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Analgesic effect and mechanism of the three TCM-herbal drug-combination Tou Feng yu pill on treatment of migraine.

ARTICLE INFO

Keywords:

Tou Feng

Yu pill

Baizhi Chuanxiong

Green tea

Migraine

Analgesia

Monoamine neurotransmitter

Vasoactive substances

Vasomotion

ABSTRACT

It is well known that pain is one of the most important characteristics of migraine. Therefore, it is important and interesting to investigate the analgesic effect and mechanism of drugs which are used to treat migraine. Tou Feng Yu pill (TFY) is a traditional Chinese herbal medicine, consisting of the three Chinese herbal drugs Radix Angelicas dahuricae (Baizhi), Rhizome Ligustici (Chuanxiong) and Fenton Cameiliae sinensis (green tea) for the treatment of migraine. In this study, we found that TFY could significantly reduce the writhing times induced by acetic acid and licking foot response induced by formalin, and extend the writhing latent period. But the analgesic effect was not observed at hot-plate test. Meanwhile, experimental migrainous model induced by nitroglycerin was used to investigate the therapeutic effect of TFY. Compared with the control group, the levels of plasma calcitonin gene related to peptide (CGRP), serum nitric oxide (NO) and contents of brain dopamine (DA) in TFY administration groups were significantly decreased, and the levels of plasma endothelin (ET) and contents of brain 5-hydroxytryptamine (5-HT) and norepinephrine (NE) were remarkably increased, also the ratio of ET/NO was clearly corrected. Furthermore, the improving effect of behavior abnormality was observed in TFY administration rats. Meanwhile, isolated vascular ring test indicated that TFY had an significant effect on vasomotion, and antagonize vasospasm; moreover TFY also could increase cerebral blood flow (CBF) and reduce cerebrovascular resistance index (RI) in normal rabbits, which verified the effect of TFY on vasomotion and abnormal hemorheology. All the results indicate that TFY has an effective therapeutical action on migraine, which may be related to three aspects as following: firstly, adjusting the level of neurotransmitters, neuropeptides and vasoactive substances, relieving neurogenic inflammation; secondly, improving vasomotion, increase cerebral blood flow, then improving hemorheology; finally, increasing pain threshold, relieving or preventing headache. These findings provide additional pharmacological information and may contribute for the further study and use of TFY as a phytomedicine.

[c] 2011 Elsevier GmbH. All rights reserved.

1. Introduction

Migraine is a kind of paroxysmal neurovascular dysfuntion, with the character of repeated attack of pulsatile severe headache in unilateral or bilateral head. The epidemiologic survey displays that the incidence of migraine headache in the world was about 8.4-28%, and the incidence in Germany was the highest (Richard et al. 2005). This disease with high incidence, low cure rate and lack of safety and effective treatment, seriously debased living quality of patients. A survey of WHO in 2000 shows that migraine has become one of the chronic diseases which heavily affect the life quality.

The pathogenesis of migraine is very complex, and the doctrines used to explain the mechanism includes vascular theory, trigeminal-vascular theory and neurotransmitter theory (Richard et al. 2005). However, the most acknowledged theory is the trigeminovascular theory. The trigeminovascular theory is one of the most persuasive theories in the pathogenesis of migraine, excessive internal and external stimulation can lead to vascular dilatation or vasomotor dysfunction, and the noxious stimulation accelerates the peripheral branch of trigeminal nerve releasing vasoactive substances and neurotransmitters, such as calcitonin gene-related peptide, P substance and 5-hydroxytryptamine, all of these will cause neurogenic inflammation, further lead to headache (Buzzi 1992; Welch et al. 2003).

In recent years, investigations on drugs for migraine have received more attention and acquired certain therapeutic effect. But there are many problems on migraine treatment, such as resistance, addiction, adverse reactions. In contrary, according to Chinese herbal theory, interactions among the different herbs in a formula exert a synergistic effect and neutralize potential toxicity and side effects of the individual constituents (Bensky and Barolet 1990; Bensky and Gamble 1993). The concept of Chinese herbal formula in phytotherapy has been paid more attention recently (Ma et al. 2009). Many Chinese herbal formulas have good clinical efficacies in the treatment of migraine (Wang et al. 2009; Liu et al. 2001). Meanwhile, the effect of many Chinese herbal formulas on treating migraine had been confirmed by experimental study (Zhao et al. 2009).

