, 8, 3 ', 4 '-kaempferol content is 78.7%, 5, 7, 8, 3 ', 4 '-penta hydroxy group chalcone derivative content is 56.8%, 6, 8, 3 ', 5 '-tetrahydroxy flavanone 54.2%, glycyrrhizin content is 99.9%, isoliquiritigenin content is 90.5%.
Embodiment 8
Butin is on the melanocytic impact of normal person:
Model is set up: normal person's melanocyte
Cell culture processes: get normal children ring cutting foreskin, select and add CT, bFGF, the conventional former culture of the DMEM culture medium of TPA one week, be inoculated in 96 orifice plates, after the cultivation 24h that goes down to posterity, the medicine and the reference substance that add variable concentrations, carry out the determination and analysis index of correlation after 48h.Positive control drug is 8-mop (Tokyo changes into), and concentration is 5 μ g/ml.
The mensuration of cell proliferation is used mtt assay; The mensuration of tyrosinase activity is used the dopa oxidase method; Melanin content NaOH method the results are shown in Table 1:
Table 1 butin on melanocytic impact (
n=6)
[notes] * compares P<0.05 with Normal group; * compares P<0.01 with Normal group.
Result shows that butin can promote melanocyte proliferation, and tyrosinase activity improves, and melanogenesis increases.
The impact of butein on the migration of melanocytes function:
Get transwell, be coated with 50 μ l people fibronectin (FN) (10 μ g/ml at upper surface,), dry 2h in dry incubator, add 600 μ l DMEM culture medium (10%FBS) in 24 well culture plates, pretreated Transwell is put into to hole, is 1 * 10 with DMEM (not containing FBS) dilution after the cell tryptase enzymic digestion that drug treating is crossed
5/ ml, get 200 μ l and add the upper chamber of Transwell cell to put into incubator cultivation 18h, takes out Transwell, wipe upper ventricular cell and FN with cotton swab, PBS washes 2 times, and cell is put into to acetic acid: the fixative of methanol=1: 3 is 30min fixedly, dry, put into the Jim Sa 10min that dyes, PBS washing 3 times, be placed on cell on microscope slide after drying, be placed in observation of cell under microscope, change the different visuals field and take pictures, 8 every group, cell counting.The results are shown in Table 2:
Table 2 butein on the impact of migration of melanocytes function (
n=8)
[notes] * compares P<0.05 with Normal group; * compares P<0.01 with Normal group.
Result shows that butein can significantly promote migration of melanocytes, and facilitation is better than positive drug.
7,8,3 ', 4 '-kaempferol is on the impact of melanocyte adhesive capacity:
Melanocyte is inoculated in to 96 orifice plates, every hole 20 μ l FN (5 μ g/ml), room temperature is dried, and the pretreated cell of medicine is prepared into to 5 * 10
4the cell suspension of/ml, every hole 100 μ l, every group of 6 repeating hole cultivated 1h, suck supernatant, add 10% trichloroacetic acid 50 μ l, 4 ℃ of temperature are 1h fixedly, distilled water flushing 5 times, dry, 2% violet staining 10min, distilled water flushing dries, and every hole adds 2%SDS100 μ l concussion 5min, 570nm measures the OD value, the results are shown in Table 3:
Table 37,8,3 ', 4 '-kaempferol on the impact of melanocyte adhesive capacity (
n=6)
[notes] * compares P<0.05 with Normal group; * compares P<0.01 with Normal group.
Result shows 7,8,3 ', 4 '-kaempferol can significantly strengthen the melanocyte adhesive capacity, and the ability of sticking increases along with the increase of the concentration of medicine, but when concentration reaches 10 μ g/ml, the ability of sticking is consistent with positive drug.
5,7,8,3 ', 4 '-the penta hydroxy group chalcone derivative is on the impact of melanocyte, co-culture system cell:
Screening model: normal person's melanocyte, HaCat cell
Cell culture processes: get normal children ring cutting foreskin, select and add CT, bFGF, the conventional former culture of the DMEM culture medium of TPA one week, the conventional DMEM of HaCat cultivates, co-culture system is with melanocyte: HaCat cell=be inoculated at 1: 3 96 orifice plates, after going down to posterity and cultivating 24h, add medicine and the contrast of variable concentrations, carry out the determination and analysis index of correlation after 48h, positive control drug is 8-mop (Tokyo changes into), and concentration is 5 μ g/ml;
The mensuration of cell proliferation is used mtt assay; The mensuration of tyrosinase activity is used the dopa oxidase method; Melanin content NaOH method the results are shown in Table 4:
Table 45,7,8,3 ', 4 '-the penta hydroxy group chalcone derivative on the impact of melanocyte, co-culture system cell (
n=6)
[notes] * compares P<0.05 with Normal group; * compares P<0.01 with Normal group.
