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Ethnopharmacological communication
Analysis of the chemical composition,acute toxicity and skin nsitivity of esntial oil from rhizomes
of Ligusticum chuanxiong
Hong Zhang a,b,1,Ting Han a,1,Cheng-Hao Yu b,Yi-Ping Jiang a,Cheng Peng b,n,Xia Ran a,Lu-Ping Qin a,nn
a Department of Pharmacognosy,School of Pharmacy,Second Military Medical University,Shanghai200433,PR China
b Key Laboratory of Standardization of Chine Herbal Medicines of Ministry of Education,Pharmacy College,Chengdu University of Traditional Chine Medicine,No.37Shierqiao Road,Chengdu610075,PR China
a r t i c l e i n f o
Article history:
Received3March2012
Received in revid form
6October2012
Accepted8October2012
Available online17October2012
Keywords:
Plant
Esntial oil
Component
Safety
Traditional Chine medicine
a b s t r a c t
Ethnopharmacological relevance:Ligusticum chuanxiong Hort.(Umbelliferae)is a plant ud as medicine
and food in China.The esntial oil(EO)extracted from its rhizomes posss many pharmacological
activities.However,there have been no scientific reports in the modern literature on the safety of EO.
Aims of the study:The objective of this study was to conduct a chemical composition analysis and
sparklevaluate acute toxicity and skin nsitivity of EO from rhizomes of Ligusticum chuanxiong.
Materials and methods:The chemical composition of hydrodistilled EO was analyzed by gas
chromatography-mass spectrometry(GC–MS)and was evaluated in animals for acute toxicity,skin
irritation and nsitization tests.
Results:Dozens of compounds were detected and the major components of EO were ligustilide and
butylidenephthalide with relative contents of67.46and 5.06%,respectively.The oral and intra-
pastoraleperitoneal lethal dos of50%(LD50)in mice were7.23g/kg(approximately14,606times of clinical
do ud)and2.25g/kg(approximately5091times of clinical do ud),respectively.The dos of
英语广告词
0.115and0.23g/kg EO(approximately232.5and465times of the respective clinical dos ud)
revealed slight irritation effects on rabbit skin,but1g/kg EO(approximately2020times of clinical do
ud)had no obrvable effect on guinea pig skin in the skin nsitization test.
Conclusions:The experimental results indicate that short term application of EO is probably safe
within the range of its clinical dos,but the do should be controlled for external u due to its slight
skin irritation.
Crown Copyright&2012Published by Elvier Ireland Ltd.All rights rerved.
upgrade esntial1.Introduction
Ligusticum chuanxiong Hort.(Umbelliferae),a plant of which
the aerial part is delicious,is ud for medicine and food in China.
Rhizoma chuanxiong,the rhizome of Ligusticum chuanxiong,is
commonly ud in traditional Chine medicines.It has warm
西点培训广州properties and is pungent inflavor.It promotes blood circulation
and qi,expels wind and alleviates pain.For the reasons it has
high medicinal value.It has been clinically ud to prevent and
cure numerous dias,such as pain,migraine,rheumatic
otaku
arthralgia,inflammation,menstrual disturbance,atherosclerosis,
hypertension,stroke,among others(China Pharmacopoeia
Committee,2005;Chan et al.,2006;Peng et al.,2009;Ran et al.,
2011).The esntial oil(EO)is considered to be the major active
part of this herbal drug responsible for the pharmacological effects
mentioned.Some investigations indicate that EO from this plant can
protect against ultraviolet B-induced DNA damage and apoptosis in
mammalian cells(Jeong et al.,2009).EO displays faster ont of
action as well as better analgesic and dative efficacy through the
nasal route than by oral administration(Guo et al.,2010).The ethyl
ether extract prevents hydrogen peroxide-induced endothelial cell
damage(Hou et al.,2004).The anti-Trichophyton effects of EO are
also substantiated(Sim and Shin,2008).
Our previous investigations indicated that EO extracted from
the plant markedly suppresd the viability of human hyper-
trophic scarfibroblasts,elicited cell apoptosis in vitro(Wu et al.,
2011a),and inhibited the formation and development of hyper-
trophic scars in the rabbit ear model(Wu et al.,2011b;Zhang
et al.,2012),suggesting its protective effects against hypertrophic
scars.However,as far as we are aware no investigations have
been carried out to evaluate safety of esntial oil extracted from
rhizomes of Ligusticum chuanxiong,which is crucial for its clinical
applications.In the prent study,we analyzed its chemical
composition and estimated its short term safety in animals.
