SEDATIVE EFFECT OF WALNUT LEAF EXTRACT AND JUGLONE, AN ISOLATED CONSTITUENT

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ABSTRACT

Sedative activity of Juglans regia L. (Juglandaceae) chloroform extract and of one of its isolated con- stituents was evaluated in mice. Dry chloroform extract and juglone, orally administered at 6.5 mg/kg and 0.125 mg/kg, respectively, induced marked seda- tion as measured by two tests (actimeter and sleep potentiation), compared with controls (diazepam, 2 mg/kg, per os). A separation method of juglone from chloroform extract was developed.

INTRODUCTION

Walnut (Juglans regia L., Juglandaceae) leaf and hull have been broadly used in traditional medicine for many years (Bruneton, 1993; Leclerc, 1976). Biblio- graphical data mention that walnut leaf has various pharmacological effects: astringent and keratolytic (Bezanger-Beauquesne et al., 1990), antidiarrhoeal (Wichtl, 1994), antifungal (Nahrstedt et al., 1981) and antihelmintic (Wichtl, 1994), hypoglycaemic (Neef et al., 1995), anti-scrofulous (Fournier et al., 1948), depu- rative and tonic (Wichtl, 1994).

Some other effects are also reported, such as an in vitro antiviral effect against VSV (Vesicularis Stomati- tis Virus) (Husson et al., 1986), a vascular protective effect (in vitro) (Perusquia et al., 1995) and an inhibitory effect on tumors (Bhargava and Westfall, 1968).

Only one report, published in 1961 (Westfall et al., 1961), indicated the sedative effect of an extract of black walnut hulls, but this activity was not compared to a reference sedative drug.

Our study aims to test a sedative activity in mice of a dry chloroform extract, obtained from fresh walnut leaves, and of one of its constituents, juglone, in comparison with diazepam control. Juglone is interesting since its chemical structure may indicate a potential pharmacological activity.

MATERIALS AND METHODS Plant Material

Fresh walnut leaves were collected in Lempdes (63370, France), altitude 380 m, in May 1996.

Preparation of a Dry Chloroform Extract

Leaves (15 g), cut in small pieces (1 cm) were extracted with chloroform in the hour following the harvesting.

Extraction consisted of maceration of leaves at room temperature for one hour. The chloroform solution was evaporated under reduced pressure, at a temperature under 50°C, up to constant weight, which assured the total elimination of solvent. The dry residue (0.1138 g) represents the chloroform dry extract (E).

Juglone Isolation

Chloroform dry extract (0.0705 g) in solution in petroleum ether (5 ml) was chromatographed on TIXOSIL (CPF 38A) (10 g) (column 20 mm i.d.) and eluted with petroleum ether/benzene, 1,2-dichloroethane and chloroform. Seven fractions were collected (first 100 ml;

second 60 ml: petroleum ether/benzene, 70/30 v/v;

third 100 ml: petroleum ether/benzene, 50/50 v/v;

fourth 50 ml; fifth 50 ml: petroleum ether/benzene, Keywords: Juglans regia L., chloroform extract, juglone,

sedative activity, mice motivity, sleep potentiation.

Address correspondence to: Dr. A.-P. Carnat, Laboratoire de Pharmacognosie et Phytothérapie, Faculté de Pharmacie, 28, place Henri Dunant, 63100 Clermont-Ferrand, France.

S ÊDATIVE E ^{FFECT OF} W ÂLNUT L ÊAF E ^XTRACT

AND J ÛGLONE , ÂN I ^SOLATED C ÔNSTITUENT

M. Gîrzu

¹

, A. Carnat

¹

, A.-M. Privat

²

, J. Fialip

²

, A.-P. Carnat

¹

and J.-L. Lamaison

¹

1Lab. Pharmacognosie et Phytothérapie, Fac. Pharmacie, Clermont-Ferrand, France 2Lab. Pharmacologie, Fac. Pharmacie, Clermont-Ferrand, France

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30/70 v/v; sixth 50 ml: 1,2-dichloroethane; seventh 50 ml: chloroform). Each fraction was evaporated under reduced pressure to give a dry residue.