Result shows: 5,7,8,3 ', 4 '-the penta hydroxy group chalcone derivative can make melanocyte proliferation, and tyrosinase activity improves, and melanin content increases; And more obvious with co-culture system; Illustrate 5,7,8,3 ', 4 '-the penta hydroxy group chalcone derivative can make in melanocyte that melanocyte is synthetic be increased by stimulating the relevant Cytokine of keratinocyte secretion.
6,8,3 ', 5 '-the tetrahydroxy flavanone is on melanocytic impact:
Model is set up: normal person's melanocyte
Cell culture processes: get normal children ring cutting foreskin, select and add CT, bFGF, the conventional former culture of the DMEM culture medium of TPA one week, be inoculated in 96 orifice plates, after the cultivation 24h that goes down to posterity, the medicine and the reference substance that add variable concentrations, carry out the determination and analysis index of correlation after 48h.Positive control drug is 8-mop (Tokyo changes into), and concentration is 5 μ g/ml.
The mensuration of cell proliferation is used mtt assay; The mensuration of tyrosinase activity is used the dopa oxidase method; Melanin content NaOH method the results are shown in Table 5:
Table 56,8,3 ', 5 '-the tetrahydroxy flavanone on melanocytic impact (
n=6)
[notes] * compares P<0.05 with Normal group; * compares P<0.01 with Normal group.
Result shows 6,8,3 ', 5 '-the tetrahydroxy flavanone can promote melanocyte proliferation, strengthens tyrosinase activity, promotes melanogenesis.
The impact of glycyrrhizin on the migration of melanocytes function:
Get transwell, be coated with 50 μ l people fibronectin (FN) (10 μ g/ml at upper surface,), dry 2h in dry incubator, add 600 μ l DMEM culture medium (10%FBS) in 24 well culture plates, pretreated Transwell is put into to hole, is 1 * 10 with DMEM (not containing FBS) dilution after the cell tryptase enzymic digestion that drug treating is crossed
5/ ml, get 200 μ l and add the upper chamber of Transwell cell to put into incubator cultivation 18h, takes out Transwell, wipe upper ventricular cell and FN with cotton swab, PBS washes 2 times, and cell is put into to acetic acid: the fixative of methanol=1: 3 is 30min fixedly, dry, put into the Jim Sa 10min that dyes, PBS washing 3 times, after drying, cell is placed on microscope slide, be placed in observation of cell under microscope, change the different visuals field and take pictures, 8 every group, cell counting the results are shown in Table 6:
Table 6 glycyrrhizin on the impact of migration of melanocytes function (
n=8)
[notes] * compares P<0.05 with Normal group; * compares P<0.01 with Normal group.
Result shows: glycyrrhizin can promote migration of melanocytes, improves the melanocyte function.
The impact of isoliquiritigenin on melanocyte, co-culture system cell:
Screening model: normal person's melanocyte, HaCat cell
Cell culture processes: get normal children ring cutting foreskin, select and add CT, bFGF, the conventional former culture of the DMEM culture medium of TPA one week, the conventional DMEM of HaCat cultivates, co-culture system is with melanocyte: HaCat cell=be inoculated at 1: 3 96 orifice plates, after going down to posterity and cultivating 24h, add medicine and the contrast of variable concentrations, carry out the determination and analysis index of correlation after 48h, positive control drug is 8-mop (Tokyo changes into), and concentration is 5 μ g/ml;
The mensuration of cell proliferation is used mtt assay; The mensuration of tyrosinase activity is used the dopa oxidase method; Melanin content NaOH method the results are shown in Table 7:
Table 7 isoliquiritigenin on the impact of melanocyte, co-culture system cell (
n=6)
[notes] * compares P<0.05 with Normal group; * compares P<0.01 with Normal group.
