Journal of Ethnopharmacology, 34 ( 1991 ) 43-48

43

Elsevier Scientific Publishers Ireland Ltd.

Myrcene mimics the peripheral analgesic activity of lemongrass tea Berenice B. Lorenzetti a, G16ria E.P. Souza b, Silvio J. Sarti c, David Santos Filho c and S6rgio H. Ferreira a aDepartment of Pharmacology, Faculty of Medicine. hSection of Pharmacology and 'Section of Pharmacognosy, Faculty of Pharmaceutical Sciences, Ribeir6o Preto Campus, 14.049 Ribeir6o Preto. S.P. ( Bra:il) (Accepted February 22, 1991)

Oral administration of a infusion of lemongrass (Cymbopogon citratus) fresh leaves to rats produced a dose-dependent analgesia for the hyperaigesia induced by subplantar injections of either carrageenin or prostaglandin E2. but did not affect that induced by dibutyryl cyclic AMP. These results indicate a peripheral site of action which was confirmed with the essential oil obtained by steam distillation of the leaves. Silica gel column fractionation of the essential oil allowed the identification of myrcene as the major analgesic component in the oil. Identification of the components was made by thin-layer chromatography and checked by mass spectrometry. The peripheral analgesic effect of myrcene was confirmed by testing a standard commercial preparation on the hyperalgesia induced by prostaglandin in the rat paw test and upon the contortions induced by intraperitoneal injections of iloprost in mice. In contrast to the central analgesic effect of morphine, myrcene did not cause tolerance on repeated injection in rats. This analgesic activity supports the use of lemongrass tea as a "sedative" in folk medicine. Terpenes such as myrcene may constitute a lead for the development of new peripheral analgesics with a profile of action different from that of the aspirin-like drugs.

Key words: myrcene analgesia: lemongrass analgesia: peripheral analgesia.

Introduction

A tea of lemongrass, Cymbopogon citratus (DC) Stapf. (family Gramineae), leaves is used in Brazil and other Third World countries as a popular remedy for various nervous and gastrointestinal disturbances. Recently, detailed studies of the pharmacological and toxicological properties of lemongrass were made in man (Leite et al., 1986) and experimental animals (Carlini et al., 1986; Formigoni et al., 1986). These studies concluded that the infusion of the plant was not toxic and that it lacked any active principle that could explain its popular therapeutic uses. However, a close analysis of those studies convinced us that the presence of a peripheral acting analgesic would pass undetected Correspondence to: S.H. Ferreira, Department of Pharmacology, Faculty of Medicine, Faculty of Pharmaceutical Sciences, Ribeirffo Preto Campus, 14.049 Ribeirffo Preto, S.P., Brazil. 0378-8741/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

in their pharmacological screening. In Third World countries, there is the anecdotal use of salicylates or dipyrone being used as a sedative (calmative) to alleviate tension resulting from malaise or minor colic of various origins. The presence of such activity in the lemongrass tea would, at least in part, support its use in folk medicine. In a previous series of studies about the site of action of analgesics, it was shown using carrageenin, prostaglandin E2 or dibutyryl cyclic AMP as nociceptive stimulants in the rat paw hyperalgesic test, that peripheral- and central acting analgesics could be differentiated (Ferreira ad Nakamura, 1979; Lorenzetti and Ferreira, 1985). Hyperalgesia was used as the primary screening tool in the present study. The oral analgesic activity of lemongrass tea made with different amounts of fresh leaves was investigated. A dose-dependent analgesic effect was observed, which was confirmed using the essential oil fraction extracted from the plant

44 infusion. The essential oil was then chromatographed and the analgesic substances present in the fractions identified by thin-layer chromatography. The major fraction with analgesic activity corresponded to myrcene, the structure of which was confirmed with mass spectrometry. Materials and Methods

