CN101664426A - Application of rhizoma chuanxiong volatile oil on preparation of medicine for curing skin scar - Google Patents
Application of rhizoma chuanxiong volatile oil on preparation of medicine for curing skin scar Download PDFInfo
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- CN101664426A CN101664426A CN200910197394A CN200910197394A CN101664426A CN 101664426 A CN101664426 A CN 101664426A CN 200910197394 A CN200910197394 A CN 200910197394A CN 200910197394 A CN200910197394 A CN 200910197394A CN 101664426 A CN101664426 A CN 101664426A
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Abstract
The invention relates to the technical field of medicine, in particular to a new application of rhizoma chuanxiong volatile oil on preparation of medicine for curing skin scar. According to test of human skin hypertrophic scar-derived fibroblasts and test in inhibiting hypertrophic scar of rabbit ears, the rhizoma chuanxiong volatile oil can obviously inhibit the proliferation of the skin scar cells, induce the apoptosis thereof and reduce scar index; therefore, the rhizoma chuanxiong volatile oil can be used for preparing the medicine for curing skin scar. The preparation method of the rhizoma chuanxiong volatile oil is simple and convenient, the price is low, and the application provides a new medicine source for the treatment of the skin scar.
Description
Technical field
The present invention relates to medical technical field, is the new purposes that Rhizoma Chuanxiong volatile oil is used to prepare treatment cicatrix of skin medicine.
Background technology
Cicatrix is the inevitable outcome of repair in trauma, and all wounds that relates to skin corium nearly all come to an end with scar healing.The cicatrix regular meeting of hyperplasia destroys the integrity of human body surface or with the dysfunction of various degree, brings dual misery on the mind ﹠ body to patient.Cicatrix of skin sickness rate height, but the medicine or the curative effect of clinical practice at present are not ideal enough, side effect is more, or the indeterminate large-scale popularization that is difficult to of effective ingredient is used.
Rhizoma Chuanxiong (Liqusticum chuanxiong Hort.) is a samphire, records in one one of Pharmacopoeia of the People's Republic of China version in 2005.This product is the Rhizoma Chuanxiong dry rhizome, irregular nodular pattern fist shape agglomerate, and diameter 2-7cm, surperficial yellowish-brown, coarse shrinkage has the wheel of most parallel ridges to save, and there is the similar round stem trace of depression on the top, on the following side wheel joint most tubercle shape root traces is arranged.Matter is solid, frangibility not, and section yellow-white or lark, loosing has the grease chamber of yellowish-brown, and it is wavy that cambium ring is, and the gas giving off a strong fragrance, slightly has a numb feeling in the tongue, flavor Hui Tian at bitter in the mouth, suffering.The Chinese medicine Rhizoma Chuanxiong is one of China's Chinese medicine, is called " the gas medicine in the blood ", has the effect of blood-activating and qi-promoting, wind-expelling pain-stopping.
