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Phytomedicine 16 (2009) 25–34

www.elsevier.de/phymed

Pharmacodynamic action and mechanism of volatile oil from

Rhizoma Ligustici Chuanxiong Hort. on treating headache

Cheng Penga,b,�, Xiaofang Xiea, Lan Wangb, Li Guoa, Tanlian Hua

aPharmacy College, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, ChinabMinistry of Education, Key Laboratory of Standardization of Chinese Medicine, Chengdu 610075, China

Abstract

The volatile oil from Rhizoma Ligustici Chuanxiong Hort. (CXVO) is likely to be the mainly active ingredient ofChuangxiong in curing headache. In this study, oral administration of CXVO (45.0, 90.0, and 135.0 ml/kg) to micesignificantly elevated the pain threshold in the hot-plate test and reduced the number of abdominal writhing caused byacetic acid. CXVO (90.0 and 135.0 ml/kg) not only reduced locomotor activity, but also prolonged the sleeping timeinduced by sodium pentobarbital (35mg/kg), and the number of mice with sleeping time over 1min by sodiumpentobarbital (25mg/kg) was markedly enlarged by CXVO (45.0, 90.0, 135.0 ml/kg) administration. The three doses ofCXVO significantly increased the pain threshold of rabbits with headache due to hot radiation and the level of plasmaET of rats with headache due to nitroglycerin injection. Besides, for the nitroglycerin-induced headache rats, the c-fosgene expression in the brain stem and hypothalamus was remarkably inhibited and the level of plasma CGRP wasreduced significantly after CXVO administration at both doses 90.0 and 135.0 mg/kg. The latter dosage could also raisethe level of plasma 5-HT markedly. The study suggests that CXVO acts probably as the active ingredient of Rhizoma

Ligustici Chuanxiong Hort. (CX) on treating headache and has potential to be an agent for treating headache.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Volatile oil from Rhizoma Ligustici Chuanxiong Hort. (CXVO); Rhizoma Ligustici Chuanxiong Hort. (CX); Headache

Introduction

As an authentic herbal medicine of Sichuan, CX isfirst recorded in the Divine Husbandman’s Classic of the

Materia Medica (Shen Nong Ben Cao Jing). It belongsto the Umbelliferae family, Ligustrum genus. CX iswarm in property and pungent in flavor, with functionsof activating blood and promoting qi, expelling windand stopping pain. As a result, it has been extensivelyapplied for many years to treat headache caused by

e front matter r 2008 Elsevier GmbH. All rights reserved.

ymed.2008.10.010

ing author at: No. 1166, Liutai Ave, Wenjiang District

huan Province, PR 611137, China.

28 61800232.

ess: pengchengchengdu@126.com (C. Peng).

various factors (e.g. blood deficiency, wind-cold, wind-hot, and wind-damp) combined with other Chineseherbal medicines and is effective definitely. Thoughsome studies have been done to find the activecomponents and the mechanism underlying of CX ontreating headache, the answer is still uncertain. Thechemical composition of CX consists of ligustrazine,CXVO and many other complex components. Amongthese, ligustrazine was studied most in the early periodand ever considered to be the mainly active componentof CX. However, it is found recently that ligustrazinehas a low content in CX compared with CXVO, which isnow instead considered to be the mainly activeingredient of CX on treating headache. Studies regard-ing the effects of CXVO and its main components

ARTICLE IN PRESSC. Peng et al. / Phytomedicine 16 (2009) 25–3426

(e.g. ligustilide, senkyunolide, and buty1-phenolphtha-lein) that include relieving fever (Li et al., 2003; Yanget al., 2003), sedation (Zhu et al., 2005), relieving spasm(Luo et al., 1996), and activating cardiovascular system(Ruan et al., 2003a, b; Wang, 1997), have been done andreported to some extent. Yet, these studies are notsystemic and some animal models used have unclearmechanisms or are still not generally accepted. Further-more, no study has been designed especially for CX ontreating headache. Therefore, none of these studies cananswer the question directly as to why CX plays asignificant role in treating headache clinically. In thecurrent study, with a variety of classic animal modelsthat have clear mechanisms, we investigated systemicallythe sedative and analgesic effects, as well as the directcurative effect of CXVO on experimental headache andpotential mechanism underlying for the purpose ofrevealing the pharmacodynamic action and mechanismof CX on treating headache and of providing pharma-cological evidence for further research and exploitationof CX.