Nociceptive tests The rat paw hyperalgesia method used was a modification of the classic Randall-Selitto technique (Ferreira et al., 1978). To determine the intensity of hyperalgesia, a constant pressure of 20 mmHg was applied to the hind paw of Wistar stock rats (140---170 g) until they presented a typical freezing reaction and the time recorded (reaction time). Measurements were made before and 3 h after the subplantar injection of a hyperalgesic substance. The volume of the subplantar injections was 0.1 ml. The intensity of hyperalgesia (delta reaction time, in seconds) was calculated by subtracting the value measured 3 h after administration of the hyperalgesic substance from the control reaction time (zero time). The mouse writhing test used was based on the method of Koster et al. (1959). Contortions were induced by intraperitoneal administration of 0.2 ml of either acetic acid 0.6%, v/v (60 mg/kg) or iloprost (10 ttg/kg) in Swiss stock mice (25--30 g). The data represent the mean of the total number ± S.E.M. of contortions observed during the 20-min period immediately after the intraperitoneal (i.p.) injection of a nociceptive stimulant. The mouse hot plate technique used in this study has been described previously by Jacob et al. (1974) and Jacob and Ramabadran (1978). Briefly, the mice were placed on a hot plate maintained at 50* + 0.5°C. The latency of the jumping reaction (time of reaction, in seconds) was determined. The "cut-off" exposure time was 20 s. In all the above tests, determinations were made by an observer who was unaware of the drug treatment. All animals were taken off food 12 h before testing but water was allowed ad libitum. Infusion preparation and essential oil extraction Lemongrass leaves were harvested in Ribeir~io

Preto and authenticated by Dr. H6rmogenes de Freitas Leit~o from the University of Campinas, SP. For the preparation of the tea, 10--40 g of fresh finely minced leaves were placed in a beaker containing 100 m l o f water and heated. The container was covered and the heating stopped after boiling the mixture for 5 min. The infusion was filtered and allowed to cool at room temperature. The essential oil was obtained by water distillation of fresh leaves (yield 0.4%, w/w).

Fractionation of the essential oil Chromatographic fractionation of 270 g of the oil was carried out using a silica gel (0.05--0.20 mm) column (15 cm diameter, 150 cm length) eluted with solvents or mixtures of solvents with increasing polarity (hexane; hexane/ethyl acetate, 99:1 to 20:80; ethyl acetate; ethyl acetate/ethanol, 99:1 to 20:80; ethanol). For the antinociceptive testing, 530 fractions of 300 ml were collected and then pooled in 12 fractions according to the profiles observed in thin-layer silica gel chromatography (hexane/ethyl acetate, 85:15). The fractions were detected by UV and by reaction with vanillin/sulphuric acid mixture. Before analgesic testing, all solvents were eliminated by reduced pressure. Isolation of potent components The most active fraction was rechromatographed using preparative thin-layer chromatography. The fraction identified as myrcene was eluted with chloroform and analyzed by mass spectrometry coupled with gas chromatography in the Chemistry Institute of Rio de Janeiro Federal University by Dr. Francisco Radler. Drugs and reagents The following drugs were utilized: prostaglandin E2 (PGE2, Upjohn, U.S.A.), iloprost (Schering, AG.; Schror et al., 1981), isoprenaline (L-isoproterenol hydrochloride, Sigma, U.S.A.), N6,2"-O-dibutyryladenosine 3',5'-cyclic monophosphoric acid (DbcAMP, bucladesine, Sigma, U.S.A.), morphine hydrochloride (Merck, Germany), carrageenin (Viscarin, Marine Colloids, U.S.A.), myrcene (Givaudan, Brazil), CremophorEL (BASF, Brazil). The routes (orally, p.o.; in-

45

traperitoneal, i.p.) and time of administration of the various drugs are indicated in the legends of the figures. Results

TABLE I ANTINOCICEPT1VE ACTIVITY OF VARIOUS CHROMATOGRAPHIC FRACTIONS FROM THE ESSENTIAL OIL OF LEMONGRASS ON PGE2-1NDUCED HYPERALGESIA Fractiona

Chromatographic identification

Antinociceptive activity (%) b

--

Essential oil Myrcene Limonene C-9 aldehyde Citronellol C-10 aldehyde Linalool Alpha-citral (geranial) Beta-citral (neral) Geranyle acetate + traces of myrcene Citronellal c Citronellal c Nerol Geraniol

