CN104825519A - Preparation method and application of vernonia anthelmintica phenolic acid part - Google Patents
Preparation method and application of vernonia anthelmintica phenolic acid part Download PDFInfo
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Abstract
The invention relates to a preparation method and an application of a vernonia anthelmintica phenolic acid part. The preparation method comprises the following steps of smashing dry vernonia anthelmintica fruits; extracting the drying vernonia anthelmintica fruits by using organic solvent; and performing resin separation and purification to obtain vernonia anthelmintica extracts which mainly comprise a phenolic acid compound. The content of phenolic acid in the vernonia anthelmintica phenolic acid part is 20%-99% in percentage by mass. The vernonia anthelmintica phenolic acid part also comprises the following main active ingredients in percentage by mass: 3-26% of 3, 4-dicaffeoyl-quinic acid, 5-34% of 3, 5-dicaffeoyl-quinic acid and 5-40% of 4, 5-dicaffeoyl-quinic acid. A preliminary experiment proves that the vernonia anthelmintica phenolic acid part obtained by the method has effects of increasing melanocyte in hair follicle and promoting melanogenesis and can be used for treating leukoderma. The preparation method is simple in technology and low in production cost; effective ingredients are stable; and the quality is easy to control. By the preparation method and the application of the vernonia anthelmintica phenolic acid part, a novel medicinal part and compounds are provided for treatment of the leukoderma.
Description
Technical field
The present invention relates to a kind of preparation method of Caulis Vernoniae andersonii phenolic acid part, and the purposes of leucoderma medicament, belong to national medicine technical field.
Background technology
Vitiligo (vitiligo, OMIM:#193200) is a kind of common posteriority depigmentation disease, and principal character is the depigmentation that skin and hair melanocyte selective destruction cause.Vitiligo has larger difference between different geographical and race, and the darker crowd of the colour of skin is more easily ill, and the leukodermic prevalence of China is 0.09%-2.7%.
Leukodermic infringement can betide the skin at any position of whole body, mucosa and hair, but exposure portion, and the position that easily rubs, all more easily occur.Skin lesion is symmetric more, or distributes along nerve segment.The complete depigmentation speckle of pigment is milky, clear border, and size and form is different, and the hair being grown on white macula district also can bleach, and severe patient white macula can general whole body.Vitiligo histopathologic characteristics is the melanocytic minimizing in skin lesion district or disappearance, melanin granule disappearance in cell, in early days can at the inflammatory cell infiltration of skin lesion periphery discovery based on lymphocyte.
Vitiligo pathogenesis is failed to understand, there is multiple pathogenesis hypothesis at present, but any one hypothesis has it to limit to, and dissimilar vitiligo pathogeny may be different.The leukodermic cause of disease and pathogenesis mainly contain autoimmune hypothesis, hereditary hypothesis, melanocyte autoclasia hypothesis etc.
Caulis Vernoniae andersonii, Vernonia anthelmintica (L.) Willd, another name India mountain Fructus Foeniculi is the annual tall and big draft of Compositae (Asteraceae) Vernonia (Vernonia), medicinal part is mellow fruit, and autumn gathers, dry.Its property three grades is xeothermic, and effect mainly removes abnormal phlegm, anthelmintic, detumescence, dispersing cold for relieving pain, is the leukodermic main classical medicine of successive dynasties tradition Uygur medicine treatment.
Contain Sesquiterpene lactones, flavonoid in Caulis Vernoniae andersonii fruit, draw together epoxy octadecenoic acid and the multiple compounds such as glyceride type, triterpenes and sterols and caffeoyl quinic acid class thereof.
Caulis Vernoniae andersonii is with a long history in medication among the people, and have clinical practice steady in a long-term, therefore its development prospect is wide.In uighur medicine theory, Caulis Vernoniae andersonii three grades is xeothermic, and effect mainly removes abnormal phlegm, anthelmintic, detumescence, dispersing cold for relieving pain.Caulis Vernoniae andersonii seed is that the marketed products of principal agent and preparation have insect-expelling saligna injection liquid, card power Fructus Cumini Cymini honey cream, compound Vernonia anthelmintica ball, compound kaliziranding, compound recipe card power Fructus Cumini Cymini sheet, drive Bai Babusi sheet etc.Flavone compound and flavone site treatment vitiligo have been reported, but phenolic acid compound and phenolic acid part treatment vitiligo have no report.