Contents lists available at SciVer ScienceDirect
journal homepage:/locate/jep
let me go什么意思Journal of Ethnopharmacology
0378-8741/$-e front matter Crown Copyright&2012Published by Elvier Ireland Ltd.All rights rerved.
/10.1016/j.jep.2012.10.010
n Corresponding author.Tel./fax:þ862887785016.
nn Corresponding author.Tel./faxþ862181871300.
E-mail address:,(C.Peng),
lpqin@(L.-P.Qin).
1The authors contributed equally to this work.
Journal of Ethnopharmacology144(2012)791–796
2.Materials and methods
2.1.Preparation of esntial oil
Rhizoma chuanxiong was collected from Xindu,Sichuan,China, and identified as the rhizome of Ligusticum chuanxiong by Lu–Ping Qin,a pharmacognosist from the Department of Pharmacognosy, School of Pharmacy,Second Military Medical University(Shang-hai,China).A voucher specimen of Lig
usticum chuanxiong was deposited with the number SY3236at the herbarium of the university.The esntial oil(EO)was extracted by hydrodistilla-tion.In accordance with our previous reports(Wu et al.,2011a; Zhang et al.,2012),the dried rhizomes were broken into grains, 200g of which were immerd in1000ml of distilled water and boiled in a distillation apparatus for6h.The yield of EO was 0.33%(v/w).
2.2.GC–MS analysis
In accordance with our previous method reported(Wu et al., 2011a),GC–MS analysis was performed for determination of EO composition with a Finnigan Voyager gas chromatographfitted with a fud silica VF-5ms capillary column(30mÂ0.25mm; coating thickness0.25m m)using the following temperature program.The initial temperature was501C for2min,raid to 3001C at151C/min,holding for10min.The injector temperature was2501C and the MS source helium was ud as the carrier gas at aflow rate of1ml/min with a split ratio of30:1(v/v).The gas chromatograph was coupled to a Finnigan Voyager mass lective detector.The ionization source temperature was2001C and the ionization energy was70eV.
2.3.Animals
ICR mice(19–21g),guinea pigs(190–210g)and New Zealand white rabbits(2.0–2.4kg),half male and half female,were obtained from the Experimental Animal Center of the Second Military Medical University(Shanghai,China)and houd in a regulated environment(20721C),with a12h light/dark cycle (08:00–20:00,light).The animals were deprived of food for12h before the experiment,but had free access to drinking water.Each animal was ud only once in the experiment.All animal treat-ments were strictly in accordance with international ethical guidelines concerning the care and u of laboratory animals (Committee for the Update of the Guide for the Care and U of Laboratory Animals,National Rearch Council,2011),and all the experiments were carried out under the approval of the Commit-tee of Experimental Animal Administration of the Second Military Medical University.
2.4.Reagents
The following reagents were ud:2,4-dinitrochlorobenzene (DCB,Yingxin biological technology(Shanghai)Co.Ltd.,China), sodium sulphide(Shanghai chunya chemical science and technol-ogy development Co.Ltd.,China),pure valine and liquid paraffin(Sinopharm Chemical Reagent Co.Ltd.,China).Basic ointment consisted of80%pure valine and20%liquid paraffin as placebo.
2.5.Acute toxicity test(LD50estimation)
2.5.1.Acute oral toxicity
This method estimated the do of EO that killed50%of a reduced sample of animals by a given route(Yao et al.,2010).In thefirst pha,a ries of dos of EO were administered to mice.Each do was tested in three mice of either x(lected randomly)each time.In the acute oral toxicity study,lethal dos of0and100%were estimated at5and10g/kg,respectively,and recorded.Thenfive different dos of EO were established according to the following formula.
Five dos:aR0¼a
ðÞ,aR1,aR2,aR3,aR4¼b
ðÞR¼
ffiffiffiffiffiffiffiffi
b=a
nÀ1
q
where R is the grouping coefficient;n is the number of divided groups(n¼1–5);a is0%lethal do;b is100%lethal do.
Five different dos were5, 5.95,7.07,8.41and10g/kg, respectively.Five groups of animals(half male and half female, n¼10)were orally treated with the abovefive dos by gastric intubation,respectively.Daily cageside obrvations were made till the end of the14-day obrvation period,post administration. If any dead mou was found it was immediately removed from the cage and the number of dead mice was registered.The lethal do of50%(LD50)was calculated using the Logit method.
2.5.2.Acute intra-peritoneal toxicity
Similarly,the acute intra-peritoneal toxicity was estimated in accordance with the method described above.The lethal dos of 0and100%,estimated as1.25and5g/kg,were divided intofive different dos which were 1.25, 1.77, 2.50, 3.54and5g/kg, respectively.Five groups of animals(half male and half female, n¼10)were given intraperitoneal injections at the abovefive dos,respectively,and then the number of dead mice was recorded for14days.The lethal do of50%(LD50)was calculated
using the Logit method.
