Antinociceptive Activity of Dyera Costulata Extract in Experimental Animals

ORIGINAL ARTICLE Antinociceptive activity of Dyera costulata extract in experimental animals Wantana Reanmongkol1 , Sanan Subhadhirasakul2 , Chittima Pairat3, Chaiya Poungsawai3 and Wiparat Choochare3 Abstract Reanmongkol, W., Subhadhirasakul, S., Pairat, C., Poungsawai, C. and Choochare, W. Antinociceptive activity of Dyera costulata extract in experimental animals Songklanakarin J. Sci. Technol., 2002, 24(2) : 227-234 The effects of the chloroform extract from the leaves of Dyera costulata Hook. f. (D. costulata) on nociceptive response using writhing, hot plate and formalin tests in mice and the antipyretic activity in yeast-induced fever in rats, were examined. General behavior was also examined using pentobarbital- induced sleep and locomotor activity in mice. The LD50 value of intraperitoneally injected D. costulata extract in mice was 0.27 g/kg. Oral administration of D. costulata extract (100-400 mg/kg) dose dependently decreased the number of contortions and stretchings induced by acetic acid and licking activity of the late phase in the formalin test but not in the heat-induced pain in mice. The extract (100-400 mg/kg, p.o.) had no significant effect on fever induced by yeast in rats. The D. costulata extract (100-400 mg/kg, p.o.) prolonged the duration of pentobarbital-induced sleep but had no significant effect on locomotor activity in mice. These results suggest that the D. costulata extract possesses marked analgesic but no antipyretic effect. Key words : Dyera costulata, antinociceptive activity, writhing test, formalin test 1Ph.D. (Pharmacology), Assoc. Prof., Department of Clinical Pharmacy , 2Ph.D. (Chemistry of Natural Pro- ducts), Assoc. Prof., Department of Pharmacognosy and Pharmaceutical Botany, 3Pharmacy Student, Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Prince of Songkla University, Hat Yai, Songkhla 90112 Thailand. Corresponding e-mail : [email protected] Received, 15 November 2001 Accepted, 2 January 2002 Songklanakarin J. Sci. Technol. Dyera costulata extract in experimental animals Vol. 24 No. 2 Apr.-Jun. 2002 228 Reanmongkol, W., et al. ∫∑§—¥¬àÕ «—π∑π“ ‡À√’¬≠¡ß§≈1 π—Ëπ »ÿ¿∏’√ °ÿ≈2 ®‘µµ‘¡“ ‰æ√—µπå1 ‰™¬– æ«ß‰ «1 ·≈– «‘¿“√—µπå ™Ÿ‡™◊ÈÕ1 º≈¢Õß “√ °—¥®“°µ’π‡ªì¥·¥ßµàÕ°“√·°âª«¥„πÀπŸ∑¥≈Õß «. ß¢≈“π§√‘π∑√å «∑∑. 2545 24(2) : 227-234 ‰¥â∑”°“√∑¥ Õ∫º≈∑“ß‡¿ —™«‘∑¬“¢Õß “√ °—¥§≈Õ‚√øÕ√å¡®“°„∫µ’π‡ªì¥·¥ß„πÀπŸ∑¥≈Õß ‚¥¬ —ß‡°µº≈ ¢Õß “√ °—¥µàÕ°“√√–ß—∫ª«¥´÷Ëß‡°‘¥®“°°√¥Õ–‡´µ‘° §«“¡√âÕπ ·≈–øÕ√å¡“≈‘π„πÀπŸ∂’∫®—°√ ·≈–º≈µàÕ°“√≈¥‰¢â ´÷Ëß‡°‘¥®“°°“√‡Àπ’Ë¬«π”‚¥¬¬’ µå„πÀπŸ¢“« ¬—ß‰¥â —ß‡°µº≈µàÕæƒµ‘°√√¡∑—Ë«‰ª‚¥¬„™â°“√‡Àπ’Ë¬«π”„ÀâÀ≈—∫¥â«¬‡æÁπ- ‚∑∫“√å∫‘µ“≈·≈–°“√‡§≈◊ËÕπ∑’Ë„πÀπŸ∂’∫®—°√ ¢π“¥¢Õß “√ °—¥∑’Ë∑”„ÀâÀπŸ∂’∫®—°√µ“¬ 50% ¡’§à“ 0.27 ¡°./°°. ‡¡◊ËÕ ©’¥‡¢â“∑“ßÀπâ“∑âÕß„πÀπŸ∂’∫®—°√ ‡¡◊ËÕªÑÕπ “√ °—¥®“°µ’π‡ªì¥·¥ß (100-400 ¡°./°°.) ‡¢â“∑“ßª“°„πÀπŸ∑¥≈Õß æ∫«à“ “¡“√∂≈¥®”π«π¢Õß°“√∫‘¥·≈–¬◊¥¢Õß≈”µ—«‡¡◊ËÕ∂Ÿ°°√–µÿâπ‚¥¬°√¥Õ–‡´µ‘° ·≈–≈¥°“√‡≈’¬„π™à«ß‡ø À≈—ß¢Õß°“√ ∑¥ Õ∫¥â«¬øÕ√å¡“≈‘π„πÀπŸ∂’∫®—°√ º≈¢Õß “√ °—¥®–‡æ‘Ë¡¢÷Èπµ“¡¢π“¥¢Õß “√ °—¥∑’Ë„™â ·µà‰¡à¡’º≈µàÕ°“√∑¥ Õ∫ ¥â«¬§«“¡√âÕπ “√ °—¥ (100-400 ¡°./°°.) ‰¡à¡’º≈„π°“√≈¥‰¢â´÷Ëß‡°‘¥®“°°“√‡Àπ’Ë¬«π”‚¥¬¬’ µå„πÀπŸ¢“« “√ °—¥ ‡ √‘¡ƒ∑∏‘Ï°“√À≈—∫¢Õß‡æÁπ‚∑∫“√å∫‘µ“≈„Àâ¬“«π“π¢÷Èπ ·µà‰¡à¡’º≈µàÕ°“√‡§≈◊ËÕπ∑’Ë„πÀπŸ∂’∫®—°√ ®“°º≈°“√∑¥≈Õßπ’È ‡ πÕ«à“ “√ °—¥µ’π‡ªì¥·¥ß¡’ƒ∑∏‘Ï·°âª«¥∑’Ë‡¥àπ™—¥ ·µà‰¡à¡’ƒ∑∏‘Ï≈¥‰¢â 1¿“§«‘™“‡¿ —™°√√¡§≈‘π‘° 2¿“§«‘™“‡¿ —™‡«∑·≈–‡¿ —™æƒ°…»“ µ√å §≥–‡¿ —™»“ µ√å ¡À“«‘∑¬“≈—¬ ß¢≈“π§√‘π∑√å Õ”‡¿Õ À“¥„À≠à ®—ßÀ«—¥ ß¢≈“ 90112 Dyera costulata Hook. f. (D. costulata) is a hot plate and formalin tests in mice, and its anti- big tree having a yellow heartwood. It occurs in pyretic activity in yeast-induced fever in rats. In primary evergreen lowland or hill forest in the addition, we also studied the general behavior family of Apocynaceae. It can be found in the using pentobarbital-induced sleep and locomotor South of Thailand, Peninsular Malaysia, Singa- activity in mice. pore, Sumatra, Borneo and intervening islands. This plant is known in Thai as “Teen-Pet Daeng” Materials and Methods (Ridley, 1923; Whitmore, 1973). The bark and leaves of D. costulata have been used in folk Plant material medicine for treatment of fever by traditional doc- The leaves of Dyera costulata (Apocyna- tors in the South of Thailand. The constituents ceae) were collected in March, 1999 from Songkhla from the leaves of D. costulata have been identi- Province, Thailand. The plant was identified by fied to contain 6 bisindole alkaloids, ochrolifuanines direct comparison with herbarium specimens in A, E, F and 18-dehydrochrolifuanines A, E, F the PSU Herbarium, Department of Biology, Fac- (Mirand, et al., 1983). ulty of Sciences, Prince of Songkla University. A Although D. costulata has been used for a voucher specimen of plant material has been de- long time as herbal medicine in the South of posited in the Faculty of Pharmaceutical Sciences, Thailand, no pharmacological studies have previ- Prince of Songkla University, Hat Yai, Songkhla, ously been conducted on analgesic and antipyretic Thailand. actions of this plant. In the present study, in order to evaluate the potential existence of analgesic Preparation of the extract from the leaves of and antipyretic activities of the extract obtained Dyera costulata from D. costulata, we investigated the antinoci- The dried coarsely powdered leaves of D. ceptive effects of the extract using the writhing, costulata (15.0 kg) were macerated with 30.0 l Songklanakarin J. Sci. Technol. Dyera costulata extract in experimental animals Vol. 24 No. 2 Apr.-Jun. 2002 229 Reanmongkol, W., et al. of methanol for five days and then filtered and was injected intraperitoneally and the number of evaporated. The marc was remacerated with me- writhings and stretchings was counted over a 20- thanol (30.0 l) for five days four times and fil- min period as previously reported (Koster et al., tered. The combined filtrate was concentrated to a 1959; Hendershot & Forsaith, 1959). The plant syrupy mass under reduced pressure, mixed with extract (100, 200 and 400 mg/kg), a reference × 2% H2SO4 (5 1000 ml) and filtered. The acidic analgesic drug aspirin (200 mg/kg) or cosolvent filtrate was washed with portions of n-hexane vehicle was orally administered 30 min before (2 × 800 ml), then made basic (pH 8) with 25% acetic acid. × NH4OH and extracted with CHCl3 (6 800 ml). 