The extract of current Caulis Vernoniae andersonii, flavone position and flavone compound for treating vitiligo have more report, but phenolic acid part and phenolic acid compound have no report.
Summary of the invention
The object of the invention is, provides a kind of preparation method and purposes of Caulis Vernoniae andersonii phenolic acid part.The method is pulverized by the Caulis Vernoniae andersonii fruit of drying, adopts organic solvent extraction, and resin separation purification obtains vernonia anthelmintica willd extract, and main component is the phenolic acid compound such as caffeoyl quinic acid, dicaffeoyl-quinic acid.Measure the mass percent 20%-99% of phenolic content.In Caulis Vernoniae andersonii phenolic acid part, the content of main active 3,4-dicaffeoyl-quinic acid, 3,5-dicaffeoyl-quinic acid and 4,5-dicaffeoyl-quinic acid is respectively 3-26%, 5-34% and 5-40%.Prove through preliminary experiment: the Caulis Vernoniae andersonii phenolic acid part obtained by method of the present invention has the effect increasing melanocyte in hair follicle, promote melanin genesis, can be used for treating vitiligo.Method technique of the present invention is simple, and production cost is low, stable effective ingredients, is easy to quality control.The present invention is that therapy of vitiligo provides new medicinal part and compound.
The preparation method of a kind of Caulis Vernoniae andersonii phenolic acid part of the present invention, follows these steps to carry out:
A. after the Caulis Vernoniae andersonii fruit of drying being pulverized, be 0-95% methanol, ethanol or aqueous solution by concentration, room temperature-95 DEG C is extracted 1-5 time, extraction time 0.5-3 hour, is cooled to room temperature, filters, merge extractive liquid, be evaporated to without alcohol taste, obtain ethanol extract, the solid-to-liquid ratio of Caulis Vernoniae andersonii fruit and methanol or ethanol water is 1:3-30;
B. the extract concentration obtained by step a is after the dispersion of 0-15% methanol, ethanol or aqueous dissolution; filter, then use resin isolation, first wash decontamination with water; be 10-95% methanol or ethanol elution by concentration again; collect methanol or ethanol elution, concentrating under reduced pressure eliminates solvent, dry; namely obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Resin described in step b is DM-130, AB-8, HPD-450, NKA-9, HPD-100, D101, HPD-400, HPD-500, HPD-600, HPD-300, HPD-417, polyamide or octadecylsilane chemically bonded silica.
Containing phenolic acid compound in the Caulis Vernoniae andersonii phenolic acid part that described method obtains is mass percent 20%-99%.
In the Caulis Vernoniae andersonii phenolic acid part that described method obtains, the content of main active 3,4-dicaffeoyl-quinic acid, 3,5-dicaffeoyl-quinic acid and 4,5-dicaffeoyl-quinic acid is respectively 3-26%, 5-34% and 5-40%.
The purposes of the Caulis Vernoniae andersonii phenolic acid part that described method obtains in the leukodermic medicine of preparation control.
The preparation method of Caulis Vernoniae andersonii phenolic acid part of the present invention and purposes, the Caulis Vernoniae andersonii phenolic acid part obtained by the method is proved through pharmacodynamics test, there is the leukodermic effect of significant treatment, and under identical clinical equivalent dosage, the effect of Caulis Vernoniae andersonii phenolic acid part is better than vitiligo capsule.
Accompanying drawing explanation
Fig. 1 is that skin tissue morphology's variation diagram is observed in HE of the present invention dyeing;
Fig. 2 is that phenolic acid part of the present invention is on impact (melanin dye, the 200 times) figure of vitiligo mouse skin containing melanin hair follicle number.