Tou Feng Yu Pill (TFY) originated from Chuanxiong Chatiao Powder which was described in the Taiping Huimin Heji Jufang, a well-known formula book edited by the office of "He Ji Ju" of the Song Dynasty (960-1279). It is composed of three herbs, Baizhi, Chuanxiong and green tea in the ratio of 14:2:1 (w/w/w), among them, the frequency of match usage of Baizhi and Chuanxiong was the highest on history of treating migraine more than 1000 years. TFY was commonly used to treat migraine It was firstly recorded in Pharmacopoeia of PRC 1977 edition (The Pharmacopoeia Commission of PRC 1977), and in drug standards of PRC health ministry (The Pharmacopoeia Commission of PRC 1994). However, the study of TFY on treating migraine was mostly used in clinic. The pharmacodynamic material bases and effect mechanism of TFY were not yet understood clearly and there was as yet a lack of rigorous scientific evaluation about it. Therefore, a classical animal model of migraine that have clear mechanisms was adopted in this study, then investigated systemically about the analgesic effects, as well as the direct curative effect of TFY on experimental migraine and potential mechanism underlying for the purpose of revealing the pharmacodynamic action and mechanism of TFY on treating migraine, and provided pharmacological evidence for further research and exploitation of TFY.

2. Materials and methods

2.1. Animal preparation

Both Kunming mice (18-22 g, Grade II) and Sprague-Dawley rats (230-250g, Grade II) with either sex were obtained from the experimental Animal Research Institute of medical scientific academy in Sichuan (Chengdu, China). Japanese white rabbits (2.0-2.5 kg, Grade II) were provided from the farm of experimental animal committee in sichuan. Animal welfare and experimental procedures were carried out in accordance with the international ethical guidelines and related ethical regulations of our university. The animals were purchased 1 week before the experiment and allowed to acclimate. They were kept in plastic cages at 24 [+ or -] 2 't with free access to pellet food and water and on a 12 h light/dark cycle.

2.2. Preparation of the extracts for Chinese herbs

TFY is composed of three herbs, Baizhi, Chuanxiong and green tea in the ratio of 14:2.1 (w/w/w), which were purchased from traditional herbal market of Hehuachi in Chengdu. All the medicinal materials were consistent with the standard of Chinese Pharmacopoeia (2005 Edition) Volume II. These three herbs were first extracted by ethanol and after evaporating ethanol, the residue was dissolved in water and then again extracted with ethylacetate. The extracts obtained has a chocolate brown and disseminated appearance with a specific odor. Pi eliminary analysis about this extracts was done by Shimazu LC-10AT series HPLC instrument (Shimazu, Japan). The chromatographic separation was carried out on Hypersil ODS-C13 (4.6 mm x 250 mm, 5 p.m) with the column temperature at 30[degrees]C. The mobile phase was acetonitrile (A) with 0.1% trifluoroacetic acid (B) in a gradient mode which was described as 0-20 min: 10-20%; 20-60 min: 20-60%; 60-65 min: 60%. The flow rate was 1.0 ml/min and the UV wavelength was 254 nm. Retention times of purified TFY were obtained directly on the chromatography.

The HPLC results were shown in Fig. 1, and the main components of TFY were determined, including caffeine, ferulic acid, epigallocatechin-3-gallate (EGCG), oxypeucedanin hydrate, oxypeucedanin, imperatorin, phellopterin, cnidilin and isoimperatorin.

2.3. Chemicals and treatment of animals

Chemical reference substance such as caffeine, ferulic acid, imperatorin and isoimperatorin were provided by the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); EGCG was purchased from Chengdu Mansite Biological Technology Co., Ltd. (Chengdu, China); Chemical standards for oxypeucedanin hydrate, phellopterin, cnidilin and oxypeucedanin were self made, identified by MS and 1 H NMR, the purity was > 98.0%.