The chemical constituent that Rhizoma Chuanxiong contains mainly contains multiple chemical compounds such as lactone, alkaloids, phenols and volatile oil, and (ten thousand is beautiful for Yang Lihong, Xie Xiuqiong, Wang Shubin, Zhan Ke. the Rhizoma Chuanxiong chemical constitution study, the time precious traditional Chinese medical science traditional Chinese medicines [J], 2007:18 (7) 1576-1577.).Rhizoma Chuanxiong volatile oil is the effective site of Rhizoma Chuanxiong.Main component is phthalide analog compound (Li HX, Ding MY, Yu JY.Separation and identification ofthe phthalic anhydride derivatives ofLiqusticum Chuanxiong Hort by GC-MS, TLC, HPLC-DAD, andHPLC-MS.Journal ofchromatographic science, 2002; 40 (3): 156-61).Ligustilide is typical case's representative of phthalide analog compound, accounts for the 50%-80% of volatile oil total amount, is main component of essential oil.Its chemical constitution is as follows:
Rhizoma Chuanxiong volatile oil has microcirculation improvement, brings high blood pressure down, cerebral blood flow increasing amount and effects such as analgesia, adjusting cardiovascular function and anticoagulation.Discover that Rhizoma Chuanxiong volatile oil can make the blood capillary spasmolytic, increase the open number of blood capillary, accelerate blood flow rate, make accumulative erythrocyte depolymerization, wherein the erythrocyte depolymerisation is particularly remarkable.(Xie Xiuqiong, Zhan Ke, Yin Rongli, Yang Lihong. the progress of Rhizoma Chuanxiong volatile oil [J]. the time precious traditional Chinese medical science traditional Chinese medicines, 2007,18 (6): 1508-1509).Rhizoma Chuanxiong volatile oil has the migrainous effect of alleviation (Cheng Peng, Xiao fang Xie, LanWang, LiGuo, Tanlian Hu.Pharmacodynamic action and mechanism of volatileoil from Rhizoma Ligustici Chuan xiong Hort.on treating headache.Phytomedicine 2009 16:25-34.), also has refrigeration function, its analgesic mechanism and inhibition Cycloxygenase (COX) activity, thus the hypothalamus prostaglandin E reduced
2Relevant (Yang Jinrong, Song Jun, Ma Xiaowen, Li Zulun, Hu Rong. Rhizoma Chuanxiong volatile oil is to the active selective inhibitory of Cycloxygenase-2 [J]. the Gansu traditional Chinese medical science, 2008,21 (2)).Lactone compound in the Rhizoma Chuanxiong volatile oil also has the smooth muscle spasmolysis effect in addition, and the vasoconstriction that NA is caused has spasmolysis.(institute of Materia Medica,Chinese Academy of Medical Sciences. Chinese herbal medicine modern study, the 2nd volume [M]. Beijing: combined publication society of China Concord Medical Science University of Beijing Medical University, 1996,6:423).But do not see that so far Rhizoma Chuanxiong volatile oil is used for the treatment of the report of cicatrix of skin aspect.
Summary of the invention
The present invention provides a kind of new purposes for the treatment of cicatrix for Rhizoma Chuanxiong volatile oil.Through human body skin fibroblasts from hypertrophic scars experiment in vitro and zoopery, find that Rhizoma Chuanxiong volatile oil has tangible apoptosis-induced effect to the cicatrix of skin fibroblast, and evident in efficacy to rabbit ear scar treatment, thereby can be used for preparing the medicine for the treatment of cicatrix of skin.
Description of drawings
Fig. 1 analyzes collection of illustrative plates for the GC-MS of Rhizoma Chuanxiong volatile oil of the present invention
Fig. 2 is the effect of vigor figure of Rhizoma Chuanxiong volatile oil to fibroblasts from hypertrophic scars
Fig. 3 is that Rhizoma Chuanxiong volatile oil is to fibroblasts from hypertrophic scars lactic acid dehydrogenase activity figure
Fig. 4 induces the streaming two dimension point diagram of fibroblasts from hypertrophic scars apoptosis for Rhizoma Chuanxiong volatile oil
Fig. 5 suppresses the cicatrix index map of rabbit ear scar hyperplasia for Rhizoma Chuanxiong volatile oil
The specific embodiment
The preparation Rhizoma Chuanxiong volatile oil
The exsiccant Rhizoma Chuanxiong decoction pieces of 1kg is ground into 24 order coarse powder, adds 6kg water, air-distillation gets Rhizoma Chuanxiong volatile oil 2.5ml routinely, and behind anhydrous sodium sulfate drying, the volatile oil that takes a morsel carries out GC-MS to be analyzed, and the results are shown in Figure 1.As seen from Figure 1, its main chemical compositions is ligustilide (67.027%) and 3-butylidene phthalide (5.831%).