Materials and methods

Plant material, chemicals and treatment of animals

In this study, CXVO was extracted by the super-critical carbon dioxide extraction. Slices of CX, pur-chased and identified by Pharmacy College, ChengduUniversity of Traditional Chinese Medicine, were driedat a temperature of 60 1C, pulverized, sifted with a sieve(mesh 16) and then extracted with the method ofsupercritical CO2 extraction twice (1 h each). Theprocess conditions were set as follows: temperature –40 1C, pressure – 15MPa and CO2 flowing rate – 30kg/h.Then mixture of the two extracts was placed in aseparator at room temperature for more than 6 h. The

Fig. 1. Fingerprint of CXVO analyzed by GC/MS. (A) 3-

oil layer in the separator was withdrawn and centrifugedat 3000 rpm for 30min to get CXVO, which was thensealed in the brown bottle. GC/MS analysis performedwith a gas chromatography-mass spectrometry system(GCMS-QP2010, Shimadzu Japan) was used for thequalitative determination of the composition of CXVO.Separation of the compounds was carried out on a DB-1quartz capillary column (30m� 0.25mm i.d., 0.25 mmfilm thickness) using the following temperature pro-gram. The initial temperature was 50 1C for 2min,increased at a rate of 4 1C/min to 140 1C holding for1min, then at a rate of 8 1C/min to 250 1C for 25min.The temperature of the injector and the MS source was250 1C. Helium was used as the carrier gas at a flow rateof 1.5ml/min with a split ratio of 30:1. The ionizationsource temperature was 200 1C and the ionizationenergy was 70 eV. The GC-MS analysis showed thatdozens of compounds were detected from CXVO andthe main components were ligustilide, 3-butylide-nephthalide and sabinene, with relative contents of58%, 5.29%, and 6.08%, respectively (Fingerprints seenin Figs. 1 and 2). Total lactones were the main activecompounds of CXVO and ligustilide was the mainactive constituent. With this method, the total extractionrate of lactones was 94.55% and the extraction rate ofligustilide (chemical structure seen in Fig. 3) was as highas 99.08%.

For the experimental testing, the preparation wasdissolved with rapeseed oil provided by Jiali food and oilindustrial Co., Ltd. (Sichuan, China) and diluted toconcentrations of 18.0, 36.0, and 54.0 ml/ml for admin-istration. Estazolam was purchased from ChangzhouFourth Pharmaceutical Co., Ltd. (Changzhou, China)and diluted to the concentration of 0.267mg/ml withdistilled water for administration. Pethidine was pur-chased from Shenyang First Pharmaceutical Co., Ltd.(Liaoning, China) and diluted to the concentration of16.67mg/ml with distilled water for administration.Ergotamine was purchased from Shanghai Xingyi

butylidenephthalide, (B) ligustilide and (C) sabinene.

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Fig. 2. MS fingerprint for the constituent B in Fig. 1.

O

Fig. 3. Chemical structure of ligustilide [3-butylidene-4,5-

dihydrophthalide or 3-butylidene-4,5-dihydro-1(3H)-isobenzo-

furanone].

C. Peng et al. / Phytomedicine 16 (2009) 25–34 27

Pharmaceutical Co., Ltd. (Shanghai, China) andwas diluted by distilled water to the concentration of0.4mg/ml. These chemicals were used as reference drugsin different animal experiments.