The TFY extracts (180.00, 90.00, 45.00 mg/kg; 108.00, 54.00, 27.00 mg/kg), were given orally to mice, rat and rabbit for administration. Morphine hydrochloride injection was purchased from Qinghai Pharmaceutical Co., Ltd. (Qinghai, China); Aspirin entericcoated tablet was purchased from Ouyi Pharmaceutical Co., Ltd. (Shijiazhuang, China); Tramadol hydrochloride tablet was provided by Ouyi Pharmaceutical Co., Ltd. (Shijiazhuang, China), ergotamine was provided from Shanghai Xingyi Pharmaceutical Co., Ltd. (Shanghai, China); flunarizine was provided from Jinhua Huixing Pharmaceutical Co., Ltd. (Shanxi China). All other chemicals were analytical grade. The control or model group received the same volume of distilled water.

2.4. Methods

2.4.1 Hot-plate test

This test was performed according to the method which announced by Lanhers et al. (1991). The hot-plate test was carried out at a constant temperature of 55 [+ or -] 0.5 The main reaction is paw licking. The time in seconds between the platform and reaction was recorded as the response latency, it is also named pain threshold. The mice pain threshold greater than 30 s or less than 5 s were eliminated. The mice were tested twice for prior to the administration and the mean pain threshold of each mice was considered as their basic pain threshold. The pain threshold was determined at 15 min, 30 min, 60 min, and 120 min after administration of drug. If the reaction time was more than 60 s, the pain threshold was recorded as 60 s. An increase of pain threshold indicates an analgesic effect.

2.4.2. Writhing reflex induced by acetic acid in mice

In the writhing test was performed according to the method described by Xu (2002). The mice received 0.6% acetic acid solution in normal saline injected intraperitoneally at the dosage of 10 ml/kg 30 min after drug administration. Then the incubation period of writhing reflex was observed and the number of abdominal writhing response for a period of 15 min was immediately recorded. Expanding hind limb, constricting abdomen and raising the croup were the typical expression of writhing reflex. A prolong of the incubation period and decrease of writhing response mean an analgesic effect.

2.4.3. The formalin test

We adopted the method previously described (Hunskaar and Hole 1987). All the mice were received 5% formalin solution in nor mal saline injected hypodermically in the right hindlimb at the dosage of 1 ml/kg 30 min after drug administration. After injection, the mice was put into the hot-plate at a constant temperature of 27[degrees]C. Then total time of licking foot response was immediately recorded for a period of 5 min. A decrease of total time shows an analgesic effect.

[FIGURE 1 OMITTED]

2.4.4. Nitroglycerin-induced migraine in rats

In this study, Sprague--Dawley rats were chosen and divided into six groups. Successive intragastric administration for 1 week. The test was clone using a previously reported technique (Tassorelli et al. 1995), nitroglycerin was injected subcutaneously at the dosage of 10 mg/kg after the last administration. Then, rat appeared the phenomena of head-twitch, ear rosacea, forelimb clawing head frequently, more climbing cage activity, dysphoria, and so on. These phenomena lasted about 3 h. Afterwards, rat manifested as an inactive state. These indicated that the migi aine model was established successfully. After model establishment, behavior and symptom of rat models were observed continuously for 3 h (30 min as a time interval), and the number of head-twitch was the main ethology index. Four hours later of model establishment, all of the rats were anaesthetized with 10% chloral hydrate injected intraperitoneally at the dosage of 3.5 ml/kg, and blood sample was collected from jugular vein, used to determine the levels of serum NO by colorimeti is method and plasma ET and CGRP by radioimmunoassay method. After that, the brain tissues of these rats were dissected and washed by saline. The brain stem and the hypothalamus were cut and prepared into homogenate, used to assay the levels of 5-HT, 5-HIAA, NE and DA with fluorometric method.

