Anxiolytic Properties of Myrica nagi Bark Extract

Pharmaceutical Biology 2008, Vol. 46, Nos. 10–11, pp. 757–761

Anxiolytic Properties of Myrica nagi Bark Extract Md. Yaseen Khan,1 H. Sagrawat,1 N. Upmanyu, and S. Siddique1 1

R.K.D.F. College of Pharmacy, Bhopal, India

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Abstract Myrica nagi Hook (syn. Myrica esculenta Buch. & Ham) (Myricaceae) is commonly known as box berry. The bark of Myrica nagi is used in the treatment of mental illness by different ethnic groups of the rural region of Orissa (India). In the present work, the anxiolytic and antidepressant effects of an ethanol extract of M. nagi were analyzed at three different dosages (100, 200, and 400 mg/kg, p.o.) by elevated plus-maze, light/dark exploration test, tail suspension test, and the forced swimming model. Oral administration of the ethanol extract (100, 200, and 400 mg/kg, p.o.) of M. nagi showed significant (p < 0.01) and dose-dependent anxiolytic activity by increasing time spent in the open arms of the elevated plus-maze and in the lit box indicating an anxiolytic effect. Nevertheless this treatment was unable to exhibit an effect identical to antidepressants in the tail suspension and forced swimming tests. Altogether, these results suggest an anxiolytic effect of the ethanol extract of M. nagi. Keywords: Antidepressant, anxiolytic, medicinal plants Myrica esculenta, Myrica nagi.

Introduction Myrica nagi Hook (syn. Myrica esculenta Buch. & Ham) (Myricaceae) is a subtropical shrub commonly known as box berry. It is a medium to large woody, evergreen, dioecious tree, 12 to 15 m high; trunk girth, 92.5 cm; bark, light brown to black; leaves are almost crowded toward the ends of branches, lanceolate, 9.2 cm long, 3.2 cm broad, with the lower surface pale green and the upper surface dark green; pistillate flowers, very small, sessile, solitary, and bracteates; sepals and petals are either absent or not visi-

ble; inflorescence, a catkin, 4.2 cm long, axillary, bearing about 25 flowers; only a thread-like style is visible with the unaided eye (Kirtikar & Basu, 1975). The medicinal uses and chemical constituents of Myrica nagi have been widely studied (Malterud et al., 1996). The constituents of M. nagi have been shown to inhibit toxicity in a number of animal model systems (Rastogi & Mehrotra, 1995; Chopra et al., 1996). A number of the chemical constituents of M. nagi have been identified as strong antioxidants (Malterud et al., 1996), and a number of pharmacological effects of M. nagi have been reported (Rastogi & Mehrotra, 1995; Chopra et al., 1996; Mathiesen et al., 1997). It is used as a remedy for various body disorders such as liver diseases, fever, asthma, anemia, chronic dysentery, ulcer, and inflammation (Nadkarni et. al., 1954; Rastogi & Mehrotra, 1995). The bark of M. nagi is used in the treatment of mental illness by different ethnic groups of the rural region of Orissa (India). M. nagi bark contains gallic acid, myricanol, myricanone, epigallocatechin 3-O-gallate, two prodelphinidin dimers [epigallocatechin(4β→8)-epigallocatechin 3-O-gallate and 3-O-galloyl epigallocatechin-(4β→8)-epigallocatechin 3-O-gallate], and the hydrolyzable tannin castalagin (Sum et al., 1988). The main objective of this work was to analyze the effect produced by the oral administration of the ethanol extract of M. nagi in an attempt to determine anxiolytic and antidepressant activity.

Materials and Methods Drugs Diazepam and fluoxetine were procured from Ranbaxy (New Delhi, India) and Sun Pharmaceuticals Ltd. (Baroda, India), respectively, and all other solvents used in this study were of analytical grade.

Accepted: 17 March, 2007 Address correspondence to: H. Sagrawat, R.K.D.F. College of Pharmacy, Bhopal (M.P.) 462036, India. E-mail: hemantsagrawat@ gmail.com DOI: 10.1080/13880200802315436

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2008 Informa UK Ltd.

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Plant material

Assessment of anxiolytic activity

The bark of M. nagi (MN) was procured from a local market in Bhopal and was authenticated by the botanists at the Department of Pharmacy, Barkatullah University, Bhopal (M.P.), India (voucher specimen no. BUPH/4017).

Anxiolytic activity was assessed by using elevated plusmaze (EPM) and the light/dark exploration test.