For animal experiments, all reference drugs as well asCXVO were administered orally (p.o.) only once at thedose of 2.5ml/kg bodyweight. Except in the experimentof hot radiation-induced headache in rabbits, thereference drug was given intravenously from the auricularvein of rabbits. Animals in the control and model groupswere treated with isovolumic distilled water instead.

Animals

Both Kunming mice (18–22 g, Grade II, Certificateno. 1999-7) and Sprague–Dawley rats (220–280 g, GradeII, Certificate no. 1999-8) with either sex were obtainedfrom the Laboratory Animal Center of ChengduUniversity of Traditional Chinese Medicine (Chengdu,China).The Japanese white rabbits (1.8–2.2 kg, Certifi-cate no. 1999-11) with either sex were supplied bythe Nursery of the Laboratory Animal ProfessionalCommittee of Sichuan Animal Society. The animals

were housed in a standard laboratory conditions (at atemperature of 2071 1C and a 12 h light/dark cycle)with food and water available. All animal treatmentprotocols were strictly in accordance with the interna-tional ethical guidelines and the National Institute ofHealth Guide concerning the Care and Use of Labora-tory Animals, and the experimental Animal Adminis-tration of the University.

Hot-plate test

The hot-plate test was carried out with Kunming mice(n ¼ 10/group) according to the method published byChen (1993): the device consists of a metal plate and ahermostatic waterbath. The mice were placed individu-ally on the hot plate with the surface temperature5570.5 1C, kept by the hermostatic waterbath. Thelatency, also named pain threshold, was recordedimmediately and manually with a timer until the typicalresponse of hind paw licking occurred. In the firstsession there was a selection of appropriate collectives.The mice were tested twice for each two days prior to theexperiment and the mean pain threshold of each mousewas considered as their basic pain threshold. Mice thathad a basic pain threshold lower than 5 s or higher than30 s were excluded. When the experiment starts, the testwas performed on mice individually at 30, 60, 90 and120min after drug administration with the methodmentioned above, once for each mouse each time. Anincrease of pain threshold indicates an analgesic effect.

Acetic acid-induced writhing test

The acetic acid-induced writhing test, performed onKunming mice (n ¼ 10/group), was another experimentused to detect the analgesic action of CXVO; therefore,

ARTICLE IN PRESSC. Peng et al. / Phytomedicine 16 (2009) 25–3428

it had the same reference drug pethidine as that of hot-plate test. As the method recorded by Xu (2002), aceticacid (0.6%) was injected intraperitoneally at the dose of10ml/kg 30min after drug administration. Then, thenumber of abdominal writhing response was immedi-ately recorded for a period of 30min and a decrease ofthe number represents an analgesic effect. The typicalabdominal writhing response manifests as expandinghind limb, constructing abdomen and raising the croup.

Locomotor activity

This experiment, performed on Kunming mice(n ¼ 12/group), aimed to study the in vivo sedativeeffect of CXVO and estazolam was used as the referencedrug. As the method published by Xu (2002), mice weresingly introduced into the cage and initially allowed toadapt to the experimental conditions for 5min beforetesting. Immediately 1 h after treatment locomotoractivity was recorded for a period of 10min with aCB3201-type digital tracing activity determinator forsmall animal manufactured by Chunbei electronicindustrial company (Sichuan, China). To avoid theenvironmental disturbance, the test apparatus wasplaced in a sound room. A reduction of locomotoractivity may be caused by central sedative drugs.

Sodium pentobarbital-induced sleeping time test

This contained two experiments with different doses(35 and 25mg/kg) of sodium pentobarbital as recordedby Xu (2002), both of which were performed onKunming mice (n ¼ 12/group) and had estazolam asthe reference drug. The sodium pentobarbital (1%) wasinjected intraperitoneally 1 h after treatment and dura-tion of loss of righting reflex was monitored for eachmouse as its sleeping time. In the experiment thatsodium pentobarbital was injected at the low dosage of25mg/kg, the accounts of mice that had a sleeping timeexceeding 1min for each group were calculated to becomparable.