2.5. Isolated vascular ring test

We adopted the method previously described (Suvitayavat et al. 2005). Bilateral common carotid arteries were took out from thetic SD rats, then vascular were cut into rings of about 4-6mm in width and mounted in organ baths containing normal Krebs physiological solution with the following composition ( mM): NaCI 118.0, KCl 4.7, CaC[1.sub.2 ]2.25, MgS[O.sub.4] 1.18, KH2P[O.sub.4] 0.118, [NaHCO.sub.3] 24.9, glucose 11.1, one section was removed vascular endothelium, vascular endothelium of another section was retained. The bath solution was maintained at 37[degrees]C and bubbled continuously with 95% 02 + 5% C[O.sub.2]. Vascular rings were equilibrated for 60 min at low tension, then equilibrated for 60 min at a resting tension of 2 g before specific experimental protocols were initiated. Responses were recorded by PowerLab recording system (AD Instruments, Australia). Then endothelial integrity was tested. Rings were allowed to achieve maximal tension by exposure to high r (90 mM), if the rings were exhibited more than 50% relaxation to acetylcholine (10 which means endothelium-intact; unrelaxation or lower than 50%, which means endothelium-deleted. Subsequently, the rings (including endothelium-intact and endothelium-deleted) were divided into five groups. The rings were precontracted with high IC. When contraction amplitude reached the maximum, and the relaxant responses were recorded by adding different doses of TFY to the tissue bath in 10 min intervals. Inhibition rate of contraction was calculated according to the following formula. Inhibition rate of contraction (%)= 100% x (mean tension of pre-administration - mean tension of post-administration)/mean tension of pre-administration.

2.6. Effect of TFY on cerebral blood flow in normal rabbits

Rabbits (fasting for 12 h) were anaesthetized with 3% pentobarbital sodium injected via ear vein at the dosage of 30 mg/kg. Mean arterial pressure (MAP) was determined via femoral artery by biological signal analysis system (Biopac, USA), and doppler flowmetry was used to measured the cerebral blood flow velocity (V) via right common carotid artery These indexes were recorded before (0 h) and after 10, 30, 60 and 120 min of duodenal administration. And vessel diameter (d) and one side brain weight (W) were measured in the end. Then cerebral blood flow (CBF) and cerebrovascular resistance index (RI) was calculated according to the following formula. Blood flow amount per unit time (Q ml/min) = 60 x V x ([pi] [d.sup.2]/4), GE (m1/100 g Wmin)= 100 x 0/W, R1= MAP/Q.

[FIGURE 2 OMITTED]

2.7. Statistical analyses

Data were expressed as mean values [+ or -] standard error. All data analysis was tested by one-way analysis of variance (ANOVA) for multiple comparisons with the Dunnett's t-test. The differences were considered to be significant if p < 0.05.

3. Results

3.1. Hot-plate test

Results of pain threshold in the hot plate test are shown in Fig. 2. Compared with the control group, all the doses of TFY could not raise the pain threshold significantly after given to the mice.

3.2. Acetic acid-induced writhing test

The numbers of writhing response are shown in Fig. 3. Compared with the control group, the administration of the three doses of TFY remarkably prolonged pain latency and educed the number of writhing response in mice caused by acetic acid, the effect of prolonging pain latency is dose dependent.

3.3. Formalin-induced pain test

The foot licking time are shown in Fig. 3. Compared with the model group, the administration of the three doses of TFY significantly reduced foot licking time, and the effect is dose dependent.

3.4. Nitroglycerin-induced migraine in rats

3.4.1. Effect of TFY on head-twitch number in nitroglycerin-induced migraine rats

The results of head-twitch number in nitroglycerin-induced migraine rat are shown in Fig. 4. Compared with the control group, nitroglycerin treatment obviously increased head-twitch number of model rats. Compared with the model group, the administration of the three doses of TFY obviously reduced head-twitch number, and this effect can last 150 min.

[FIGURE 3 OMITTED]

3.4.2. Effect of TFY on vasoactive substances in nitroglycerin-induced migraine rats

As shown in Table 1, the levels of plasma CGRP and serum NO of rats in the model group were increased significantly due to the injury of nitroglycerin, and the ET/NO ratio in the model rats were reduced significantly, compared with that of the control group. But the levels of plasma ET were not changed remarkably.
Table 1

Effect of TFY on vasoactive substances in nitrogfyccrin-induced
migraine rats.