2. Hot plate test The combined CHCl3 extract was washed with The hot plate test was carried out accord- × water (2 1000 ml), dried over anhydrous Na2SO4 ing to the method described by Woolfe & Mac- and evaporated to yield the crude alkaloids Donald (1944). Mice were placed on a hot plate 18.058 g. The dried chloroform extract was used maintained at 55 oC ± 1 oC. Latency of nociceptive as the test extract. One gram of the extract was response such as licking, flicking of a hind limb equivalent to 830.66 g of dried powdered leaves or jumping was measured. Starting thirty minutes of D. costulata. All doses were expressed in after p.o. administration of the test agents except terms of dried crude extract (mg/kg body weight). morphine (15 min after administration), the nociceptive response was measured every 15 min over Animals a 60 min period. Morphine sulfate was injected All animals used in this study were ob- subcutaneously. The cut-off time was 45 sec. tained from the Animal House, Faculty of Sciences, Only the mice that showed nociceptive responses Prince of Songkla University, Hat Yai, Songkhla, within 15 sec were used for the experiments. Thailand. Male Swiss mice with the weight rang- 3. Formalin test ing from 28-38 g were used for all experiments Thirty minutes after administration of except for yeast-induced fever test, in which the D. costulata extract (100, 200 and 400 mg/kg, male Wistar rats with the weight ranging from p.o.), aspirin (200 mg/kg, p.o.) or cosolvent, 20 µl 150-220 g were used. The rats were handled for of 2.5% formalin in saline was injected subcuta- 5-10 min daily for several days before experi- neously to a hindpaw of the mice. The time spent ments. The animals were housed for at least one licking the injected paw was recorded and the data week in the laboratory animal room prior to test- were expressed as total licking time in the early ing. Food and water were given ad libitum unless phase (0-5 min) and the late phase (15-30 min) otherwise specified. after formalin injection (Hunskaar et al., 1985). Acute toxicity Antipyretic activity The 50% lethal dose of the D. costulata Antipyretic actvity of drug was measured extract was estimated by the up-and-down method by slightly modifying the method described by in mice (Bruce, 1985). Doses were adjusted by a Adams et al. (1968). Male Wistar rats were fasted constant multiplicative factor; viz. 4, for this ex- overnight with water ad lib before the experi- periment. The dose for each successive animal ments. Pyrexia was induced by subcutaneously was adjusted up or down depending on the previ- injecting 20% (w/v) brewer’s yeast suspension ous outcome. (10 ml/kg) into the animals’ dorsum region. Nine- teen hours after the injection, the rectal tempera- Antinociceptive activity ture of each rat was measured using a digital ther- 1. Writhing test mometer (SK-1250MC, Sato Keiryoki Mfg. Co., Writhing behaviour was tested, in which Ltd., Japan). Only rats that showed an increase in o 0.6% acetic acid solution (10 ml/kg body weight) temperature of at least 0.7 C were used for the Songklanakarin J. Sci. Technol. Dyera costulata extract in experimental animals Vol. 24 No. 2 Apr.-Jun. 2002 230 Reanmongkol, W., et al. experiments. Test agent or cosolvent vehicle was were prepared immediately before starting the administered orally and the temperature was experiments.

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