Hot radiation-induced headache in rabbits

The experiment was conducted on rabbits (n ¼ 6/group) that had a basic pain threshold less than 60 s.The pain threshold was tested by the method publishedin the Compilation of Guiding Principle for PreclinicalResearch of New Drugs (Chemical Drugs) (DrugAdministration of Ministry of Health, 1994). First ofall, hairs covering the upper part of nose were removedfollowed by ink painting on the skin of this region.Then, this ink-painted area was radiated by hot radio-meter until head throwing reaction was triggered. Thelatency was measured manually for each rabbit as its

pain threshold. The rabbits were tested twice beforeexperiment and the mean value of each one wasconsidered as its basic pain threshold. Then aftertreatment, the rabbits were measured for pain thresholdcontinuously at 30, 60, and 120min, and the maximumvalue was confined to 180 s, which implied that radiationof rabbits with a pain threshold longer than 180 s werestopped.

Nitroglycerin-induced headache in rats

Sprague–Dawley rats were chosen to conduct thisexperiment (n ¼ 20/group) and ergotamine was thereference drug. As method recorded by Tassorelli et al.(1995) and Peng (2000), nitroglycerin was injectedsubcutaneously after pre-treatment for a period of30min to build the animal model of headache. Fourhours later of model establishment, 10 rats of eachgroup were anaesthetized with pentobarbital (1%) givenintraperitoneally at the dosage of 50mg/kg. They werefixed dorsally and operated to open the chest. Then atube was put into the aorta for perfuse first with 100mlaseptic sodium chloride fluid and then with 100mlphosphate buffer saline of paraformaldehyde (4%) afterthe right auricle cutting. After that, the brain tissues ofthese rats were dissected and put into the 4% phosphatebuffer saline of paraformaldehyde for 24 h before putinto 20% PBS of sucrose till the tissues dipped down tothe bottom. The brain stem and the hypothalamus werecut constantly for 10 slices with the same thickness of40 mm at the temperature of �20 1C with the MICROW-500-type cryostat (made in Germany). Then slices werekept in 20% sucrose–paraformaldehyde solution andhandled as required by the ABC immunohistochemistryreagent: they were first washed with 0.01mol/l PBS forthree times (10min each), 0.3%Tritonx-100 for 30min,0.3%H2O2–PBS for 15min and 0.01mol/l PBS for threetimes (10min each) again. After that, they wereincubated in 0.01mol/l PBS of normal sheep serum for20min, the rabbit antibody of c-fos (1:1500) for 1 h, thesheep antibody of rabbit IgG marked by biotin for 0.5 h,washed by 0.01mol/l PBS as before and finallyincubated in the ABC compound for 0.5 h. Washedwith 0.01mol/l PBS for three times (10min each) again,the slices were stained with DAD for 15min andterminated dyeing with fumigate distilled water for10min. The slices were then stuck on glass slide with Cr-gelatin, open-air dried overnight, dehydrated orderlywith gradient concentration of ethanol 80%, 95%,100%, and 100% (10min each), followed by transpar-ented with xylene twice (15min each), and finallymounted with neutral gum. The handled slices were atlast read with the Olympus inverted microscope (madein Japan) to counter the cells that manifested as reddishbrown or yellowish brown granules, which indicated

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Table

1.

EffectofCXVO

onpain

threshold

ofmicecausedbyhotstim

ulus(X̄�

SD).

Group

Anim

al(n)

Dose

(ml/kg)

Before

administration

(s)

After

administration(s)

30min

60min

90min

120min

Control

10

–16.7575.34

16.6076.19

19.4075.23

18.70710.86

17.4075.44

Pethidine

10

33.33mg/kg

16.9573.59

55.40713.53��

,DD

45.10719.54��

,DD

43.50717.24��

,DD

44.50717.24��

,DD

CXVO

10

135.00

17.4572.15

27.40715.19

28.30710.06��

,D29.2077.55��

,D38.20714.30��

,DD

10

90.00

17.1072.79

17.5076.43

22.60710.37

30.50711.34��

,D30.9079.06��

,DD

10

45.00

17.3573.15

20.10712.96

23.80710.97

29.90712.08��

,D33.60712.88��

,DD

Data

are

presentedasmean7SD.