Group Dose CGRP NO(|xmol/l) F.T(PG/mi) ET/NO
 (mg/kg) (PC/ml)

Control - 160.16[+ or 23.04 [+ or 117.09 [+ 5.07 [+
 -] 28.58 -] 3.50 or -] or -]
 21.20 0.65

Model - 213.41 [+ 126.73 [+ or 113.82 [+ 0.92
 or -] -] 18.03 ## or -] 9.10 -0.16 **
 15.22 ##

Ergotamine 1.00 87.45 [+ or 72.57 [+ or 143.16 [+ 1.72 -t
 -] 21.36 ** -] 6.72 ** or -] 0.31 **
 13.13 **

 108.00 99.86 + 71.71 [+ or 152.88 [+ 2.16 +
 23.27 ** -] 8.76 ** or 0.33 **
 -] 9.39 **

TFY 54.00 81.25 [+ or 89.68 [+ or 146.48 [+ 1.63 +
 -] 18.76 ** -] 6.34 ** or -] 0.22 **
 23.23 **

 27.00 98.15 [+ or 96.31 [+ or 132.90 [+ 1.39 [+
 -]20.20 ** -] 10.79 ** or -] or -]
 8.27** 0.17 **

Data were presented as mean [+ or -] SD, (n = 10).
## p < 0.01 significantly different compared with the control group.
* p < 0.05 significantly different compared with the model group.
** p<0.01 significantly different compared with the model group.


The administration of TFY at all doses reduced the levels of plasma CGRP and serum NO, increased the levels of plasma ET and the ET/NO ratio, compared with the model group, which were treated by distilled water. The effect of TFY increasing ET and reducing NO shown to be dose dependent.

3.4.3. Effect of TFY on monoamine neurotransmitter in brain of nitroglycerin-induced migraine rats

From Table 2, compared with that of the control group, we can see that the contents of monoamine neurotransmitter in rat brain, including 5-HT and NE were decreased significantly due to the nitroglycerin treatment in the model group, while the contents of 5-HIAA and DA were no significant change after the treatment.
Table 2

Effect of TFY on monoamine neurotransmitter in brain of
nitroglycerin-inducecl migraine rats.

Group Dose 5-HT(ng/g) 5-HIAA(ng/g) DA(ng/g) NE(ng/g)
 {mg/kg)

Control 1227.90 = 823.47 + 2484.06 521.55 [+
 88.00 89.92 [+ or -] or -]
 410.92 34.22

Model _ 1045.89 + 854.23 x 2125.87 455.23 [+
 97.18 ** 116.66 [+ or or -]
 -]337.98 28.63 ##

Ergotamine 1.00 1138.34 [+ 619.32 [+ or 93739 [+ 587.34 [+
 or -] 29.42 ** or -] or -]
 -] 47.27 * 197.23 ** 34.73 **

 108.00 1336.55 [+ 858.69 [+ or 982.36+ 619.12 [+
 or -] -] 31.72 137.10 ** or
 44.39 ** -]40.68 **

TFY 54.00 1284.16 [+ 761.34 [+ or 898.77 [+ 639.48 [+
 or -] -] 58.41 or -] or -]
 154.91 * 202.83 ** 38.56 **

 27.00 1278.40 x 871.83 [+ or 1034.10 616.08 +
 94.14 ** -] 87.76 [+ or -] 48.99 **
 175.03 **

Data were presented as mean [+ or -] SD (n = 10).
## p<0.01 significantly different compared with the control group,
* p < 0.05 significantly different compared with the model group.
** p<0.01 significantly different compared with the model group.


[FIGURE 4 OMITTED]

Three doses of TFY could all increase the contents of 5-HT and NE remarkably, and reduced the contents of DA. But the contents of 5-HIAA had no significant change at all doses of TFY.

3.4.4. Effect of TFY on vasospasm induced high IC' in isolated vascular

As shown in Table 3, whether the endothelium was intact or not, strong responses were observed treated with high IC' stimulation. After treating with TFY, vasospasm induced high ICE were improved obviously. Moreover, the improvement of TFY shown to be a dose dependent and endothelium-independent.
Table 3

Effect ofTFYon vasospasm induced high K* in isolated vascular.