Detailed description of the invention
Embodiment 1: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii fruit medical material 10kg of drying, add the aqueous solution of 30 times of volumes, heating and refluxing extraction 3 times, temperature 95 DEG C, 3 hours time, be cooled to room temperature, filter, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 5% ethanol is dispersed to containing crude drug 100g/L by the ethanol extract concentration obtained; filter, then use AB-8 type macroporous adsorptive resins, first use 1 times of column volume water elution; be 40% ethanol elution by 8 times of column volume concentration again; collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 390g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 2: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii fruit medical material 10kg of drying, adding 3 times of volumetric concentrations is 95% ethanol, heating and refluxing extraction 5 times, and room temperature, is cooled to room temperature at 3 hours time, and filter, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 5% ethanol is dispersed to containing crude drug 50g/L by the ethanol extract concentration obtained; centrifugal (4000 revs/min), DM-130 type macroporous adsorptive resins, first uses 3 times of column volume water elutions; be 95% ethanol elution by 1 times of column volume concentration again; collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 420g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 3: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii fruit medical material 10kg of drying, adding 15 times of volumetric concentrations is 30% ethanol, heating and refluxing extraction 3 times, temperature 75 DEG C, and time 2 h, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 10% methanol is dispersed to containing crude drug 80g/L by the extract concentration obtained; centrifugal (4000 revs/min), HPD-100 type macroporous adsorptive resins, first uses 4 times of column volume water elutions; be 50% ethanol elution by 6 times of column volume concentration again; collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 440g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 4: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii fruit medical material 10kg of drying, the concentration adding 18 times of volumes is 60% methanol, heating and refluxing extraction 2 times, and temperature 50 C 0.5 hour time, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 15% methanol is dispersed to containing crude drug 100g/L by the extract concentration obtained; centrifugal (4000 revs/min), NKA-9 type macroporous adsorptive resins, first uses 5 times of column volume water elutions; be 60% methanol-eluted fractions by 5 times of column volume concentration again; collect meoh eluate, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 460g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 5: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii fruit medical material 10kg of drying, adding 12 times of volumetric concentrations is that 10% alcohol heating reflux extracts 2 times, temperature 80 DEG C, and time 2 h, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 10% ethanol is dispersed to containing crude drug 30g/L by the extract concentration obtained; filter, then use D101 type macroporous adsorptive resins, first use 8 times of column volume water elutions; be 80% ethanol elution by 6 times of column volume concentration again; collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 564g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 6: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii fruit medical material 10kg of drying, adding 18 times of volumetric concentrations is 95% ethanol, heating and refluxing extraction 2 times, and temperature 60 C 1 hour time, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
By the extract aqueous dispersion obtained to containing crude drug 50g/mL; centrifugal (4000 revs/min), with HPD400 type macroporous adsorptive resins, first use 6 times of column volume water elutions; be 65% methanol-eluted fractions by 6 times of column volume concentration again; collect meoh eluate, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 490g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 7: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii medicine fruit material 10kg of drying, adding 30 times of volumetric concentrations is 95% methanol, heating and refluxing extraction 2 times, temperature 65 DEG C, and time 2 h, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 10% ethanol is dispersed to containing crude drug 60g/mL by the extract concentration obtained; centrifugal (4000 revs/min), HPD600 type macroporous adsorptive resins, first uses 3 times of column volume water elutions; be 95% ethanol elution by 8 times of column volume concentration again; collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 524g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 8: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii medicine fruit material 10kg of drying, adding 15 times of volumetric concentrations is 95% methanol, heating and refluxing extraction 2 times, temperature 55 DEG C, and time 2 h, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 10% ethanol is dispersed to containing crude drug 80g/mL by the extract concentration obtained; centrifugal (4000 revs/min), HPD500 type macroporous adsorptive resins, first uses 3 times of column volume water elutions; be 75% ethanol elution by 6 times of column volume concentration again; collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 498g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 9: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii medicine fruit material 10kg of drying, adding 20 times of volumetric concentrations is 75% methanol, heating and refluxing extraction 2 times, temperature 60 C, and time 2 h, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 15% ethanol is dispersed to containing crude drug 60g/mL by the extract concentration obtained; centrifugal (4000 revs/min), HPD450 type macroporous adsorptive resins, first uses 1 times of column volume water elution; be 75% ethanol elution by 4 times of column volume concentration again; collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 433g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 10: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii medicine fruit material 10kg of drying, adding 30 times of volumetric concentrations is 55% methanol, heating and refluxing extraction 2 times, and temperature 70 C 3 hours time, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 10% ethanol is dispersed to