��Po0.01,significantlydifferentcomparedwiththevalues

before

administration.

DPo0.05.

DD

Po0.01,significantlydifferentcomparedwiththecontrolgroup.

C. Peng et al. / Phytomedicine 16 (2009) 25–34 29

positive cells of c-fos gene expression. The remainingrats were sacrificed and the blood sample was collectedfrom the femoral artery, used to assay the levels ofplasma 5-HT, ET, and CGRP with radioimmunoassaymethod.

Statistical analysis

All statistical procedures were performed by using theSPSS statistical package (version 11.5). All data analysiswas performed by one-way analysis of variance (ANO-VA) for multiple comparisons with the Dunnett’s t-test.Except for the data on number of sleeping mice, theanalysis was performed by Chi-square test. Po0.05 wasconsidered statistically significant and data were pre-sented as mean7SD.

Results

Hot-plate test

Results of pain threshold in the hot plate testare shown in Table 1. The doses of 45.5, 90.0 and135.0 ml/kg raised the pain threshold significantly aftergiven to the mice for 90min and the highest dosageincreased the pain threshold as early as 60min aftergiven to the mice.

Acetic acid-induced writhing test

The numbers of writhing response are shown inTable 2. Compared with the control group, the admini-stration of the three doses of CXVO remarkablyreduced the number of writhing response in mice causedby acetic acid and the effect is dose dependent.

Locomotor activity

Results of motility experiments are shown in Table 3.Compared with the control group, the number oflocomotor activity of mice pretreated with CXVO atthe dosage of 135.0 ml/kg declined significantly.

Sodium pentobarbital-induced sleeping time test

The results of the experiment that sodium pentobar-bital was given at the dosage of 35mg/kg are shown inTable 4 and the results of the experiment that sodiumpentobarbital was given at the dosage of 25mg/kg areshown in Table 5. As shown in Table 4, sleeping time ofmice prolonged remarkably after treatment with CXVOat the dosage of 135.0ml/kg. The two lower doses of 45.0and 90.0 ml/kg of CXVO also prolonged the sleeping timebut without statistical significance. On the contrary, in

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Table 2. Effect of CXVO on acetic acid-induced writhing

response in mice (X̄� SD).

Group Animal

(n)

Dose

(ml/kg)Number of

writhing response

(times/30min)

Control 10 – 44.20714.92

Pethidine 10 33.33mg/kg 0.6071.26��

CXVO 10 135.0 24.50716.24�

10 90.0 25.9079.64��

10 45.0 29.0074.00��

Data were presented as mean7SD.�Po0.05, significantly different compared with the control group.��Po0.01, significantly different compared with the control group.

Table 3. Effect of CXVO on locomotor activity of mice

(X̄� SD).

Group Animal

(n)

Dose

(ml/kg)Number of

locomotor activity

(times/10min)

Control 12 – 794.17770.58

Estazolam 12 0.67mg/kg 671.507111.57��

CXVO 12 135.0 32.67766.32�

12 90.0 751.58796.04

12 45.0 777.25735.75

Data were presented as mean7SD.�Po0.05, significantly different compared with the control group.��Po0.01, significantly different compared with the control group.

Table 4. Effect of CXVO on sleeping time of mice caused by

sodium pentobarbital at a high dosage (X̄� SD).

Group Animal (n) Dose (ml/kg) Sleeping time (min)

Control 12 – 47.76722.12

Estazolam 12 0.67mg/kg 70.60723.98�

CXVO 12 135.0 67.08721.45�

12 90.0 57.19726.94

12 45.0 50.50732.09

Data were presented as mean7SD.�Po0.05, significantly different compared with the control group.

