The Effect of Crude Drug Extracts Bathing (III) - The effect of phthalides from Cnidii rhizoma- Hidenori YOROZU, Hirotaka SATO Tochigi Research Laboratories, KAO Corporation Yoshiaki KOMOTO Department of Cardiovascular Surgery, Okayama University Medical School
124 H. YOROZU et al. INTRODUCTION Cnidii rhizoma and Angelicae radix are common herbs used in traditional Chinese medicine, and are used as medical bath additives to improve circulation1,2). Our previous studies3,4) indicated that bathing in a solution containing crude drug extracts from Cnidii rhizoma and Angelicae radix was effective in increasing dermal blood circulation and maintaining body temperature after bathing, and that the mechanism of the thermal effect from bathing with crude drug extracts was not only from a direct action on dermal blood circulation but also from an indirect action due to inhalation or absorption of the volatile fraction. In the present study, the effect of phthalides, volatile components of Cnidii rhizoma, on rat aorta contraction, skin blood flow and pentobarbital-na induced sleeping time were studied to clarify the active substances of Cnidii rhizoma and Angelicae radix. METHODS Preparation of phthalides Senkyunolide A, ligustilide, cnidilide, butylidenephthalide, and butylphthalide, which are representative volatile components of Cnidii rhizoma, were used as phthalides (Fig. 1). Senkyunolide A, ligustilide, and cnidilide were separated from Cnidii rhizoma by extraction5). The chemical purity of the senkyunolide A, ligustilide, and cnidilide were 99.7%, 99.1%, and 99.0%, respectively. Butylidenephthalide was synthesized by the methods described by Lin et al6). The chemical purity of the butylidenephthalide was 99.5%. Butylphthalide was obtained by hydrogenation of butylidenephthalide7). The chemical purity of the butylphthalide was 99.3%. Effect on rat aorta contraction Adult Sprague-Dawley rats were intraperitoneally anesthetized with 35 mg/kg sodium pentobarbital (Abbott Lab., U.S.A.). The thoracic portion of the aorta was flushed with physiological saline solution, and carefully excised. The aorta was removed of fat and connective tissue, and cut into rings approximately 3mm in length. The tissue was placed in a warm physiological saline solution (PSS) of the following composition (in mm); NaCI, 140; KCl, 4.6; CaCl2, 1.5; MgC12, 1.0; D-glucose, 10.0; N-2- hydoxyethylpiperazine-n'-2- ethanesulfonic acid (Hepes), 5.0. The ph of the buffer was adjusted to 7.40. The temperature was tion was oxygenated by equilibration with 100% O2. Fig. 1 Chemical structures of phthalides The contractile tension was
The effect of crude drug extracts bathing 125 measured isometrically. Aortic rings were mounted on two stainless steel hooks, with the lower hook attached to an aerator and the upper hook connected to a force transducer. The rings were immersed in 30 ml of PSS, and stretched to a resting tension of 1.0 gram. High potassium buffer (80mM) was prepared by substitution of KCl for NaCl. Measurement of skin blood flow on abdomen of rabbits Rabbits each weighing about 2 kg were intubated after tracheotomy under intravenous anesthesia with 25 mg/kg sodium pentobarbital (Abbott Lab., U.S.A.). The animals were ventilated with room air at 250ml/kg/min. Under intravenous maintenance of anesthesia with 15-20 drops/min of 0.3-0.4% sodium pentobarbital by a constant micro-drip infusion set (Terufusion(R), 470L, Terumo, Tokyo), heparin was administered at doses of 1mg/kg, and a catheter for measurement of arterial blood pressure was introduced into the carotid artery. The mean arterial blood pressure was controlled at about 100-80mmHg. After thorough shaving of the chest and abdomen, the rabbits were placed in a supine position, and changes in the blood flow of the abdominal skin were evaluated using a laser Doppler flowmeter (PERIFLUX PF 1, Perimed, Sweden) (output: mv). After the adaptor of this flowmeter was applied, each test sample was dropped into the adaptor4). The phthalide solutions were used without dilution. As a control, tap water was dropped into the adaptor. Effects on pentobarbital-na induced sleeping time cycles (light period:7:00-19:00) for a week or more. Water and food were provided ad libitum. The mice were intraperitoneally injected with 50mg/kg of sodium pentobarbital and the middle of the silicon tube for air delivery, and 0.1g of each phthalide was infused into the glass tube4). As a control, only deodorized room air treated with charcoal was ventilated. Pentobarbital-Na induced sleeping time was defined as disappearance of the righting reflex. The duration of the absence of this reflex was measured. RESULTS Effect on rat aorta contraction Table 1 shows the ED50(50% effective dose) value of the vasodilative effect of phthalides on rat aorta contraction by 80mM KCl. Phthalides affected the contractile responses elicited by 80mM KCl. Butylidenephthalide was found to be the most potent chemical among these phthalides.
