ANIMAL MODELS TO INVESTIGATE//VALIDATE THE TRADITIONAL USAGE OF L. CHUANXIONG

V. ANIMAL MODELS TO INVESTIGATE//VALIDATE THE TRADITIONAL USAGE OF L. CHUANXIONG

A. Pharmacokinetics of Ligusticum Mixtures The pharmacokinetics of most herbs and formulae used in traditional Chinese

medicine (TCM) are not well understood. Mixing many different herbs together into complex formulations is a common practice in TCM. The effects of mixing different herbs together on the pharmacokinetics of individ- ual compounds are to a large extent unknown. One of the rare efforts in this area was the attempt to investigate herb-herb interactions and the pharma- cokinetics of L. wallichii in rat models with the use of both healthy and diseased rats (21). The results demonstrated that the pharmacokinetics of the

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properties. The investigators compared the presence in serum of two known constituents (tetramethylpyrazine and ferulic acid) of the herb after oral administration of Ligusticum alone and after administration of a mixture of Ligusticum and Salvia miltiorrhiza. Tetramethylpyrazine and ferulic acid were detected with HPLC, MS, and nuclear magnetic resonance (NMR). The

absorption (Ka), transport (K 21 ), and distribution (Vc/F) of these two compounds were significantly lower when the second herb was present. Since Ligusticum and S. miltiorrhiza are commonly used together, the pharmaco- kinetics of these and other components in each herb are likely to be mutually affected when they are combined, thereby affecting their eventual toxicity and efficacy.

B. Efficacy Studies with the Crude Extract

A limited number of studies have been performed in Chinese laboratories to demonstrate the efficacy of the herb on a particular illness/treatment based on the use of the crude extracts. Most of these studies were unable to demonstrate either the specific molecular mechanism or the mode of action of the herbal extracts. The nature of the bioactive compound was also not known.

1. Muscle Function Chuanxiong was administered to dogs to test its efficacy on muscle function,

and specifically to see whether the use of a local or systemic treatment on muscle graft will increase the function of the transplanted muscle (22). The investigators subjected adult dogs to an orthotopic replantation of their bilateral rectus femoris muscles by microneurovascular anastomoses and split the subjects into a postoperative treatment group (with/without administra- tion of chuanxiong) and a control group. They reported that therapy improved muscle function and morphology and maximal tetanic tension of the transplanted muscle. This study concluded that the combination of localized therapy and systemic injection of chuanxiong had a favorable effect on nerve regeneration as well as muscle function after a muscle transplant.

2. Effects on Central Nervous System Chuanxiong was also reported to reduce cerebral ischemia (23) in a rabbit

carotid occlusion model. Occlusion of the bilateral carotid arteries of rabbits produced bilateral partial cerebral ischemia, resulting in increased plasma and cerebrospinal fluid levels of the marker, dynorphin. L. wallichii Franch (ligusticum) pretreatment to the test group resulted in definite improvement of dynorphin levels in plasma and cerebrospinal fluid, suggesting that the severity of brain ischemic damage and neurological dysfunction in ligusticum- treated animals was less than that of the saline-treated group. Neither the active component nor mechanisms of action were investigated.

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3. Renal Effects L. wallichii has been reported to reduce the toxicity of cyclosporine A on renal

function (24). Infusion of cyclosporine A to Sprague-Dawley rats resulted in a significant fall in glomerular filtration rate and renal plasma flow as well as an increase of plasma renin activity, angiotensin II level, and percentage of platelet aggregation. Treatment with 20% L. wallichii before cyclosporine A infusion significantly prevented the decline of glomerular filtration rate and renal plasma flow as well as enhancement of platelet aggregation induced by cyclosporine. These results suggested that L. wallichii may be beneficial for the treatment of acute nephrotoxicity induced by cyclosporine A.

4. Other Effects Asthma andBronchial Smooth Muscle. L. wallichii mixture was also

reported to inhibit bronchospasm induced by histamine and acetylcholine in guinea pigs. The incubation period from antigen inhalation to asthma attack could be delayed by the L. wallichii mixture and the incidence of asthma and its mortality were reduced significantly in guinea pigs compared with controls (25).

Skin Permeability. Ligustici chuanxiong rhizoma (senkyu) ether extract has been reported to enhance permeability of moderately lipophilic compounds into the skin (26,27).

C. Efficacy Studies with the Use of Pure Compounds Derived from Ligusticum

With the characterization of components from the L. chuanxiong, some experiments have been performed to identify their biological function. The most well documented are tetramethylpyrazine, ferulic acid, and butylide- nephthalide.

1. Cardiovascular Effects Many investigators have focused on the cardiovascular effects of ligusticum

since the traditional use of chuanxiong was to improve blood circulation. Tetramethylpyrazine isolated from L. wallichii Franch has been shown to have positive effects on portal-hypertensive rat models (28). Sprague-Dawley rats were used and portal hypertension was induced by partial ligation of the portal vein. Two weeks after the ligation, the rats were cannulated for measurement of mean arterial pressure, portal venous pressure, cardiac index, and heart rate. After the administration of tetramethylpyrazine to the rats, a dose-dependent reduction of portal venous pressure was observed and the total peripheral resistance was also significantly reduced.

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Ferulic acid displayed a dose-responsive bradycardiac effect on pento- barbital-anesthetized Wistar rats, with no significant effects on blood pres- sure. Other effects observed from these experiments were the inhibitive effect on tachycardia and lack of activity on arterial pressure induced by phenyl- ephrine. These results indicated that ferulinolol was a h-adrenoreceptor competitive antagonist, as it could competitively antagonize isoproterenol. Ferulinolol also demonstrated relaxant properties with isolated atria and trachea from reserpine-treated guinea pig, and at higher concentrations the compound had partial h2 agonist activity (29).