The second fraction (J) (0.0035 g) contained juglone. This constituent was identified by TLC (sol- vents: benzene-ethyl formiate: 90/10 v/v; reagent:

methanolic solution of potassium hydroxide, 5% w/v) and HPLC (Waters apparatus, photodiode array detec- tor, column RP-18 endcapped, Merck, 125 3 4 mm, 5 µ, Lichrospher 100).

Animals

Male Swiss albino mice (Dépré Laboratoire, 18230- Saint Doulchard, Fr.) weighing 20–22 g were used. All the animals were confined in standard plastic boxes (5 mice per box) (box size: 25 320 310 cm). They were fed a laboratory diet (croquettes Extralabo, Provins, Fr.) ad libitum and allowed free access to drinking water;

they were kept in a 12/12 hour light/dark cycle.

Study of Sedative Activity

Experiments were carried out in two steps, in strict blind conditions. First, we tested for sedative activity using an Apelex type 01–1668B actimeter. This test afforded the most effective doses.

We then tested for hypnosedative activity in mice using a specific test, sleep potentiation (Winter, 1948).

We also carried out additional behavior tests in mice to check for any neuromuscular toxicity: the chimney test (Boissier et al., 1960), the traction test (Boissier &

Simon, 1960) and the rota-rod test (Dunham & Miya, 1957); neuromuscular toxicity induces an impairment of animals’ performance in these tests.

Actimeter Test

Test description. Sedative activity was tested by using an Apelex type 01–1668B actimeter as described by Boissier and Simon (1965). Twenty-five minutes after oral administration, each animal was placed in a plexi- glass box (26 321 310 cm) fitted with two perpen- dicular light beams, 1 cm above the floor. Motor activity was quantified by the number of times the light beams were interrupted over a 10-min period, after a 5- min exploration phase. Sedative activity corresponded to a decrease in the percentage of motor activity versus control group.

Experimental procedures. Two experimental procedures were followed with chloroform extract and juglone. Dispensed doses were calculated in concordance with the percentages of each constituent in the plant material, and for the juglone in concordance with

the LD₅₀(LD₅₀5 2.5 mg/kg) (Westfall et al., 1961).

Diazepam (2 mg/kg, per os) was chosen as the reference compound. Doses followed a ratio two geometri- cal progression. Each fraction was dissolved in neutral olive oil and orally administered (with a gastric probe) in a volume of 0.5 ml/20 g of body weight. Mice in the control group were given neutral olive oil in a volume of 0.5 ml/20 g of body weight.

(1) Effect on mice motivity of the chloroform extract (3.25, 6.5, 13 and 26 mg/kg) compared with diazepam (Valium^®, Roche) (2 mg/kg).

(2) Effect on mice motivity of juglone (0.0625, 0.125, 0.25 and 0.5 mg/kg). For each group, inhibition percentage was calculated with the formula:

(N '–N) 3100/N where:

N ' 5 number of times the light beam was interrupted in the 10-min period for the treated mice group;

N5number of times the light beam was interrupted in the 10-min period for the control mice group.

Sleep Potentiation Test

Test description. This test consisted of studying the influence of drugs on the duration of sleep induced by a hypnotic, pentobarbital. The criterion used to define sleep was loss of righting reflex. Sedative effect was assessed by measuring increase in sleep duration. It was considered that the potentiation threshold was reached when sleep duration was increased 1.5-fold.

Experimental procedure. One experimental procedure was followed using fractions E and J, which had previously shown a significant sedative activity. These two fractions were given in the same conditions as in the actimeter test.

- The control group was given neutral olive oil per os, - The reference group was given diazepam (Valium^®, Roche) administered at 2 mg/kg body weight, per os,

- Three treated groups were given fraction E, administered per os, at the following doses: 1.625, 3.25 and 6.5 mg/kg.

- Three treated groups were given fraction J, administered per os, at the following doses: 0.03125, 0.0625 and 0.125 mg/kg.

- Pentobarbital was intraperitoneally administered at 35 mg/kg body weight to all groups, 30 min after neutral olive oil, diazepam, or fractions E or J.