Result shows that isoliquiritigenin is little on melanocyte, the synthetic impact of melanocyte, but can strengthen tyrosinase activity, and significantly promote the co-culture system cell proliferation, melanogenesis significantly increases, tyrosinase activity obviously strengthens, and illustrates that isoliquiritigenin may increase by stimulating the relevant Cytokine of keratinocyte secretion to make melanocyte synthesize in melanocyte.
Embodiment 9
Pharmacodynamic study in the body of butin, butein mixture
Observe butin, butein mixture to black guinea pig skin basal layer melanocyte by gastric infusion, the impact that tyrosinase activity is expressed.
The foundation of animal model
The black guinea pig back is smeared 5% hydroquinone every day, and each 0.5ml, every day 2 times, smear 30 days; The conventional paraffin section of making is observed discovery, black guinea pig skin basal layer melanocyte, and basal layer is containing the melanin granule cell, and tryrosinase is expressed and is weakened.Show the model success.15.2 the butin of various dose, butein mixture are to black guinea pig skin hyperchromic effect
72 of black Cavia porcelluss, be divided into 6 groups at random; Slough back black hair 5cm * 5cm, wherein smear normal saline for the 1st group, all the other 5 groups adopt equivalent 5% hydroquinone decolouring, smear continuously 30 days.
Normal saline 0.5ml is smeared in I group (Normal group) animal unnairing district, every day 2 times, smears continuously 30 days.Then every day gavage normal saline 2ml, continuous 30 days.
5% hydroquinone 0.5ml is smeared in II group (model group) animal unnairing district, every day 2 times, smears continuously 30 days.
Hydroquinone 5%0.5ml is smeared in IV group (positive controls) animal unnairing district, every day 2 times, smears continuously 30 days.Then every other day gavage 8-methoxsalen sheet 0.93mg/ only, continuous 30 days.
Hydroquinone 5%0.5ml is smeared in V group (low dose group) animal unnairing district, every day 2 times, smears continuously 30 days.Then every day gavage butin, butein mixture 0.30mg/ only, continuous 30 days.
Hydroquinone 5%0.5ml is smeared in VI group (middle dosage group) animal unnairing district, every day 2 times, smears continuously 30 days.Then every day gavage butin, butein mixture 0.60mg/ only, continuous 30 days.
Hydroquinone 5%0.5ml is smeared in VII group (high dose group) animal unnairing district, every day 2 times, smears continuously 30 days.Then every day gavage butin, butein mixture 0.90mg/ only, continuous 30 days.
Result treatment:
After administration finishes, put to death animal, the bark fetching skin tissue, the routine paraffin wax embedding, carry out respectively the dyeing of Dopa-oxidase, Lillie dyeing, DAB-H after section
2o
2dyeing and streptomycin avidin-peroxidase connects (S-P) normal dyeing, and under light microscopic, (400 *) are observed.
The melanocytic observation of table sheet:
The dyeing of skin biopsy Dopa-oxidase is placed under light microscopic to be observed, and each specimen is observed 10 visuals field, calculates the par of MC in every 100 epidermal basal cells.The SPSS12.0 software statistics is analyzed, and the results are shown in Table 8:
Table 8 butin, butein mixture on the impact of guinea epidermis melanocyte number (
n=10)
[notes]
△for with model group, comparing P<0.05;
△ △for with model group, comparing P<0.01.
Basal cell counting containing melanin granule:
Skin biopsy Lillie dyeing is placed under light microscopic to be observed, and each specimen is observed 10 visuals field, calculates the par that contains the basal cell of melanin granule in every 100 epidermal basal cells, and the SPSS12.0 software statistics is analyzed, and the results are shown in Table 9:
Table 9 butin, butein mixture on guinea epidermis containing the impact of melanin granule basal cell number (
n=10)
[notes]
△for with model group, comparing P<0.05;
△ △for with model group, comparing P<0.01.
Tryrosinase content is observed:
Adopt the secondary scoring method to be judged tryrosinase positive expression situation, the tryrosinase positive products is positioned in the Cytoplasm of cell, is yellow or brown yellow granule.Positive cell counting<5% is 0 minute; 5%-25% is 1 minute; 25%-50% is 2 minutes; 50%-75% is 3 minutes;>75% is 4 minutes.Press the staining power classification: faint yellow 1 minute; Yellow or deep yellow 2 minutes; Brown or brown color 3 minutes.Both additions are less than 2 minutes negative (-), 2-3 divides positive (+), 4-5 is divided into the medium positive (2+), 6-7 is divided into strong positive (3+), positive rate between each treated animal skin more all adopts the Wilcoxon rank test, P≤0.05, for difference has significant difference, the results are shown in Table 10:
Table 10 butin, butein mixture on the impact of guinea epidermis tryrosinase intensity (
n=10)
[notes] * compares P<0.05 with model group; * compares P<0.01 with model group.