Group Dose Inhibition rate of Inhibition rate of
 (mg/ml) contraction contraction
 (endothelium-intact) (endothelium-deleted)

Control - 5.071 [+ or -] 7.992 7.743 [+ or -] 6.486

Flunarizine 6.00 69317 [+ or 78.239 [+ or -]
 -] 12.289 ** 20.065 **

 300.00 94.395 [+ or -] 86.276 [+ or -]
 6.136 ** 9.689 **

TFY 200.00 65.573 [+ or -] 67.724 [+ or -]
 4.448 ** 6.193 **

 100.00 66.507 [+ or -] 66.312 [+ or -] 14.187 **
 16.495 **

Data were presented as mean [+ or -] SO (n = 6), * p<0.05 significantly
different compared with the control group. ** p<0.01 significantly
different compared with the control group.


3.4.5. Effect of 'FEY on CBF and RI in normal rabbits

The results are shown in Tables 4 and 5. Compared with the control group, CBF were increased obviously at all times after administration of TFY at high and middle dose; and TFY also could reduce RI at all times after administration except 30 min. These results indicated that TFY had an improving effect on cerebral blood flow.
Table 4 Effect of TFY on CBF in normal rabbits.

Group Dose (m Pre-administration Post-administration/change
 g/kg) (ml/min) rate (%)

 10 min

Control - 256.00 [+ or -] -1.74 [+ or -]6.82
 136.25

Flunarizine 6.00 168.60 [+ or 13.14 [+ or -] 4.30 **
 -]29.86

 108,00 174.71 [+ or 12.41 [+ or -]4.76 **
 -]39.76

TFY 54.00 163.24 [+ or -] 11.60 [+ or -] 6.99 **
 27.95

 27.00 154.98 [+ or -] -0.28 [+ or -] 2.87
 20.48

Group

 30 min 60 min 120 min

Control 2.76 [+ -2.55 [+ -2.12 [+
 or -] or -] or -]
 13.60 19.72 15.44

Flunarizine 38.24 [+ 51.05 [+ 69.76-
 or -] or 19.74 **
 10.12 ** -] 9.59 **

 39.32 [+ 48.44 [+ 40.84 [+
 or -] or -] or -]
 17.93 ** 20.71 ** 18.09**

TFY 19.30 [+ 28.87 [+ 24.79 [+
 or -] or -] or -]
 9.29 * 12.68 ** 13.12 **

 3.04 [+ 5.55 [+ 8.82 [+
 or -] or -] or -]
 6.45 11.61 15.43

Data were presented as mean [+ or -] SD, (n-6); Change rate= 100 x
(post-administration value-before administration value)/
pre-administration value. * p<0.05 significantly different compared with
the control group. ** p<0.01 significantly different compared with the
control group.

Table 5

Effect of TFY on Rl in normal rabbits.

Group Dose Pre-administration Post-administration/
 (mg/kg) (ml/min) change rate (%)

 10 min

Control - 0.399 [+ or 5.75 [+ or -] 9.61
 -]0.165

Flunarizine 6.00 0.523 [+ or -16.04 [+ or -] 8.83 **
 -]0.116

 108.00 0.540 [+ or -13.86 [+ or
 -]0.091 -] 13.48 **

TFY 54.00 0.627 [+ or -8.79 [+ or -] 4.6l **
 -]0.123

 27.00 0.692 [+ or -1.70 [+ or -] 4.41
 -]0.081

Group

 30 min 60 min 120 min

Control 1.81 [+ 4.12 [+ or 7.79 [+ or
 or -] 18.05 -] 13.73
 -] 16.36

Flunarizine -36.49 [+ -46.93 [+ -55.19 [+
 or or or -]
 -] 13.94 * -] 13.57 ** 11.23 **

 -27.60 [+ -32.00[+ -26.25 [+
 or or -] or
 -] 14.04 14.15 ** -] 15.16 **

TFY -15.32 [+ -20.05 [+ -15.09 [+
 or or or -]
 -] 5.06 -] 6.16 * 10.00 **

 -8.08 [+ -8.30 [+ -10.64 [+
 or or -] 8.99 or
 -] 6.41 -] 8.2l **

Data were presented as mean [+ or -] SD (n = 6); formula is same as
Table 4.
* p < 0.05 significantly different compared with the control group.
** p < 0.01 significantly different compared with the control group.