Rhizoma Chuanxiong volatile oil is induced the experiment of human hypertrophic scar apoptosis of fibroblasts
(1) reagent
1. hyclone (FBS Hyclone)
2. phosphate buffer (PBS Hyclone)
3. dimethyl sulfoxide (DMSO Sigma)
4. methyl tetrazolium blue (MTT Amresco) is mixed with 0.5%MTT5.0.25% pancreatin (Sigma) with PBS
6.DMEM cell culture fluid (Sigma)
7. apoptosis test kit (the triumphant basic biotech development company limited in Nanjing)
8. (Chengdu Japan hundred believes pharmaceutical Co. Ltds, authentication code: the accurate word Z20050438 of traditional Chinese medicines) to the antipruritic ointment for softening of cicatrix
9. Rhizoma Chuanxiong volatile oil solution: after the Rhizoma Chuanxiong volatile oil of getting above-mentioned preparation is dissolved in an amount of dimethyl sulfoxide, with the DMEM cell culture fluid dilution that contains 10%FBS, make the final concentration of Rhizoma Chuanxiong volatile oil be respectively 25 μ g/ml, 50 μ g/ml, 100 μ g/ml (DMSO content all is less than 1%) are standby
(2) preparation human hypertrophic scar fibroblast
Get 16-30 year patient skin hypertrophic cicatrix, under aseptic condition, remove epidermis and subcutaneous tissue, normal saline cuts into 0.5-1.0mm after washing repeatedly
3Piece of tissue, be inoculated on the disposable culture bottle wall, piece of tissue spacing 0.3-0.5mm, it is an amount of to add FBS, after in cell culture incubator, being inverted cultivation 2h routinely, add DMEM culture fluid (containing penicillin 100U/ml, the streptomycin 100U/ml) 5ml that contains 20%FBS and continue to cultivate, change liquid weekly 2 times.When treating that the primary cell growth is fused into sheet substantially, absorb culture fluid, add the about 1min of 0.25% trypsinization after the PBS liquid rinsing 2 times, when seeing kytoplasm retraction, intercellular substance increase under the inverted microscope, pancreatin is abandoned in suction, adds the DMEM culture fluid contain 10%FBS and stops digestion, and suction pipe is blown and beaten a bottle parietal cell gently and made and be cell suspension, go down to posterity in ratio packing in 1: 3, use 3-6 to carry out following experiment for cell.
(3) Rhizoma Chuanxiong volatile oil is tested the fibroblasts from hypertrophic scars effect of vigor
The experiment of MTT colorimetric is a kind of method that detects cell survival and growth.The measured absorbance value of enzyme-linked immunosorbent assay instrument has reflected cell viability indirectly.This experiment is divided into 4 groups, and the 1st group is the blank group, and the 2nd, 3,4 groups are respectively the basic, normal, high dosage group of Rhizoma Chuanxiong volatile oil.
The take the logarithm fibroblasts from hypertrophic scars of trophophase is mixed with 5 * 10 with the DMEM culture fluid that contains 10%FBS
4The cell suspension of individual/ml is inoculated in 3 96 well culture plates respectively with every hole 100 μ l, puts CO
2After incubator is cultivated 24h, culture fluid is abandoned in suction, the 2nd of every block of plate, 3,4 groups add the low of above-mentioned preparation respectively, in, the Rhizoma Chuanxiong volatile oil solution 100 μ l/ holes of Senior Three kind variable concentrations, the 1st group of blank group adds the DMEM culture fluid that contains 10%FBS of equivalent, and every group of 10 parallel holes continue to cultivate, 3 blocks of plates are respectively at 12,24,48h adds 0.5%MTT20 μ l dyeing, continue to cultivate 4h, culture fluid is abandoned in suction, and every hole adds DMSO150 μ l, vibration 10-15min, automatically the enzyme immunoassay instrument is measured absorbance A (wavelength 570nm), record the variable concentrations Rhizoma Chuanxiong volatile oil respectively and cultivate the cell viability of different time, (cell viability=experimental group absorption value/matched group absorption value) repeats to do three batches of cell experiments, the results are shown in Figure 2.As seen from Figure 2,12,24, three different time points of 48h, each dosage group of Rhizoma Chuanxiong volatile oil is compared with the blank group, and cell viability all has decline, and is tangible dose-effect and time-effect relationship.Particularly middle and high dosage group suppresses very obvious to fibroblast proliferation, has compared significant difference with the blank group.Illustrate that Rhizoma Chuanxiong volatile oil can obviously suppress the propagation of fibroblasts from hypertrophic scars.