75 88 17 38 8 29 I0 41 40

Rat paw hyperalgesia In Fig. 1 (left panel), a d o s e - d e p e n d e n t analgesic effect is s h o w n for 3 ml o f three different concent r a t i o n s o f l e m o n g r a s s infusion u p o n c a r r a g e e n i n i n d u c e d hyperalgesia. T h e m i d d l e a n d right panels show t h a t 3 ml o f a 20% infusion b l o c k e d a b o u t 50% o f the h y p e r a l g e s i a i n d u c e d b y p r o s t a g l a n d i n E 2 a n d h a d no effect u p o n the h y p e r a l g e s i a induced by D b c A M P . T a b l e 1 shows t h a t the several p o o l s from fract i o n a t i o n o f the essential oil possessed v a r y i n g degrees o f a n t i n o c i c e p t i v e effect o n P G E 2 - i n d u c e d hyperalgesia. P o o l A h a d the m o s t p o t e n t antinociceptive effect. T h i s p o o l was c h r o m a t o g r a p h ically identified as m y r c e n e a n d further c o n f i r m e d by m a s s s p e c t r o m e t r y . As shown in Fig. 2 ( u p p e r panel), the essential oil o f l e m o n g r a s s p r o d u c e d a s t r o n g analgesic effect on the h y p e r a l g e s i a i n d u c e d b y p r o s t a g l a n d i n u

25

,5 I

Cg

20

b.

i

PGE2

DbcAMP

15

.t5

10

,10 .5

5

C 10 20 40

I

J K L M

62 33 32 5 0

aEach letter indicates a pool of several similar fractions as revealed by thin-layer silica gel chromatography. t~rhe percent of inhibition was calculated taking the control PGE 2 hyperalgesia as 10ft'/,,. The oil and all fractions were given in a fixed dose of 90 mg/kg/po. cj and K pools had the same predominant chromatographic pattern but contained small proportions of different contaminants.

.20

.x-

0

A B C D E F G H 1

C 20 Inlusion

%

c

0

2o

t

Fig. I. Antinociceptive action of oral administration of an infusion from fresh lemongrass leaves. The left panel shows the effect of increasing concentrations of the infusate (3 ml of IlY'/,,. 21Y'/,,and 40% infusions, w/v) upon the hyperalgesia induced by the subplantar injection of 100 #g of carrageenin (Cg). The middle and right panels show respectively the effect of 21Y¼,infusion (3 ml) on the hyperalgesia induced by PGE 2 (100 ng/paw) and DbcAMP (100 #g/paw). All treatments were given 30 rain before the subplantar injections. The results are the means 4- S.E.M. (5 animals per group) of measurements made 3 h after the intraplantar injections of the hyperalgesic substances.

E 2 o r isoprenaline in the rat paw. No effect was detected upon the hyperalgesic effect of DbcAMP. Figure 2 (lower panel) shows the strong analgesic effect of orally administered myrcene alone on the hyperalgesia induced by prostaglandin E2 or isoprenaline in the rat paw. Like the essential oil, myrcene had no effect on DbcAMP-induced hyperalgesia.

Mouse writhing F i g u r e 3 ( u p p e r panel) shows the d o s e - d e p e n d ent a n t i n o c i c e p t i o n o f the essential oil on b o t h acetic acid- a n d i l o p r o s t - i n d u c e d writhing in mice. F i g u r e 3 (lower panel) shows the d o s e - d e p e n d e n t a n t i n o c i c e p t i o n o f c o m m e r c i a l l y available m y r cene in these same tests.

46 ~'~ 25-

50. Essential

Essential

Oil

DbeAMP

15.

0

.,... ®

Myrcene mimics the peripheral analgesic activity of lemongrass tea.

Oral administration of an infusion of lemongrass (Cymbopogon citratus) fresh leaves to rats produced a dose-dependent analgesia for the hyperalgesia i...
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