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Preparation of plant extract The bark was coarsely powdered in an electrical grinder and stored at 5◦ C until further use. Powdered bark (500 g) was defatted with 1 L petroleum ether (40–60◦ C) to remove lipid constituents by soxhlation for 24 h not exceeding the boiling point of solvent. It was then filtered with filter paper, and the residue was air-dried at 30◦ C and extracted with 95% ethanol (1 L) by soxhlation for 48 h, not exceeding the boiling point of solvent. Ethanol was evaporated in a rotary evaporator at 40–50◦ C under reduced pressure. The yield of extracts was calculated as 119 g/500 g dried bark. Phytochemical analysis Chemical tests were carried out on ethanol extract of MN using standard procedure to identify the constituents (Harborne, 1973; Trease & Evans, 2002). Thin-layer chromatography of ethanol extracts of MN was also performed using toluene, ethyl acetate (7:3) as mobile phase and silica gel as stationary phase. Animals Male Swiss albino mice (25–35 g) were procured from the College of Veterinary and Animal Husbandry, Mhow, Indore (M.P.), India, and were acclimatized for at least 7 days prior to commencement of experiments. Animals were randomly distributed into different groups (five) and placed in plastic cages. Each group was composed of six animals and each mouse was numbered. They were fed standard laboratory diet and water ad libitum, except when the experiment was started. Animals were maintained at 22 ± 2◦ C and 55 ± 5% relative humidity in a light-controlled room (14 h light/10 h dark cycle). The experiments were carried out in accordance with the guidelines given by the Indian Council for Medical Research, and the institutional animal ethical committee (CPSA no. 780/03/C/CPCSEA) approved all the experimental protocols.

Elevated plus-maze The EPM as described earlier by Lister (1987) was used in this study. Mice were placed individually in the center of the EPM facing an enclosed arm. The time spent by the mouse during the next 5 min on the open and closed arms was recorded. Mice (n = 6) were treated with ethanol extract of MN (100, 200, and 400 mg/kg, p.o.) 60 min before, and diazepam (1 mg/kg p.o.) 30 min before, their placement on the maze. Light/dark exploration test The apparatus consisted of two boxes (24 × 21 × 21 cm) joined together. One box was made dark by covering its top with plywood, and a 10 W lamp illuminated the other box. The light source was placed 25 cm above the open box. The mice were placed individually in the center of the lit box and observed for the next 5 min for the time spent in lit and dark boxes (Crawley & Goodwin, 1981). Group I mice (n = 6) were treated with vehicle, group II, III and IV with 100, 200, and 400 mg/kg, p.o. of MN respectively 60 min before and group V with diazepam. (1 mg/kg, p.o.) 30 min before, their placement in the lit box. Assessment of antidepressant activity Tail suspension test The method described by Steru et al. (1985) was used. Mice were divided into five groups of six each. They were suspended by tying a thread to their tails from a height of 50 cm above the tabletop. The duration of immobility was recorded for 6 min (after discarding activity in the first 2 min because animals try to escape during this period). Mice were considered immobile only when they hung passively and remained motionless. Group I mice were treated with vehicle group II, III and IV with 100, 200, and 400 mg/kg, p.o. of ethanol extract of MN respectively 60 min before the test and Group I mice with fluoxetine (10 mg/kg, p.o.) 30 min before the test. Forced swimming model

Administration of material Ethanol extract of MN and standard drugs were suspended in 0.2% gum acacia in distilled water. Mice were divided into five groups of six mice each. Group I served as control and was given vehicle only. Groups II, III, and IV were given 100, 200, and 400 mg/kg of ethanol extract orally. Group V was kept as positive control and received standard drug suspension orally.

This test was based on the method described by Porsolt et al. (1978). In brief, animals were forced to swim individually for 15 min in a glass beaker (12 cm diameter, 15 cm height) containing fresh water up to 8 cm at a temperature of 22 ± 1◦ C. Twenty-four-hours after this “pre-test” session, the animals of respective groups were treated either with a drug (test group) or vehicle (control group), and each animal was once again forced to swim in a similar environment

Anxiolytic effect of Myrica nagi for a period of 6 min in “test session.” The attempts by the mice to get out of the beaker were accompanied by periods of immobility, which were significantly “behavioral despair.” The total duration of the immobility during the last 4 min of the 6-min test was recorded. Statistical analysis

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The results were statistically analyzed by using GraphPad Instant software (ver. 2.02, San Diago, California, USA) package. The values were analyzed by ANOVA test followed by Dunnet’s test. All the results were expressed as mean ± SEM for six animals in each group. A p value < 0.05 was considered significant.

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Table 2. Effect of MN on time spent in lit/dark box in light/dark exploration test. Time spent (s) Treatment

Lit box

Control EEMN (100 mg/kg) EEMN (200 mg/kg) EEMN (400 mg/kg) Diazepam (1 mg/kg)

Dark box

74 ± 4.3 138 ± 6.81∗ 156.33 ± 7.51∗ 178 ± 6.92∗ 151 ± 6.4∗

226 ± 4.7 152 ± 6.5∗ 139.75 ± 8.1∗ 121.01 ± 5.57∗ 149 ± 6.7∗

Values are given as mean ± SEM, six animals in each group. Experimental groups were statistically compared with the corresponding values of control group. EEMN, ethanol extract Myrica nagi. ∗ Values are statistically significant at p < 0.01.