The observation of melanophorin (a-MSH) positive expression number:
Adopt the secondary scoring method to be judged a-MSH positive expression situation, the a-MSH positive products is positioned in melanocyte endochylema and extracellular matrix inner cell matter, with melanocyte endochylema or extracellular matrix under microscope occur the brown yellow granule shape or lumps person positive.Positive cell counting<10% is 0 minute; 11%-40% is 1 minute; 41%-70% is 2 minutes;>71% is 3 minutes.According to the shallow classification of color depth: faint yellow 0 minute; Yellow or deep yellow 1 minute; Brown or brown color 2 minutes; Dark-brown is 3 minutes.Both additions are less than 2 minutes negative (-), and 2-3 is divided into the weak positive (+), and 4-5 divides positive (2+), and 6-7 is divided into strong positive (3+); Positive rate between each treated animal skin more all adopts the Wilcoxon rank test, and P≤0.05, for difference has significant difference, the results are shown in Table 11:
Table 11 butin, butein mixture on the impact of guinea epidermis a-MSH positive expression number (
n=10)
[notes] * compares P<0.05 with model group; * compares P<0.01 with model group.
Interpretation of result:
Show that by above-mentioned experiment butin, butein mixture can strengthen melanocyte synthetic by the activation melanocyte, activity of tyrosinase reaches the effect that pigment recovers.Melanophorin is also had to obvious facilitation, prompting butin, butein mixture may be secreted melanophorin by stimulation and reach melanocyte proliferation and migrate to focal zone simultaneously, thereby cure vitiligo.
Embodiment 10
Glycyrrhizin, isoliquiritigenin mixture are to the leukodermic effectiveness study of hydroquinone decolouring C57BL/6 mouse experiment
Laboratory animal
The C57BL/6 mice, 18-22g, 144, male and female half and half, buy in Yangzhou University's comparative medicine center, the animal quality certification number: SCXK (Soviet Union) 2007-0001.
The reagent dosage
Glycyrrhizin, isoliquiritigenin mixture dosage: low dosage: 30mg/kg; Middle dosage: 60mg/kg; High dose: 120mg/kg.
The dosage of positive drug 8-MOP converts: known according to " methoxsalen sheet description ", the vitiligo adult, calculate by body weight 0.5mg/kg, and the adult is 25mg-30mg each serving consumption, 2-3 time weekly, converts to mice kg body weight dosage:
0.5mg/kg×(0.059/0.1)×(70/0.02)1/3＝4.25mg/kg
Concrete experimental technique:
After buying the C57BL/6 mice, raise the SPF Animal Lab in Nanjing Southeast China University, certified feed and drinking-water are provided, temperature 21-24 ℃, humidity 50-60%, light dark period is 12h/12h (light application time 6:00-18:00), bedding and padding of every 3d replacing and cage box.Animal conforms 3 days, and the 4th, Colophonium/wax mixture was sloughed each mouse back hair 2 * 2cm
2, starting modeling after 24h, the modeling cycle is 50 days, animal grouping and processing method are in Table 14; Since the 21st day, every group of gastric infusion, the dosage setting is in Table 15, and successive administration 30 days, carry out skin conditions and take pictures every day.Experimental session close observation mouse skin change color.
Result:
After treatment finishes, the curative effect of naked eyes gross examination of skeletal muscle to animal model, after the last administration, get each 3 mices of male and female, get the blood sampling of EDTA-2Na anticoagulant tube and measure routine blood test, all the other each animals, pluck eyeball and get blood execution, get 4 ℃ of placement 3h of blood and be placed on refrigerated centrifuger 3500r/min, 4 ℃ of centrifugal 10min, draw each Mus serum, temperature-20 ℃ preservation, cholinesterase activity and mda content in serum to be detected, press the operation of test kit description; After putting to death animal, win each mouse spleen and thymus, carry out the mensuration of immune organ coefficient; Cut medication centre skin 1cm * 1cm, by 10% neutral formalin, fix, paraffin embedding, counted containing melanic hair follicle by normal dyeing after section, detects the expression of respectively organizing m-Tyrosine pheron, tyrosinase related protein-1 by SABC.