2.6.Skin irritation test
As described in the literature(State Food and Drug Administration,2005),rabbits were divided into two groups,a skin-intact group and skin-damaged group consisting of20 animals per group given4different dos of EO(0.029,0.058, 0.115and0.23g/kg).24h before EO treatment,the hair (5Â5cm)was removed on both sides of the back of rabbits using a depilatory agent of6%sodium sulfide solution.In the skin-damaged group,after iodine disinfection,the left side of the denuded skin was slightly damaged with a No.5pinhead to induce mild blood oozing and then4different dos of EO were applied directly to the skin.Following that,the treated skin was covered with gauze and cellophane andfixed with bandages. As control,basic ointment(BO)was applied to the denuded skin on the opposite side.After4h,EO and BO were removed with warm water.This once daily treatment lasted one week.On day 7after removal of EO and BO,any signs of erythema,edema, pigmentation or blood oozing at the sites of EO or BO treatment were obrved and recorded after1,24,48and72h.At the same time,erythema and edema symptoms were given scores accord-ing to Table1.In the skin-intact group of animals,erythema and edema symptoms were estimated in the same way.The average score was calculated,that is,total scores of erythema or edema were divided
by the number of animals given the same do of EO.Subquently the irritation intensity of EO was evaluated according to Table2.
2.7.Skin nsitization test
In accordance with literature(State Food and Drug Administration,2005),guinea pigs were randomly divided into 6groups,consisting of a negative control group,four treatment groups(0.125,0.25,0.5and1g/kg)and positive control group, each of which consisted of10animals.24h before treatment,the hair(3Â3cm)was removed on both sides of the back of guinea
H.Zhang et al./Journal of Ethnopharmacology144(2012)791–796 792
pigs with a depilatory agent of 8%sodium sulfide solution.The denuded skins of animals in the treatment groups were treated with 0.125,0.25,0.5and 1g/kg EO,respectively.The negative
controls received basic ointment and the positive controls 2,4-dinitrochlorobenzene (DCB,0.1%,0.2ml).After applying the respective treatments the treated skin was covered with sterile bandages.After 6h,the treatments were removed with warm water.On days 7and 14,each group of animals were given the corresponding treatments mentioned above.After 14days of nsitization,bas
ic ointment,EO (0.125,0.25,0.5and 1g/kg)and DCB (0.1%,0.2ml)were applied to the corresponding group of animals for excitation of skin allergy.6h later,the treatments were removed,erythema and edema symptoms were estimated accord-ing to Table 1after 1,24,48and 72h,respectively,and the average score was recorded for each group,followed by evaluation of skin allergy intensity according to Table 2.The ratio of allergic animals in each group was calculated to estimate the allergic intensity in accordance with Table 3.At the same time,animals was cloly obrved for signs of ,asthma,standing instability,shock,or other vere systemic allergic reactions.
3.Results
3.1.Esntial oil components
GC–MS analysis detected dozens of compounds from the esntial oil,of which the major components were ligustilide and butylidenephthalide with relative contents of 67.46and 5.06%,respectively (Fig.1,Table 4).3.2.Acute toxicity study (LD 50)
Bad on the first pha test for acute oral toxicity,a range of five oral EO dos (5,5.95,7.07,8.41,and 10g/kg)between the lethal dos of 0and 100%were calculated by the given formula.After oral administration of the above EO dos,a marked reduction in spontaneous activity
of mice was obrved in each do group.The group of mice given a do of 10g/kg EO began to die after 1h and all died within 24h.The groups of mice treated with dos of 7.07and 8.41g/kg died 4h after treatment.Logit analysis revealed the oral LD 50of the esntial oil was 7.23g/kg,95%confidence limits 6.66–7.79g/kg (Table 5).