Table 5. Effect of CXVO on sleeping time of mice caused by

sodium pentobarbital at a low dosage (X̄� SD).

Group Animal (n) Dose

(ml/kg)Amount (n)

Sleeping

time

o1min

Sleeping

time

41min

Control 12 – 11 1

Estazolam 12 0.67mg/kg 0 12��

CXVO 12 135.00 0 12��

12 90.00 3 9�

12 45.00 3 9�

�Po0.05, significantly different compared with control group.��Po0.01, significantly different compared with control group.

C. Peng et al. / Phytomedicine 16 (2009) 25–3430

the experiment that sodium pentobarbital was given atthe low dosage of 25mg/kg, three doses of CXVOsignificantly all increased the accounts of mice that had asleeping time longer than 1min, as shown in Table 5.

Effect of CXVO on hot radiation-induced headache

in rabbits

As shown in Table 6, pain threshold value (PTV) ofthe rabbits treated with CXVO at doses of 45.0, 90.0,

and 135.0 ml/kg was raised significantly 30min after drugadministration, compared either with the values ofthemselves before drug administration or with thecontrol group treated with saline.

Effect of CXVO on nitroglycerin-induced headache

in rats

As shown in Table 7 and Fig. 4, the number of c-fos-positive cells in brain stem and hypothalamus of rats inthe model group were increased significantly due to theinjury of nitroglycerin, compared with that of thecontrol group. The administration of CXVO at dosesof 90.0 and 135.0 ml/kg obviously inhibited c-fosexpression. Results of plasma 5-HT, ET, and CGRPare shown in Table 8.The levels of plasma 5-HT and ETsignificantly declined and CGRP remarkably raised inrats exposed to pentobarbital, compared with thecontrol group. The administration of CXVO at thedosage of 135.0 ml/kg significantly retrieved the levels ofplasma 5-HT, ET, and CGRP. CXVO given doses 45.0and 90.0mg/kg significantly increased the level ofplasma ET, and the latter dosage also significantlydecreased the level of CGRP in plasma, compared withthe model group, which were treated by distilled water.

Discussion

As a common symptom in the clinic, headache hasbadly reduced the quality of patients’ daily life. Itappears in many diseases or as the side effect evoked bysome drug. In the International Classification of Head-ache (second edition) issued in 2004, the InternationalHeadache Society classifies headache into two types –the primary headache and the secondary headache,which are further divided into 16 subtypes for differentpathogenesis (Huang and Liu, 2000). Etiology andpathomechanism are complicated, of different subtypes;

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Table 6. Effect of CXVO on PTV of rabbits with headache caused by hot radiation (X̄� SD).

Group Animal (n) Dose (ml/kg) Before

administration (s)

After administration (s)

30min 60min 120min

Control 6 – 37.33713.14 50.00716.11 52.67713.00 55.33720.08

Pethidine 6 16.67mg/kg 39.17711.87 156.17727.79��,DD 138.67754.37��,DD 103.17762.66�

CXVO 6 135.00 37.50711.58 127.83758.77��,D 143.00760.97��,DD 135.50768.36��,D

6 90.00 39.50714.83 124.50738.79��,D 138.83765.48��,D 133.00758.07��,D

6 45.00 37.92716.00 123.17770.18�,D 126.67767.43��,D 130.33772.13�,D

Data were presented as mean7SD.�Po0.05, significantly different compared with the values before administration.��Po0.01, significantly different compared with the values before administration.DPo0.05, significantly different compared with the control group.DDPo0.01, significantly different compared with the control group.

Table 7. Effect of CXVO on the number of c-fos-positive cells in brain tissue of rats exposed to nitroglycerin (X̄� SD).