Perusal normal group change of skin:
In the modeling process, perusal is visible, it is slow that the tested district of each animal of modeling group skin follicle enters trophophase speed compared with normal group, grow the speed of new hair also the compared with normal group is slow simultaneously, and tested district skin is obviously partially white, after modeling finishes, blank group mouse hair follicles grows into resting stage, and the tested district of each model group skin all grows white hair; After administration finishes, each treatment group white hair and model group relatively, obviously reduce.
The impact on acetylcholine esterase (CHE) vigor in C57BL/6 mice vitiligo model serum of glycyrrhizin, isoliquiritigenin mixture
Get mice serum 50 μ L, press acetylcholine esterase and measure the operation of test kit description, measure cholinesterase activity in microplate reader 520nm place;
As a result, model group CHE vigor is starkly lower than blank group (P<0.01); And each administration group CHE vigor is all apparently higher than model group (P<0.01), in Table 12:
Table 12 glycyrrhizin, isoliquiritigenin mixture on the impact of CHE vigor in C57BL/6 mouse model serum (
n=14)
[notes] * * P<0.01VS model group.
16.4.3 glycyrrhizin, the isoliquiritigenin mixture impact on malonaldehyde (MDA) content in C57BL/6 mice vitiligo model serum
Get mice serum 100 μ l, press the operation of MDA test kit description, by microplate reader, in 532nm place colorimetric, measure MDA content in serum, in serum
carry out the processing of t test statistics with SPSS13.0 software.
As a result, model group MDA content is apparently higher than blank group (P<0.01); And methoxsalen sheet group, in glycyrrhizin and the middle and high dosage group of isoliquiritigenin mixture mice serum, MDA content is starkly lower than model group (P<0.01), in Table 13:
Table 13 glycyrrhizin, isoliquiritigenin mixture on the impact of MDA content in C57BL/6 mouse model serum (
n=14)
[notes] * P<0.05VS model group.
Glycyrrhizin, the impact of isoliquiritigenin mixture on counting containing the melanin hair follicle in C57BL/6 mice vitiligo model skin:
Along the skin of mice midvertebral line clip 1cm * 1cm size, institute's bark fetching skin is fixed to the routine paraffin wax embedded section by 10% neutral formalin, after HE dyeing, carry out skin histology's observation, hair follicle counting, with 50 hair follicles of om observation, calculate melanic hair follicle number is wherein arranged.
Result: blank group skin is containing melanin hair follicle number apparently higher than model group (P<0.01), and each administration group contains melanin hair follicle number also apparently higher than model group (P<0.01), in Table 14:
Table 14 glycyrrhizin, isoliquiritigenin mixture on C57BL/6 mouse model skin containing the impact of melanin hair follicle number (
n=14)
[notes] * P<0.05, * * P<0.01VS model group, +++show more than 90 that melanin is arranged; ++ show more than 1/2 melanin is arranged; + show that 1/3-1/2 has melanin; ± show accidental melanin.
The impact on tyrosinase protein (TYR) in C57BL/6 mouse model skin and tyrosinase related protein-1 (TYR-1) content of glycyrrhizin, isoliquiritigenin mixture:
Adopt streptomycin avidin-peroxidase to connect (SP) normal dyeing method, first antibody is TYR and TRP-1 monoclonal antibody, working concentration is 1: 100, second antibody is biotin labeling, the DAB colour developing, concrete operation step is as follows: each antibody the positive expression of yellow or brown yellow granule or lumps occurs in cell under aobvious mirror.Adopt secondary scoring method result of determination, positive cell counting<5% is 0 minute; 5%-25% is 1 minute; 25%-50% is 2 minutes; 50%-75% is 3 minutes;>75% is 4 minutes; Press the staining power classification: faint yellow 1 minute; Yellow or deep yellow 2 minutes; Brown or brown color 3 minutes; Both additions are less than 2 minutes negative (-); 2-3 divides positive (+); 4-5 is divided into the medium positive (2+); 6-7 is divided into strong positive (3+); Positive rate between each treated animal skin more all adopts the Wilcoxon rank test, and there is significance P≤0.05 for difference; Result: model group animal TYR and TYR-1 obviously reduce than the blank group, and each administration group and model group relatively, obviously increase, in Table 15, and table 16:
The impact that table 15 glycyrrhizin, isoliquiritigenin mixture are expressed tyrosinase protein in C57BL/6 mouse model skin (
n=14)
[notes] * P<0.05, * * P<0.01VS model group
The impact that table 16 glycyrrhizin, isoliquiritigenin mixture are expressed tyrosinase related protein-1 in C57BL/6 mouse model skin (
n=14)
[notes] * P<0.05, * * P<0.01VS model group.