containing crude drug 100g/mL by the extract concentration obtained; centrifugal (4000 revs/min), HPD417 type macroporous adsorptive resins, first uses 1 times of column volume water elution; be 85% ethanol elution by 8 times of column volume concentration again; collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 603g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 11: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii medicine fruit material 10kg of drying, adding 30 times of volumetric concentrations is 45% methanol, heating and refluxing extraction 2 times, and temperature 55 DEG C 1 hour time, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 15% ethanol is dispersed to containing crude drug 90g/mL by the extract concentration obtained; centrifugal (4000 revs/min), HPD300 type macroporous adsorptive resins, first uses 2 times of column volume water elutions; be 65% ethanol elution by 6 times of column volume concentration again; collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 491g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 12: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii medicine fruit material 10kg of drying, adding 30 times of volumetric concentrations is 95% methanol, heating and refluxing extraction 2 times, temperature 45 C, and time 2 h, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Be that 10% ethanol is dispersed to containing crude drug 90g/mL by the extract concentration obtained; centrifugal (4000 revs/min), polyamide resin column, first uses 1 times of column volume water elution; be 85% ethanol elution by 8 times of column volume concentration again; collect ethanol elution, concentrating under reduced pressure, vacuum drying becomes dry powder; obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part 586g of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
Embodiment 13: the preparation of Caulis Vernoniae andersonii phenolic acid part:
Pulverized by the Caulis Vernoniae andersonii medicine fruit material 10kg of drying, adding 30 times of volumetric concentrations is 55% methanol, heating and refluxing extraction 2 times, temperature 65 DEG C, and time 2 h, is cooled to room temperature, merge extractive liquid, is evaporated to without alcohol taste, obtains ethanol extract;
Adopt octadecylsilane chemically bonded silica, by preparation liquid phase carry out purification repeatedly, respectively obtain purity be 3, the 4-dicaffeoyl-quinic acid of 99%, 3,5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid;
Take respectively 2.6 milligram of 3,4-dicaffeoyl-quinic acid, 3.4 milligram of 3,5-dicaffeoyl-quinic acid and 4 milligram of 4,5-dicaffeoyl-quinic acid mix homogeneously, namely obtain the Caulis Vernoniae andersonii phenolic acid part that total phenolics content is 99%.
Embodiment 14: the component analysis of Caulis Vernoniae andersonii phenolic acid part:
Carry out assay with spectrophotography to obtained Caulis Vernoniae andersonii phenolic acid part, result shows: in Caulis Vernoniae andersonii phenolic acid part, total phenolics content mass percent is 20%-99%.Employing liquid-phase chromatographic analysis shows: in Caulis Vernoniae andersonii medicine fruit material raw material 3, 4-dicaffeoyl-quinic acid, 3, 5-dicaffeoyl-quinic acid and 4, 5-dicaffeoyl-quinic acid content mass percent is respectively 0.3-3%, 0.5-3%, 0.6-3%, the Caulis Vernoniae andersonii phenolic acid part 3 obtained by the method for the invention, 4-dicaffeoyl-quinic acid, 3, 5-dicaffeoyl-quinic acid and 4, in 5-dicaffeoyl-quinic acid, content mass percent is respectively 3-26%, 5-34%, 5-40%, result shows: the method for the invention can significantly improve 3, 4-dicaffeoyl-quinic acid, 3, 5-dicaffeoyl-quinic acid and 4, the content of 5-dicaffeoyl-quinic acid.
Embodiment 15: the anxious toxicity assessment of Caulis Vernoniae andersonii phenolic acid part:
20 cleaning grade kunming mouses, in 5 week age, weight 20g, equilibrium is divided into 2 groups (administration group and matched groups) at random, often organizes 10, male and female half and half.After water 16h is can't help in fasting, administration group mouse stomach gives the phenolic acid part of concentration 0.6g/mL, and administration volume is 40mL/kg, gastric infusion 1 time.Naive mice gavage equivalent distilled water.Continuous Observation in 4h after administration, later every morning observes 1 time, Continuous Observation 14 days.Experimental session measures Mouse Weight every day, and equal no difference of science of statistics before and after administration group and the administration of matched group body weight, the results are shown in Table 1; Observe the outward appearance of animal, diet, behavioral activity, secretions, Excreta etc. all without significantly abnormal simultaneously; Duration of test has no dead mouse; After off-test, mice is sacrificed by decapitation respectively, dissects, and each internal organs color is normal, organizes no abnormality seen, the results are shown in Table 2; The maximum dosage-feeding (MLD) calculating phenolic acid part is: 24g/kg/d (being equivalent to crude drug amount 564.7g crude drug/kg), is 6776.67 times of clinical adult's dosage.The basic avirulence of phenolic acid part is described, the safety of clinical application is large;
Table 1 phenolic acid part acute toxicity test Mice Body mass change (g,
n=10)
Table 2 phenolic acid part is to acute toxicity test in mice result
Embodiment 16: Caulis Vernoniae andersonii phenolic acid part is to the effect of vitiligo model mice:
C57BL/6 mice 70, body weight 18-22g, male and female half-and-half, shave off back hair with shaver, area is about 2cm × 2cm, and equilibrium is divided into 6 groups at random: blank group, model group, positive drug (vitiligo capsule) matched group, the basic, normal, high dosage group of extract, often organizes 12.Model group and each administration group smear the hydroquinone of 5% in district of losing hair or feathers, each 0.5mL, every day 2 times, and blank group smears equivalent distilled water in depilation district; Continuous 50 days, set up vitiligo mice model, during this period, each group mice is tested skin zone leveling depilation in every 3 days 1 time; Hydroquinone modeling is after 20 days, and decolouring phenomenon appears in mouse skin and hair; From modeling the 21st day, each group is smearing 5% hydroquinone after 1 hour respectively, by setting dosage, starts gastric infusion: blank group, model group gavage gives equal-volume distilled water, and administration group gavage gives test medicine, every day 1 time, successive administration 30 days, after experiment terminates, puts to death mice.