4. Discussion

Migraine is induced by the long-term stimulation of spiritual factors and drug side effect (Schafer 1990). These harmful stimulations stimulate trigeminal nerve terminals to release vasoactive peptides, which leads to vasomotion dysfunction and plasma protein extravasation. And more seriously it will develop into neurogenic inflammation. All of these noxious stimulations induce pain by nerve reflex. According to these problems, ergotamine, non-steroidal anti-inflammatory drugs, angiotensin receptor antagonist, estrogen, triptans, and so on. However, these drugs need to be taken chronically, and is most likely associated with side effect and addiction. Furthermore, as high incidence, low cure rate and random attack, migraine has badly reduced the quality of patients' daily life.

The theory of TCM believes that migraine is attributed to the pathogenic wind-cold, blood stasis and failure in nourishing, also this is the reason of migraine repeated attack. TFY consists of herbs known in TCM theory to dispel wind, activate blood and relieve pain (The Pharmacopoeia Commission of PRC 1994). Li Dongyuan, the famous Chinese physician of Yuan Dynasty, ever said: "Chuanxiong is indispensable in curing headache, if the patient still doesn't recover, Baizhi should be added". Modern pharmacological research showed that Chuanxiong and Baizhi have the effect of analgesia, spasmolysis (Liu et al. 2002; Nie et al. 2002; Wang et al. 2005) and improving abnormal blood Theology. Chuanxiong and green tea can protect cardio-cerebrovascular (Ling et al. 2008; Finger et al. 1992).

Long-lasting chronic pain is the main symptom of migraine. In our study, the model of formalin-induced pain was established to evaluate the analgesic effect of TFY, because of the persevering neuropathic pain induced by formalin is similar to Long-lasting chronic pain which is close to migraine. The results showed that the mice appeared pain sensitive symptoms after formalin injection, such as foot licking, limping, jumping and bray. The administration of TFY could significantly reduce the time of foot licking and improve per severing nociceptive behavioral response. Meanwhile, we adopted two classical methods of HAc-induced writhing response and hot plate test, and the effects of prolonging writhing latent period and decreasing writhing frequency were observed after the administration of TFY. Based on the above results, TFY could increase pain threshold and showed analgesic effects. But unusually, TFY had no obvious effect on pain threshold of mice caused by hot stimulus, and similar experiment was repeated three times.

To investigate the therapeutic effects of migraine, we chose the experimental migraine model induced by nitroglycerin that was widely accepted. Interestingly, the rats would appear a series of discomfort on head after being injected with nitroglycerin, which resembles the attack of human migraine. In these discomfort symptoms, "head-twitches" was more obvious than other symptoms, which was higher frequency and measurement. Therefore, we considered that behavioral evaluation was an important observation index in experimental migraine models, and also was the signal of success of migraine models. The experimental results indicated that the number of head-twitches were obviously decreased with TFY administration, the behavior abnormality was significantly improved.

As we know, neurogenic inflammation is the core link in the process of migraine, which also is the core of trigeminal-vascular theory. And the cerebral vascular dysfunction which is induced by disorder of vasoactive substances play an important role in neurogenic inflammation. For instance, as the strongest vasodilator substance, CGRP also is endogenous algogenic substance (Zhang and Zeng 2000), which is widely distributed in trigeminal nerve. CGRP is released in huge quantity when trigeminal nerve is stimulated. The CGRP released has the function of vasodilatation, plasma protein extravasation and mast cells degranulation. Then neurogenic inflammation is caused by these changes (Julia and Bueno 1997); CGRP has the inhibition of protease (Schaible et al. 2006), which can slowdown the elimination of kinins and substance P and aggravate pain action. Besides that, CGRP release is enhanced by the bioactive substances such as NO in trigeminal nerve. And it is noteworthy that the decreased secretion of neurotransmitters (including 5-HT and NE) weaken the descending inhibitory effect of the modulation of nociceptive transmission. Meanwhile, the sensitivity of dopamine system plays the same effect in pain modulation. These abnormal phenomenon was also observed in migraine rats by nitroglycerin (Peng et al. 2009).