Group Animal (n) Dose (ml/kg) Number of c-fos-positive cells (n)

Brain stem Hypothalamus

Control 10 – 23.80714.28 30.60719.91

Model 10 – 156.00774.55�� 211.707119.06��

Ergotamine 10 1.00mg/kg 70.60727.37D 117.20761.32D

CXVO 10 135.00 90.70747.67D 107.50754.28D

10 90.00 98.90729.14D 120.50726.77

10 45.00 119.20728.13 148.30740.90

Data were presented as mean7SD.��Po0.01, significantly different compared with the control group.DPo0.05, DPo0.01, significantly different compared with the model group.

C. Peng et al. / Phytomedicine 16 (2009) 25–34 31

however, the functional disorders of cranial nerves andblood vessels, and the content changing of relatedbiochemical transmitters are usually involved (Mao,1996). Up to date, besides classic analgesic agents, drugson treating headache also include cycloxygenase-2inhibitors, angiotensin a inhibitors, triptan, droperidol,botulinum toxin and so on, which either need long-termtaking or cause apparent side effects.

The theory of traditional Chinese medicine considersthat headache is attributed to the obstruction of clearorifices due to vessel and collateral contraction andobstruction, or attributed to the failure in nourishingcaused by external evil or internal impairment. Head-ache is also mainly classified into two types in TCMaccording to the etiology and pathomechanism –external headache and inner headache, treating reason-ably by pattern identification with the combination ofpain arresting herbal medicines and pain-killing herbalmedicines. CX is used most, as Zhu Danxi, the famousChinese physician of Yuan Dynasty, ever said: ‘‘CX isindispensable in curing headache. If the patient stilldoesn’t recover, channel guidance herbal medicineshould be added’’, which not only emphasizes theimportance of CX on headache treatment but also

indicates that CX is likely to have pain arresting andkilling actions that make it useful for all patterns ofheadache.

The chemical composition of CX mainly includesCXVO (including ligustilide, senkyunolide, cnidiummlactone, 3-butylphthalide, etc.), alkaloid (includingligustrazine, black ergotine, etc.), and phenols, andorganic acid (including ferulaic acid, chrysophanol,etc.). Ligustrazine was studied most in the previousstudy of CX and has been proved to have strongfunctions of activating cardiovascular system, protectingcerebral and cardiac tissues from damaging by ischemia,and improving hemorheology. However, ligustrazinedid not show the effect of arresting or killing pain.Moreover, it has a very low content in CX. Therefore,ligustrazine is hardly responsible for the curative effectof CX on headache. Then, the chemical compositionand pharmacal effect of CXVO are gradually concernedand studied. Chemical compositions and proportion ofCXVO will change as methods for extraction change, asits main component, known as ligustilide, has quiteunstable structures, which may be easily brokendue to different solvent or high temperature (Li andWang, 2003; Zhou and Li, 2001). The extraction rate of

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Fig. 4. Effect of CXVO on c-fos gene expression in the brain tissue of rats exposure to nitroglycerin (size 10� 40). (a) C-fos gene

expression in brain of rats from the control group, (b) C-fos gene expression in hypothalamus stem of rats from the control group,

(c) C-fos gene expression in brain of rats from the model group, (d) C-fos gene expression in hypothalamus stem of rats from the

model group, (e) C-fos gene expression in brain of rats treated by ergotamine, (f) C-fos gene expression in hypothalamus stem of rats

treated by ergotamine, (g) C-fos gene expression in brain of rats treated by CXVO, (h) C-fos gene expression in hypothalamus stem

of rats treated by CXVO.

C. Peng et al. / Phytomedicine 16 (2009) 25–3432

CXVO also changes simultaneously when the extractionapproach changes (Ruan et al., 2003a, b). Therefore, it isof great significance to control the preparative processand identify ingredients as well as their proportions inCXVO prior to studying it. According to our study, withthe supercritical carbon dioxide fluid extraction, thevolatile oil is well extracted from CX at a high ratewithout ligustilide broken, compared with the hydro-

distillation extraction and organic solvent extraction.Ligustilide obtained from the CXVO by further separa-tion and purification was identified using UV, IR,13C-NMR, and 1H-NMR spectroscopies. The purity ofligustilide gotten by our process is so high as 98.56%that it can be used as reference of ligustilide.