1. the leukodermic curative effect of perusal Drug therapy:
Experimental result shows, and in modeling process, respectively group modeling mice tested district skin color is pale as seen for naked eyes, and hair growth is slow, with modeling time lengthening, occurs white macula, and new piliation becomes white; After treatment, naked eyes are visible, and vitiligo capsule group and phenolic acid part each dosage group can recovery vitiligo mice tested district skin colors in various degree, and new piliation also shows and reverts to black.Under identical clinical equivalent dosage, the heavy dose of group of phenolic acid part is better than vitiligo capsule group, the results are shown in Table 3;
Table 3 phenolic acid part is to the observation of curative effect of vitiligo mice caused by hydroquinone
Note: compare with model group,
*p<0.05.
2. histological observation
Skin tissue morphology's change is observed in 2.1 HE dyeing:
Blank group: mouse skin is methodically arranged, hair follicle structure is complete, and number is many, and pigment content is many and color is dark;
Vitiligo model group: mouse skin is obviously thinning, and horny layer slightly thickens, the visible edema of prickle cell layer, hair follicle quantity is few, and structure is imperfect, sebaceous gland disintegrate, hair shaft depigmentation.Subcutaneous tissue has no hair follicle distribution;
Vitiligo capsule group: mouse skin caliper recovery is normal, and keratodermatitis has no and obviously thickens, and epidermis and corium are without edema, and basal layer cell has pigmentation, and hair follicle increases, structural integrity, pigment content increases, and color and luster is deepened;
Phenolic acid part low dose group: mouse skin is obviously thinning, part cuticle thickening, epidermis cell Mild edema, the visible edema of prickle cell layer, skin corium and the visible a small amount of hair follicle distribution of subcutaneous tissue;
Dosage group in phenolic acid part: mouse skin caliper recovery is normal, and epidermis and corium have no obvious edema, and basal layer cell has pigmentation, and hair follicle structure is complete, and its number and pigment content obviously increase, skin corium and the visible hair follicle distribution of subcutaneous tissue;
Phenolic acid part high dose group: mouse skin caliper recovery is normal, and keratodermatitis is thinning, and epidermis and corium are without obvious edema, basal layer cell has pigmentation, and hair follicle structure is complete, and number obviously increases, pigmentary colours are obviously deepened, and skin corium and the visible hair follicle distribution of subcutaneous tissue, be shown in Fig. 1.
Dermal melanin distribution situation is observed in 2.2 melanin dyeing:
Model group mice, compare with blank group, not only hair follicle is few, and most of hair follicle is hardly containing melanin; Each administration group mice, compare with model group, hair follicle increases, and wherein phenolic acid part middle and high dosage group melanin obviously increases.Under identical clinical equivalent dosage, the effect of Caulis Vernoniae andersonii phenolic acid part is better than vitiligo capsule, the results are shown in Table 4, Fig. 2;
Table 4 phenolic acid part causes the impact of vitiligo mice skin containing melanin hair follicle number to hydroquinone
Note: compare with blank group,
#p<0.05; Compare with model group,
*p<0.05.
3. mice serum and skin histology tryrosinase assay:
Experimental result shows, model group mice, compares with blank group, and in serum, the content of TYR significantly increases, and in skin histology, the content of TYR significantly reduces; The each dosage group of phenolic acid part obviously can reduce the content (P<0.05) of TYR in vitiligo mice serum, and content (P<0.05) effect increasing TYR in vitiligo mice skin histology is better than vitiligo capsule, the results are shown in Table 5;
Table 5 phenolic acid part is on the impact of vitiligo mice serum caused by hydroquinone and skin TYR
Note: compare with blank group,
#p<0.05; Compare with model group,
*p<0.05.