Phthalide dimers from L. chuanxiong (tokinolide B, levistolide A, ligustilide, and senkyunolide P) were able to relax KCl-induced contraction of rat thoracic aorta and reduced KCl-induced perfusion pressure of rat mesenteric arteries (30). Coniferylferulate reduced methoxamine-induced perfusion pressure on rat mesenteric arteries. The phthalide dimers tokino- lode B and senkyunolide P and the phthalides senkyunolide, buthylphtalide, and cnidilide can decrease blood viscosity. These suggested that the herb may have important functions for activating the blood circulation and removing blood stasis. The chloroform fraction of Cnidium rhizome (Senkyu) exerted potent negative inotropic and chronotropic effects on isolated guinea pig atria (31). Contraction was attenuated by two major components in the chloroform fraction, ligustilide and senkyunolide, although heart rates remained unaf- fected by these components. Other effects of the chloroform fraction include changes in resting potential and the upstroke velocity of the action potentials.

Butylidenephthalide was reported to have a selective antianginal effect without changing blood pressure. Experiments performed to determine the mechanism of this action suggest that butylidenephthalide inhibited calcium release from calcium stores more selectively than calcium influx from extra- cellular space via voltage-dependent calcium channels (32). The inhibition by butylidenephthalide of calcium release from KCl-sensitive calcium stores might be similar to its inhibition of calcium release from phenylephrine- sensitive calcium stores. Senkyu phthalides have also been described as having antiarteriosclerotic properties (33). Synthetic butylidenephthalides have also been described to have inhibitory effects on mouse aorta smooth muscle cells in vitro (34).

Butylidenephthalide have been reported to strongly increase the pe- ripheral blood flow in rats (35) and may increase the dermal absorption of radiolabeled butylidenephthalide in hairless mice (36). The results of these studies indicated that upon dermal application, the radiolabeled compound quickly permeated through the skin into the peripheral circulation. The half- life was approximately 1 hr, while the plasma concentration peaked at 2 hr postapplication. About 80% of the butylidenephthalide was excreted in the urine 24 hr postapplication, as a cystine conjugate and 5% was excreted in the

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feces, indicating that butylidenephthalide was not accumulated in the skin. If substantiated in humans, these studies show that butylidenephthalide may be developed into a form of treatment by skin application for an increase in blood flow.

The above studies indicate that extracts from ligusticum have potent biological activity and bioactive compounds contained therein may be developed into novel therapies for patients with high risk of cardiovascular diseases.

2. Antiplatelet Properties Tetramethylpyrazine may have antiplatelet activity. The effect of this agent

on platelet activation, aggregation, and thrombus formation occurring under various flow conditions was thought to be mediated by von Willebrand factor and platelet receptor proteins GP IBa and GP IIb/IIIa (37). Inhibition of platelet activation, aggregation, and thrombus formation under high sheer rates were observed. These findings suggest that tetramethylpyrazine may be effective for treating patients with thrombotic disease (38).

3. Anti-Inflammatory Properties Tetramethylpyrazine and ferulic acid were reported to display anti-inflam-

matory and analgesic effects (16). When administered to guinea pigs with histamine/acetylcholine-induced bronchospasm, Ligusticum was found to decrease plasma levels of thromboxane B2, relax tracheal muscle, increase the forced expiratory volume, and inhibit the synthesis and release of thromboxane A2 with no adverse side effects. It has recently been reported that tetramethylpyrazine had an inhibitory effect on the elevation of aqueous

flare induced by transcorneal application of prostaglandin E 2 agonists (39). Tetramethylpyrazine was able to inhibit prostaglandin E 2 -induced flare. Thus tetramethylpyrazine may have applicability to treat aqueous flare; as prosta- glandin E 2 is an important modulator in ocular inflammation.

4. Uterine Effects Tetramethylpyrazine and ferulic acid have been suggested as therapeutic

agents for woman with uterine hypercontractility and primary dysmenorrhea. These suggestions were based on rat uterine experiments conducted by Ozaki and Ma in 1990. In their experiments, inhibition of uterine contraction in rats was investigated by oral administration as well as intravenous administration of either tetramethylpyrazine, ferulic acid, or a combination of both agents. Their inhibitory activity was compared to that of papaverine. The results showed that tetramethylpyrazin and ferulic acid, either alone or in combina- tion, had an inhibitory effect on spontaneous uterine contractions in estrous

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rats. Their therapeutic index was found to be more effective than papaverine, suggesting that these compounds may have utility as tocolytic agents (40).

5. Other Effects CNS andAnticonvulsive Activities. Early reports suggest that 3-n-

butyl phthalide may facilitate learning and memory in rats (41) and that it has

a protective effect on rat brain cells (42). Recently, Chen et al. (43) investigated whether administration of tetramethylpyrazine led to any improvement in injuries related to the spinal cord. The group used rabbits and induced spinal cord ischemia via infrarenal aortic occlusion. Their results demonstrated that neurological and histopathology status of the animals in the treatment groupwas better than in those not treated, suggesting that tetramethylpyrazine can reduce neurological injury from the spinal cord (43).

Antibacterial/Antifungal Effects. Ligusticum demonstrated in vitro ef- fects against several strains of pathogenic bacteria including Pseudomonas aeruginosa , Shigella sonnei, Salmonella typhi, and Vibrio cholera (44). The es- sential oil butylphthalide has been shown to inhibit dermatophytes in vitro (45).