It has been reported in the literature that CXVOhas effects of inhibiting cerebral activity, reducing

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Table 8. Effect of CXVO on the levels of plasma 5-HT, ET, and CGRP of rats exposure to nitroglycerin (X̄� SD).

Group Animal (n) Dose (ml/kg) 5-HT (ng/ml) CGRP (pg/ml) ET (pg/ml)

Control 10 – 145.20719.86 106.86727.62 189.23748.23

Model 10 – 112.07728.09�� 159.67732.39�� 128.17711.51��

Ergotamine 10 1.00mg/kg 140.70722.28D 115.07748.37D 173.07755.97D

CXVO 10 135.00 138.74724.22D 94.78719.55DD 149.85722.46DD

10 90.00 126.14728.00 108.08734.11DD 158.67729.98DD

10 45.00 116.17719.39 131.63745.89 163.81735.86DD

Data were presented as mean7SD.��Po0.01, significantly different compared with the control group.DPo0.05, significantly different compared with the model group.DDPo0.01, significantly different compared with the model group.

C. Peng et al. / Phytomedicine 16 (2009) 25–34 33

autonomic activity of animal, inhibiting activities ofvasomotor center (VMC), respiratory center (APC), andspinal reflex in the medulla oblongata, and is effectivefor experimental epilepsy. In this study, the results thatCXVO could significantly raise the pain threshold ofmice in the hot-plate test and reduce the number ofwrithing response caused by chemical materials show itscentral analgesic effect. The centrally sedative effect issupported by the results that CXVO reduced thespontaneous locomotor activity and had the effect ofsynergism with sodium pentobarbital to prolong thesleeping time in mice. The effect of CXVO on painthreshold of rabbits exposed to hot radiation proves thedirect therapeutic effect of CXVO on treating headache.The opioid peptide gene has been proved to be the targetgene of c-fos protein (Wang et al., 2001); therefore, theimpacts of CXVO on c-fos gene expression in the braintissue and on levels of 5-HT, ET, and CGRP in plasmaof rats with headache caused by nitroglycerin indicatenot only that CXVO is effective for headache, but alsothat the mechanism of analgesic effect may relate toadjusting the level of opioid peptide in the CNS byimpacting c-fos gene expression in the brain tissue,raising levels of 5-HT and ET and reducing CGRP levelin plasma. As contents of 5-HT, ET, and CGRP vary indifferent stages of headache, for example, the 5-HT leveldeclines in the intermediate stage while constantlyascending during the period of onset, and there arecomplicated relationships among the biochemical para-meters. Thus, further research is needed to reveal theexact mechanism.

All the animal models applied in this study havefeatures of clear mechanism, good repeatability, highcontrollability of index and widely used in correlatedstudies. Therefore, the results of our study are stable andreliable enough to effectually prove the efficacy andprobable mechanism of CXVO in treating headache.

To conclude, the present study indicates that CXVOis definitely effective to experimental headache andprobably acts as the main active ingredient of CX incuring headache, the mechanism of which is related to

its effects of sedation, relieving pain and adjusting thelevels of related biochemical transmitters in the tissueand blood. In addition, it is found that CXVO hasactions of relieving fever and promoting blood flow(Chai et al., 1997; Shi et al., 1995), which indicates thatCXVO can simultaneously treat both the root andbranch of headache caused by fever, cerebral ischemia,or disorder of hemorheology and hemodynamics. Aboveall, CXVO is considerably worth studying clinically andtheoretically and is a potential agent for the therapy ofheadache.

Acknowledgements

The authors would like to express their thanks to theKey Laboratory for Standardization of Chinese HerbalMedicine of Ministry of Education and PharmacyCollege of Chengdu University of Traditional ChineseMedicine for financial support, as well as to ProfessorTang Wu-sheng, Professor Jia Ming-ru, and ProfessorLiu Chuang-jie for technical support.

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