Statistical Evaluation

Statistical comparison of the results was carried out

SEDATIVE EFFECT OF WALNUT LEAF EXTRACT AND JUGLONE 281

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Fig. 1. Influence on mice motivity of Juglans regia L. chloroform extract (E) administered at different doses (3.25, 6.5, 13 and 26 mg/kg) and of diazepam (2 mg/kg) compared with control group (neutral olive oil). n58 mice/group.

**p,0.005 versus control group (neutral olive oil).

*p,0.05 versus control group (neutral olive oil).

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Fig. 2. Influence on mice motivity of juglone (J) administered at different doses (0.0625, 0.125, 0.25 and 0.5 mg/kg) compared with control group (neutral olive oil). n58 mice/group.

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Fig. 3. Influence of chloroform extract (1.625, 3.25 and 6.5 mg/kg), juglone (0.03125, 0.0625 and 0.125 mg/kg) and diazepam (2mg/kg) on the sleep duration induced by pentobarbital, compared with control group (neutral olive oil). n510 mice/group.

*p,0.05 versus control group (neutral olive oil).

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ance, followed by Student’s t-test. In every case the sig- nificance level was p ,0.05.

RESULTS AND DISCUSSION

Actimeter Test

Activity of chloroform extract (E). Results, in Figure 1, show a significant decrease (p,0.005) of 64.4% in mouse motor activity for the group treated at 6.5 mg/kg, compared to the control group. There was a significant decrease (p , 0.05) in mouse motivity of 39.4% for the 13 mg/kg treated group, and of 41% (p ,0.005) for the 26 mg/kg treated group. The less concentrated dose (3.25 mg/kg) treated group showed significant decrease in mouse activity (p , 0.005) of 39.8%. For the reference group, there was a significant decrease (p,0.005) in mouse activity of 56.9%.

Activity of juglone (J). Results, in Figure 2, show a significant decrease (p , 0.005) of 68% in mouse activity for the group treated at 0.125 mg/kg, compared to the control group. There was significant decrease (p ,0.005) in mouse activity of 22.4% for the 0.25 mg/kg treated group and of 41% (p,0.05) for the 0.5 mg/kg treated group. The less concentrated dose (0.0625 mg/kg) treated group showed a significant decrease in mouse activity (p,0.005) of 38%.

Sleep Potentiation Test

Activity of chloroform extract (E). Results, in Figure 3, show a significant increase in sleep duration (p , 0.005) for animals treated with chloroform extract (E) (2.89- and 1.64-fold for 6.5 and 3.25 mg/kg, respectively). For animals treated at 1.625 mg/kg, the increase in sleep duration was less important (1.25-fold) and nonsignificant.

Activity of juglone (J). There was a significant increase (p,0.005) in sleep duration (2.47- and 1.5- fold for 0.125 and 0.0625 mg/kg, respectively). For the 0.03125 mg/kg treated group, the increase in sleep duration was significant, but less than for other doses (1.35-fold; see Fig. 3).

Maximal effect was obtained for the diazepam treated group (3.71-fold for 2 mg/kg, p , 0.005) (Fig. 3).

Behaviorial Muscular Tests

In all tests, animals treated with E or J showed the same skill to perform the test as the reference group, for any dose, indicating no neurotoxicity. On the other hand, 3 animals out of 10 treated with diazepam did not exhibit

the required performance in the traction test, and 4 out of 10 did not exhibit the required performance in the chimney test.

In conclusion, the two main tests showed an important sedative activity for the dry chloroform extract and for one of its constituents, juglone. The most important pharmacological activity has been found for 6.5 and 0.125 mg/kg doses of chloroform extract and juglone, respectively. This activity increased up to these doses, then decreased for higher doses. As seen on the behaviorial muscular tests, no neurotoxic effect such as ataxia or decrease in neuromuscular tone could account for the sedative activity. Therefore, the chloroform extract from walnut fresh leaves shows an important sedative activity due to juglone; this activity may be useful in therapeutics.

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Accepted: March 27, 1998