(4) Rhizoma Chuanxiong volatile oil is to the active influence experiment of hypertrophic cicatrix fibroblast lactic acid dehydrogenase (LDH)
LDH is the sign of membrane integrity, also is the index that detects cell death, and the LDH of release is many more, and the LDH activity is high more, illustrates that cell injury is serious more.The get it filled cell culture fluid of thing effect 24 and 48h of the present invention, the automatic biochemistry analyzer analysis.Experiment is divided into 4 groups, and the 1st group is the blank group, and the 2nd, 3,4 groups are respectively the basic, normal, high dosage group of Rhizoma Chuanxiong volatile oil.
With people's fibroblasts from hypertrophic scars of exponential phase, be mixed with 5 * 10 with the DMEM culture fluid that contains 10%FBS
4The cell suspension of individual/ml is inoculated in 2 96 well culture plates respectively with every hole 100 μ l, puts CO
2After incubator is cultivated 24h, culture fluid is abandoned in suction, and every block of plate is by the Rhizoma Chuanxiong volatile oil solution 100 μ l/ holes of organizing the basic, normal, high three kinds of variable concentrations that add above-mentioned preparation respectively, and the blank group adds the DMEM culture fluid that contains 10%FBS of equivalent, 10 parallel holes of each concentration, continue to cultivate, 2 blocks of plates are respectively at 24,48h, draw culture fluid, automatic biochemistry analyzer is analyzed the LDH activity, repeats to do three batches of cell experiments, the results are shown in Figure 3.As seen from Figure 3,24, the time point that 48h is different, low dose group is compared with the blank group, the active no significant difference of LDH.And middle and high dosage group increases permeability of cell membrane, lactic acid dehydrogenase discharges, therefore the LDH in the culture fluid is active significantly raises, with the blank group significant difference is arranged more all, and the increase with activity and time increases, and illustrates that Rhizoma Chuanxiong volatile oil can impel the apoptosis of fibroblasts from hypertrophic scars.
(5) Rhizoma Chuanxiong volatile oil is to the influence experiment of hypertrophic cicatrix apoptosis of fibroblasts
The various functional statuses that flow cytometry can be studied cell comprise cell proliferation, apoptosis, cell differentiation etc.This experiment is divided into 4 groups, and the 1st group is the blank group, and the 2nd, 3,4 groups are respectively the basic, normal, high dosage group of Rhizoma Chuanxiong volatile oil.
With the fibroblasts from hypertrophic scars of exponential phase, be mixed with 1.5 * 10 with the DMEM culture fluid that contains 10%FBS
5The cell suspension of individual/ml is inoculated in 16 well culture plate respectively with every hole 2ml, puts CO
2After incubator is cultivated 24h, culture fluid is abandoned in suction, the 2nd, 3,4 groups of Rhizoma Chuanxiong volatile oil solution 2ml/ holes that add basic, normal, high three kinds of variable concentrations of above-mentioned preparation respectively, the blank group adds the DMEM culture fluid that contains 10%FBS of equivalent, after continuing to cultivate 12h, culture fluid is abandoned in suction, 0.25% trypsinization, the centrifugal 5min of 1500rpm, PBS buffer washing 2 times, the Binding Buffer suspension cell that adds 500 μ l respectively adds each 5 μ l of Annexin V-FITC and Propidium Iodide, mixing; Room temperature lucifuge reaction 5-15min, the flow cytometry analysis apoptosis rate.Its apoptosis two dimension point diagram can be divided into four quadrants, and upper left is dead cell; The lower-left is a living cells; Upper right is apoptosis and dead cell in late period; The bottom right is a viable apoptotic cell; Apoptosis rate is upper right portion and lower right-most portion apoptosis and dead cell percentage rate sum, the results are shown in Figure 4.As seen from Figure 4, the Rhizoma Chuanxiong volatile oil effect is after 12 hours, and apoptosis is obvious, increases with administration concentration, and apoptosis rate significantly increases, and the apoptosis rate of middle and high dosage group is respectively 36.42% and 48.01%, and apoptosis rate is significantly higher than the blank group.Illustrate that Rhizoma Chuanxiong volatile oil can suppress the propagation of fibroblasts from hypertrophic scars and can induce its apoptosis.