126 H. YOROZU et al. Skin blood flow in rabbits Table 2 shows the percentile changes in skin blood flow. The skin blood flow in the rabbits increased 20 minutes after application of senkyunolide A (P <0.05), ligustilide (P<0.05), cnidilide (P<0.05), butylidenephthalide (P<0.01) and butylphthalide (P< 0.05). The skin blood flow in rabbits increased in the order: butylidenephthalide> 7min during exposure to room air. The duration was significantly increased during exposure to senkyunolide A, ligustilide, cnidilide, butylidenephthalide, and butylphthalide. No significant difference in the increase was found among these 5 phthalides. DISCUSSION Our previous studies3,4) revealed that hot water extracts of Cnidii rhizoma and Anglicae radix increased the dermal blood flow and subcutaneous tissue perfusion rate, and that the volatile fraction of Cnidii rhizoma increased the skin blood flow. Kimura et al.8) reported that the extracts of these two plants dilated the isolated mouse portal vein. In this study, we evaluated the effects of representative volatile components of Cnidii rhizoma; senkyunolide A, ligustilide, cnidilide, butylidenephthalide, and butylphthalide, on rat aorta contraction and skin blood flow. The findings showed that phthalides affected the rat aorta contractile responses elicited by 80 mm KCl and increased the skin blood flow, and that butylidenephthalide was the most potent chemical among the phthalides we used in these experiments. Ko9) reported that butylidenephthalide inhibited the contraction of the smooth muscle caused by acetylcholine, KCl, BaCl2 or norepiniphrine. Teng et al.10) reported that butylidenephthalide suppressed the aggregation of platelets, and that the antiplatelet effect of butylidenephthalide is mainly due to an inhibitory effect on cyclo-oxygenase and may be bue partly to interference with calcium mobilization. Ko et al.11) reported that the alkyl group substituted to the C-3 position of the phthalide was very important for the inhibitory effect on rat uterine contraction induced by prostaglandin Fla. Our findings also showed that the double bond of the C-3 position is very important from the results that butylidenephthalide was significantly more active than butylphthalide. Recently, Namba et al.12) reported that ether and methanol extracts, and some essential oils of Senkyu (i. e. ligustilide, neocnidilide and butylidenephthalide) markedly enhanced the skin penetration of benzoic acid. Their findings suggest that the increase in skin blood flow observed in our study is due to dilation of skin blood vessels by percutaneously absorbed phthalides. Bjeldanes et al.13) reported that in mice, intraperitoneal injection of butylphthalide prolonged pentobarbital-na induced sleeping time. Ozaki et al.5) reported that senkyunolide A depressed the crossed extensor reflex in anesthetized rats and considered that this muscle relaxation induced by senkyunolide A originated in the central nervous
The effect of crude drug extracts bathing 127 Table 1 ED50 (50% effective dose) value of the vasodilative effect of phthalides on rat aorta contraction elicited by 80 mm KCI. Table 2 Changes in skin blood flow in rabbit abdomen 20 minutes after application of the samples. Table 3 Effect of phthalides on pentobarbital-na induced sleeping time in mice.
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