Based on above views, we aimed on vasoactive substances and neurotransmitters, the investigated the effect of TFY on migraine. The results obtained in the present study showed that TFY could significantly decrease the plasma CGRP and increase the plasma ET in the migraine model, correct deviation of the ET/NO ratio; furthermore, we also found that levels of 5-HT and NE of treated rats were higher than the migraine rats, and the content of DA was lower than the model rats. The results obtained in the present study showed that the level of plasma ET increased and the level of plasma CGRP decreased in each TFY treated rats with a negative correlation between the levels of plasma ET and CGRP, and the ET/NO ratio tended to be normal; furthermore, we also found that levels of 5 HT and NE of treated rats were higher than the migraine rats, and the content of DA was lower than the model rats. The analgesic effect of experimental migraine may relate to adjusting the level of bioactive substances and neurotransmitters, which is conducive to alleviate neurogenic inflammation. But what was noteworthy is that the contents of CGRP, 5-HT and ET change in different stages of headache, for example, the level of 5-HT increased during the onset while accordingly decreased during the intermediary stage. And there were complicated relationships among the biochemical parameters, which was needed to further research and reveal the exact mechanism.

Moreover, numbers of clinical studies found that cerebral vascular dysfunction usually accompanied with migraine (Brennan and Andrew 2010). And it appears a series symptoms, such as cerebral angiospasm, overstretch, abnormal hemorheology, cerebral pial microcirculation disturbance, low compliance and so on. These abnormal phenomenon would stimulate cerebral vessels and trigeminal nerves to release harmful substances, which would induce or aggravate migraine (Denuelle et al. 2008; Dalkara et al. 2010); and also some bioactive substances like CGRP and NO would stimulate cerebrovascular and trigeminal nerve, lead to vascular dysfunction and neurogenic inflammation; which would fall into a vicious circle (Williamson and Hargreaves 2001.). In this study, we also adopted vascular test in vitro and vivo, then the improvement Of TFY on vasomotion was observed in these test. We found that TFY had a significant spasmolysis effect on vasospasm of rat common carotid artery induced by high le, also could increase cerebral blood flow, reduce cerebral vascular resistance. And the previous study showed that TFY could improve mice cerebral pial microcirculation disturbance induced by macromolecule dextran (Zeng et al. 2008). These results also indicated that the therapeutic action may relate to adjusting the vasomotor function and abnormal hemorheology. Meanwhile, this effect is corresponding to the regulation of TFY on neurotransmitters and vasoactive substances, and which also verified the important position of trigeminal-vascular theory in migraine.

In summary, WY, a formula comprised of Baizhi, Chuanxiong and green tea, showed an anti-migraine effect via regulation the levels of neurotransmitters, neuropeptides and other bioactive substances to down-regulate the releasing of proinflammatory-propain substances and vasoactive substances, correct relative disequilibrium, consequently lessens the further damage of vascular and neurons, and relieve the neurogenic inflammation. Besides, the effect of increasing pain threshold and improving vasomotion ofTFY for amelioration of migraine could not be excluded.This suggested that TFY may be beneficial as an adjuvant to conventional drugs in the treatment of migraine and related vascular headaches.

Acknowledgement

The present study was supported by the fund of Key Projects in the National Science and Technology Pillar Program during the Eleventh Five-Year Plan Period (2006BA106A18-15, 2007BA140B03 and 2007BA140B05).

0944-7113/$ - see front matter[C] 2011 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2011.01.008

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Jia-chuan Li, Xiao-fei Shen, Xian-li Meng *, Yi Zhang, Xian-rong Lai

Chengdu University of Traditional Chinese Medicine. No. 1166, Liu-tai Road, Wenjiang District, Chengdu 61 11 7.3, China

* Corresponding author. Tel.: +86 028 61800158; fax: +86 028 61800158. E-mail address: 163.com (X.-I. Meng).
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Author:Li, Jia-chuan; Shen, Xiao-fei; Meng, Xian-li; Zhang, Yi; Lai, Xian-rong
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jun 15, 2011
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