Brief summary and discussion:
Each treated animal of perusal, find that the tested district of modeling group mice skin follicle enters trophophase speed slow than the blank group, and the speed that grows new hair is also slack-off than the blank group, and tested district skin is obviously partially white.After modeling finishes, blank group mouse hair follicles grows into resting stage, and the tested district of each model group skin all grows white hair, and after administration finishes, each treatment group white hair and model group relatively, obviously reduce.
(CHE) vigor of acetylcholine esterase in serum and malonaldehyde (MDA) content, the model group cholinesterase activity is starkly lower than the blank group, and in each administration treated animal serum, the CHE vigor is significantly higher than model group, the model group mda content is apparently higher than the blank group, and the middle and high dosage group of methoxsalen sheet group and mixture all can significantly reduce mda content.
Be starkly lower than the blank group containing melanin hair follicle number in the model group animal skin, and each administration group obviously can promote increasing containing the melanin hair follicle.
Model group animal tryrosinase and tyrosinase related protein-1 expression intensity are starkly lower than the blank group, and each administration group and model group relatively, obviously increase.
By above experimental result, shown: adopt the hydroquinone decoloring method to copy C57BL/6 mice vitiligo animal model, also can simulate the indices of clinical observation; The comparison of each administration group and model group, glycyrrhizin and isoliquiritigenin mixture all can improve the indices that modeling changes to some extent, show to play therapeutical effect.
Claims (2)
1. the flavone composition of a Caulis Vernoniae andersonii, is characterized in that this flavonoid component is butin, butein, 7,8,3 ', 4 '-kaempferol, 5,7,8,3 ', 4 '-penta hydroxy group chalcone derivative, 6,8,3 ', 5 '-tetrahydroxy flavanone, glycyrrhizin and isoliquiritigenin, wherein the flavonoid component total content is 50-99.9%, each flavonoid component content is 20-99.9%, and this flavonoid component is that concrete operations follow these steps to carry out in dimension medicine Caulis Vernoniae andersonii, carrying out separation and purification:
A, the Caulis Vernoniae andersonii seed meal is broken to the 5-80 order, the solvent of doubly measuring with 6-12 is that the ethanol water that water or concentration are 20-95% extracts 1-4 time, temperature 40-100 ℃, each extraction time 1-3 hour, merge extractive liquid,, reclaim solvent, concentrated, vacuum drying, obtain dry extract, obtains total crude extract;
B, the ethanol water that is 30-90% by the total crude extract water of step a or concentration fully dissolve, then with petroleum ether, chloroform or ethyl acetate extraction 2-7 time, each extractant consumption is with liquor capacity than being 1:1-4, and extract concentrates and to obtain dry extract;
C, the ethanol water that chloroform-methanol solution or concentration is 40-90% of take are eluent, adopt polyamide, silica gel chromatography or polydextran gel to carry out separation and purification, eluting 6-30 times column volume, collect respectively eluent, by the eluent concentrating under reduced pressure, drying, obtain each component of Caulis Vernoniae andersonii flavone.