Similarly,after intra-peritoneal administration of dos of 1.25,1.77,2.50,3.54and 5g/kg EO,respectively,a marked reduction in
Table 1
Grading standard for skin irritation or allergic respon.Irritation or allergic respon
Score
Erythema No
0Slight (barely visible)1Moderate (visible)2Severe
3Purple erythema to mild eschar 4Edema No
0Slight (barely visible)
1Moderate (apparent swelling,edge higher than surrounding skin)2Severe (1mm of swelling and clear contour)
3Extremely vere (over 1mm of swelling or blisters or ulceration)4Highest total value
8
Table 2
Evaluation standard for skin irritation intensity.Score Evaluation 0–0.49No irritation 0.5–2.99Slight 3.0–5.99Moderate 6.0–8.00
Severe
Table3
Evaluation standard for skin allergy intensity.Incidence of allergic respon (%)Evaluation 0–10No 11–30Slight 31–60Moderate 61–80Severe
81–100
Extremely
vere
Fig.1.Fingerprint of the esntial oil analyzed by GC–MS.inprogress
H.Zhang et al./Journal of Ethnopharmacology 144(2012)791–796
793
Table 4
Chemical compounds of the esntial oil from rhizomes of Ligusticum chuanxiong Hort.(Xindu China).Compound
Apex RT (min)%Area Butanal
1.550.181-lsopropyl-4-methylbicyclo[3.1.0]hex-2-ene 4.990.061R-a -Pinene 5.120.274(10)-Thujene
5.620.72(1S,5S)-(-)-2(10)-Pinene 5.710.02b -Pinene
japane foot
5.750.11a -Phellandrene
6.030.06a -Terpinen 6.160.24o-Cymene 6.250.59Limonene
6.310.17b -Phellandrene 6.350.11t -Terpinen
6.65 1.13cis-b -Terpineol
6.810.03p-Mentha-1,4(8)-diene
6.97 1.231-methyl-4-(1-methylethenyl)-Benzene
7.040.06b -Linalool
7.080.03cis-b -terpineol
7.180.02trans-1-methyl-4-(1-methylethyl)-2-Cyclohexen-1-ol 7.440.05(S)-3-Ethyl-4-methylpentanol
7.550.08cis-1-methyl-4-(1-methylethyl)-2-Cyclohexen-1-ol 7.640.066-Butyl-1,4-cycloheptadiene 7.79 1.42(R)-(-)-p-menth-1-en-4-ol 8.08 1.99p-Cymen-8-ol
8.110.13p-menth-1-en-8-ol
8.210.08Trans-p-menth-1-en-3-ol 8.350.041,3,5-Dodecatriene
8.550.08Dodecamenthylcyclohexasiloxane
8.79 1.571,7,7-Trimethyl-bicyclo[2.2.1]hept-2-yl 9.120.022-Methoxy-4-vinylphenol 9.380.102,4-Decadienal 9.420.03Phthalic anhydride
9.490.07p-Mentha-1,4-dien-7-ol
9.540.031,4-Cyclohexadiene-1,2-dicarboxylic anhydride 9.800.43Valerophenone
9.820.27Methyl benzoylformate 9.930.16(E)-3-Decen-2-ol
9.990.121-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-cyclohexane 10.140.25Dodecanal
10.220.021-Methoxyadamantane
10.280.03Tetradecanmethyl-cycloheptasiloxane
10.460.901,2,3,4,4a ,7-hexahydro-1,6-dimethyl-4-(1,-methylethyl)-naphthalene 10.520.88b -Farnene 10.600.13Epiglobulol
10.660.129,12-Octadecadienal 10.790.04a -Caryophyllene
10.820.042-lsopropenyl-4a,8-dimethyl-1,2,3,4,4a,5,6,7,-octahydronaphthalene 10.920.17-10.970.13b -phellandrene 11.13 3.06b -chamigrene 11.170.65Copaene
11.280.27[(Z)-1,2-Diphenyleth-1enyl]-trimethylsilan 11.440.104-isopropylidene-1-winyl-o-menth-8-ene 11.730.05Diepi-a -cedrene epoxide 11.770.17(-)-Spathulenol 11.87 1.67Globulol 11.950.09Carotol 12.090.09Cedrol
12.200.06(-)-Spathulenol
12.290.26cis,trans-4-methyl-3-oxabicyclo[4.4.0]decane
12.330.092-chloro-1-(2,4-dimethylphenyl)-2-methyl-1-propanone 12.46 3.01Humulane-1,6-dien-3-ol 12.540.18Butylidenephthalide
12.63 5.061,1-Dimthyl-1,2,3,5,7,8,9,9a -octahydro-benzocyclohepten-6-one 12.670.43cis-3-buty-4-vinyl-cyclopentene 12.78 1.16Ligustilide
13.1867.462-octyl-phenol
13.460.045,7,8-trimethyl-dihydrocoumarin 13.600.751-hexadeanol
13.970.13Methyl ester hexadecanoic
14.240.062,5,5,8a-tetramethyl-3,5,6,7,8,8a-hexahydro-1(2H)-naphthalenone 14.300.263,7-dihydro-7-(2-hydroxyethyl)-1,3-dimethyl-1H-Purine-2,6-dione 15.070.30Ethyl linoleate 15.79
0.04Total content 99.91
–,not detected.
H.Zhang et al./Journal of Ethnopharmacology 144(2012)791–796
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