Rhizoma Chuanxiong volatile oil is to the inhibition experiment of rabbit ear hypertrophic cicatrix
This experiment is divided into 5 groups: (1) blank group, (2) Rhizoma Chuanxiong volatile oil low dose group, dosage group in (3) Rhizoma Chuanxiong volatile oil, (4) Rhizoma Chuanxiong volatile oil high dose group, (5) positive controls
Said medicine is unguentum, drug component and the percentage by weight such as the following table of its 1-4 group:
The 5th group of positive control medicine is the antipruritic ointment for softening of cicatrix
Get 5 of new zealand rabbits, male and female are regardless of, cleaning level, rabbit 3-4 in age month, body weight 2.2-2.6kg.Provide by Second Military Medical University, PLA's Experimental Animal Center.18-22 ℃ of receptacle temperature, relative humidity 60%-75%, artificial mixed fodder is fed.Animal divides the cage adaptability to raise after 3 days, and the quiet notes of medication 25% urethane are anaesthetized routinely.Behind the rabbit ear 75% alcohol disinfecting, avoiding blood vessel both sides at rabbit ear facies ventralis center line along major axis with circular brill cutter is the circular incision of 7mm as diameter, and complete excision holostrome skin is removed perichondrium, cartilage-preserving.Every ear 6 places, 2cm at interval.Postoperative wound surface is smeared chlorotetracycline ointment and is protected from infection.Postoperative the 7th day is divided into 5 groups at random with 60 cicatrix wound surface, i.e. the high, medium and low dosage group of Rhizoma Chuanxiong oil ointment, and positive controls and blank group, every group of 12 wound surface are coated with corresponding drug ointment outward respectively, and gently rub up to drug absorption.Every day 1 time, the about 50mg of every place cicatrix, continuous 30 days.The 31st day, with vernier caliper measurement rabbit ear scar thickness and cicatrix edge normal ear thickness, cicatrix index=(scar thickness+normal ear thickness)/normal ear thickness.Record and respectively organize the cicatrix index, compare, the results are shown in Figure 5.As seen from Figure 5, middle and high dosage group drug effect is obvious, compares with the blank group, and rabbit ear cicatrix index obviously reduces, and has significant difference.High dose group particularly, drug effect is not worse than positive controls.
Experimental result shows that Rhizoma Chuanxiong volatile oil can significantly suppress the cicatrix of skin fibroblast proliferation and induce its apoptosis, can reduce the cicatrix index, therefore, can be used for preparing the medicine for the treatment of cicatrix of skin.
Claims (1)
1. the application of Rhizoma Chuanxiong volatile oil in preparation treatment cicatrix of skin medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105079809A (en) * | 2015-07-09 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Composition, preparation, method for preparing same, application and method for applying preparation |
| CN113476489A (en) * | 2020-12-31 | 2021-10-08 | 成都中医药大学 | Ligusticum wallichii volatile oil plaster for treating dysmenorrhea as well as preparation method and application thereof |
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2009
- 2009-10-20 CN CN2009101973943A patent/CN101664426B/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105079809A (en) * | 2015-07-09 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Composition, preparation, method for preparing same, application and method for applying preparation |
| CN113476489A (en) * | 2020-12-31 | 2021-10-08 | 成都中医药大学 | Ligusticum wallichii volatile oil plaster for treating dysmenorrhea as well as preparation method and application thereof |
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