2. the purposes of the flavone composition of Caulis Vernoniae andersonii as claimed in claim 1, is characterized in that the flavonoid component of described Caulis Vernoniae andersonii is treated the medicine in vitiligo in preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210045334 CN102526153B (en) | 2012-02-27 | 2012-02-27 | Vernonia anthelmintica flavone components, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210045334 CN102526153B (en) | 2012-02-27 | 2012-02-27 | Vernonia anthelmintica flavone components, preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102526153A CN102526153A (en) | 2012-07-04 |
| CN102526153B true CN102526153B (en) | 2013-12-25 |
Family
ID=46334974
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210045334 Expired - Fee Related CN102526153B (en) | 2012-02-27 | 2012-02-27 | Vernonia anthelmintica flavone components, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102526153B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103342653B (en) * | 2013-07-30 | 2015-04-01 | 中国科学院新疆理化技术研究所 | Preparation method and application of chalcone amide derivatives |
| CN104825519B (en) * | 2015-05-29 | 2019-10-29 | 中国科学院新疆理化技术研究所 | The Preparation method and use of vernonia anthelmintica phenolic acid part |
| CN105837540B (en) * | 2016-03-18 | 2018-06-15 | 中国林业科学研究院林产化学工业研究所 | Method of the extraction with the sulphuretin and butein for inhibiting tyrosinase activity from lacquer tree wood powder |
| CN105801509B (en) * | 2016-05-03 | 2018-01-05 | 中国科学院新疆理化技术研究所 | The preparation method and purposes of a kind of chalcone derivative |
| CN106421717A (en) * | 2016-11-26 | 2017-02-22 | 新疆维吾尔自治区维吾尔医药研究所 | Uighur medicine compound effective parts and effective part group for treating leucoderma and preparation method thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101559050A (en) * | 2004-11-23 | 2009-10-21 | 尚靖 | Use of butin and derivatives thereof for preparing drugs having inhibitive effect on immune system |
| CN1778324A (en) * | 2004-11-26 | 2006-05-31 | 乌鲁木齐瑞普药物研究开发有限公司 | Effective part of nightshadeleaf ironweed for antiscolic medicines and its preparation and use |
| CN101721445B (en) * | 2009-12-28 | 2011-06-01 | 中国科学院新疆理化技术研究所 | Preparation method of vernonia anthelmintica compound silver nanometer colloid |
| CN101962376A (en) * | 2010-10-19 | 2011-02-02 | 南京荣世医药科技有限公司 | Method for preparing vernodalol |
-
2012
- 2012-02-27 CN CN 201210045334 patent/CN102526153B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN102526153A (en) | 2012-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102526153B (en) | Vernonia anthelmintica flavone components, preparation method and application thereof | |
| CN103893345B (en) | Composition containing a natural extract | |
| CN102038765B (en) | Qingpeng plaster for relieving pain and eliminating swelling and preparation method thereof | |
| CN103222988B (en) | A kind of American-cockroach-extract and its preparation method and application | |
| CN101810812B (en) | Use of rhizoma galangae effect part for preparing medicament for treating leucoderma | |
| CN109662904A (en) | It is a kind of to have effects that the skin care compositions of whitening antioxidation and its application and preparation method | |
| CN102988264A (en) | Ginkgo leaf freckle removing whitening cream and preparation method thereof | |
| CN108530430B (en) | Ester type catechin pyrrolidine alkaloid and preparation method and application thereof | |
| CN101352464B (en) | Quality control method of Chinese medicinal composition | |
| CN102846499A (en) | Composition having effects of whitening, removing spots and refreshing skin | |
| CN105012329B (en) | It is a kind of for treating the drug of type-II diabetes | |
| CN102258598A (en) | Medicine prepared by active part of pawpaw, its preparation method and its application | |
| CN100453063C (en) | Frost-like powder possessing effects of beautification and nourishing face | |
| CN109364073A (en) | The purposes in drug that a kind of piperlongumine alkaloid promotes melanin production in preparation treatment leucoderma | |
| CN103301110B (en) | Application for galangin derivatives in preparation of medicines for preventing and treating vitiligo | |
| KR101026873B1 (en) | Mixed medicinal herb extracts having anti-inflammatory activity and cosmetic composition containing the same | |
| CN103585196A (en) | Application of silvery aleuritopteris herb extract in preparation of anticancer drugs | |
| CN103230003B (en) | Health food with immunity boosting function and preparation method thereof | |
| KR101503583B1 (en) | Compositions for anti-obesity comprising extract of Vitis amurensis ruprecht | |
| KR101998685B1 (en) | A cosmetic composition comprising arenaria serpyllifolia extract | |
| KR20150024502A (en) | A cosmetic composition for preventing and improving skin winkle | |
| CN103083523B (en) | Traditional Chinese medicine composition for treating chronic prostatitis and preparation method of composition | |
| CN103739657B (en) | A kind of Sasanguasaponin compound, its preparation method, the antitumor drug applying and prepare | |
| CN106421717A (en) | Uighur medicine compound effective parts and effective part group for treating leucoderma and preparation method thereof | |
| CN207445232U (en) | For maintaining the epidermal stem cells of skin and there is company's neck facial mask of multiple functions |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131225 Termination date: 20200227 |