After the modeling of C57 mice hydroquinone, there is white macula in model group mouse skin; The visible spongiosis of histological observation, hair shaft depigmentation, reduces containing melanin hair follicle; In skin histology, hair follicle structure is imperfect, sebaceous gland disintegrate; In serum, TYR content significantly increases, and in skin histology, TYR content significantly reduces, and shows melanocyte in hydroquinone damage model group mouse hair follicles organizational structure and hair follicle.
Compare model group, phenolic acid part effect group can improve the impact of hydroquinone modeling on C57 mice, recovers vitiligo model mice tested district skin color; Histological observation is without obvious edema, and hair shaft depigmentation, recovers normal range containing melanin hair follicle quantity; In skin histology, hair follicle structure is complete, and number obviously increases; In serum, TYR content recovers normal range, and in skin histology, the content of TYR significantly increases.Phenolic acid part can increase melanogenesis protein content in skin histology, increases containing melanin hair follicle quantity, better recovers mice tested district skin color.
In sum, Caulis Vernoniae andersonii phenolic acid part can increase melanocyte quantity in vitiligo mice hair follicle, promote melanin genesis, can be used for preparing the purposes in the leukodermic medicine for the treatment of.
Claims (5)
1. a preparation method for Caulis Vernoniae andersonii phenolic acid part, is characterized in that following these steps to carry out:
A. after the Caulis Vernoniae andersonii fruit of drying being pulverized, be 0-95% methanol, ethanol or aqueous solution by concentration, room temperature-95 DEG C is extracted 1-5 time, extraction time 0.5-3 hour, is cooled to room temperature, filters, merge extractive liquid, be evaporated to without alcohol taste, obtain ethanol extract, the solid-to-liquid ratio of Caulis Vernoniae andersonii fruit and methanol or ethanol water is 1:3-30;
B. the extract concentration obtained by step a is after the dispersion of 0-15% methanol, ethanol or aqueous dissolution; filter, then use resin isolation, first wash decontamination with water; be 10-95% methanol or ethanol elution by concentration again; collect methanol or ethanol elution, concentrating under reduced pressure eliminates solvent, dry; namely obtain containing 3; the Caulis Vernoniae andersonii phenolic acid part of 5-dicaffeoyl-quinic acid, 4,5-dicaffeoyl-quinic acid and 3,4-dicaffeoyl-quinic acid.
2. the preparation method of Caulis Vernoniae andersonii phenolic acid part according to claim 1, is characterized in that the resin described in step b is DM-130, AB-8, HPD-450, NKA-9, HPD-100, D101, HPD-400, HPD-500, HPD-600, HPD-300, HPD-417, polyamide or octadecylsilane chemically bonded silica.
3. containing phenolic acid compound in the Caulis Vernoniae andersonii phenolic acid part that method obtains according to claim 1 is mass percent 20%-99%.
4. the content of main active 3,4-dicaffeoyl-quinic acid, 3,5-dicaffeoyl-quinic acid and 4,5-dicaffeoyl-quinic acid is respectively 3-26%, 5-34% and 5-40% in the Caulis Vernoniae andersonii phenolic acid part that obtains of method according to claim 1.
5. the purposes of Caulis Vernoniae andersonii phenolic acid part in the leukodermic medicine of preparation control of the method for claim 1 acquisition.
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| CN106083882A (en) * | 2016-06-04 | 2016-11-09 | 中国科学院新疆理化技术研究所 | Sesquiterpene dimers compounds in Caulis Vernoniae andersonii and preparation method and purposes |
| 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 |
| CN114350527A (en) * | 2022-01-17 | 2022-04-15 | 中国科学院新疆理化技术研究所 | Separation and purification method of endophytic fungi in overground part tissue of Vernonia anthelmintica and application of endophytic fungi |
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| CN106083882A (en) * | 2016-06-04 | 2016-11-09 | 中国科学院新疆理化技术研究所 | Sesquiterpene dimers compounds in Caulis Vernoniae andersonii and preparation method and purposes |
| 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 |
| CN114350527A (en) * | 2022-01-17 | 2022-04-15 | 中国科学院新疆理化技术研究所 | Separation and purification method of endophytic fungi in overground part tissue of Vernonia anthelmintica and application of endophytic fungi |
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