Results

Assessment of antidepressant activity

Phytochemical studies revealed that alkaloids, tannins, glycosides, and saponins are present in ethanol extracts of MN. Two compounds having Rf 0.6 and 0.31 were found on thin-layer analysis.

Tail suspension test

Assessment of anxiolytic activity

The total duration of immobility in vehicle-treated mice was 165.23 ± 9.27 s. Oral administration of MN only at 400 mg/kg significantly (P < 0.01) increased the duration of immobility, whereas fluoxetine (10 mg/kg) significantly (p < 0.01) reduced the duration of immobility. The observations are recorded in Table 3.

Elevated plus-maze Vehicle-treated mice spent 63.75 ± 7.7 s in the open arm. The oral administration of ethanol extract of MN bark (100, 200, and 400 mg/kg, p.o.) dose-dependently and significantly (p < 0.01) increased the time spent in the open arm. The results are similar to those of the standard group as shown in Table 1.

Forced swimming test Oral administration of MN (100, 200, and 400 mg/kg, p.o.) ethanol extract increased the duration of immobility, whereas fluoxetine (10 mg/kg) significantly (P < 0.01) reduced the duration of immobility compared with the control group. The observations are presented in Table 4.

Light/dark exploration test The vehicle-treated mice spent 74 ± 4.3 s in the lit box and 226 ± 4.7 s in the dark box. The ethanol extracts of MN bark significantly (p < 0.01) increased the time spent in the lit box and decreased time spent in the dark box in a dose-dependent manner. The observations are summarized in Table 2. Table 1.

Effect of MN on time spent in open/closed arm in EPM. Time spent (s)

Treatment Control EEMN (100 mg/kg) EEMN (200 mg/kg) EEMN (400 mg/kg) Diazepam (1 mg/kg)

Open arm

Enclosed arm

63.75 ± 7.77 144 ± 6.2∗ 155 ± 4.7∗ 161 ± 5.92∗ 156 ± 6.25∗

237 ± 7.8 156 ± 6.5∗ 139.25 ± 3.9∗ 124.01 ± 4.57∗ 133 ± 6.7∗

Values are given as mean ± SEM, six animals in each group. Experimental groups were statistically compared with the corresponding values of control group. EEMN, ethanol extract Myrica nagi. ∗ Values are statistically significant at p < 0.01.

Discussion Plants have been used by human beings since time immemorial to cure diseases and to promote relief from ailments. There were times when they were the most important sources of medicines for people. However, beginning in the late 1940s, this old form of therapeutics began to Table 3.

Effect of MN on time of immobility in tail suspension test.

Treatment Control EEMN (100 mg/kg) EEMN (200 mg/kg) EEMN (400 mg/kg) Fluoxetine (10 mg/kg)

Time of immobility (s) 165.23 ± 9.27 189.12 ± 17.18∗∗ 203.5 ± 13.5∗∗ 231.5 ± 16.2∗∗ 101.25 ± 8.9∗

Values are given as mean ± SEM, six animals in each group. Experimental groups were statistically compared with the corresponding values of control group EEMN, ethanol extract Myrica nagi. ∗ Values are statistically significant at p < 0.01. ∗∗ Values are statistically significant at P > 0.05.

M.Y. Khan et al.

760 Table 4. Effect of MN on time of immobility in forced swimming test. Treatment Control EEMN (100 mg/kg) EEMN (200 mg/kg) EEMN (400 mg/kg) Fluoxetine (10 mg/kg)

Time of immobility (s) 131.4 ± 5.95 140 ± 11.97 153.4 ± 10.2 179.37 ± 7.95∗ 77.4 ± 10.75∗

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Values are given as mean ± SEM, six animals in each group. Experimental groups were statistically compared with the corresponding values of control group. EEMN, ethanol extract Myrica nagi. ∗ Values are statistically significant at p < 0.01.

lose its importance, being more and more replaced by synthetic remedies. The lessons of millennia were forgotten and considered “unscientific.” On the other hand, the ancient use of plants was a lead for scientists in their search for new substances endowed with therapeutic properties. It is estimated that nearly 25% of modern drugs directly or indirectly originated from plants (De Smet, 1997). Anxiety is one of the most common mental disorders affecting mankind. Its prevalence is increasing in recent years due to the rather tense “man’s zest to win nature” (Dhawan et al., 2001); that is, the rather tense lifestyle imposed on man by the competitive and inhumane atmosphere pervading everyday life. Anxiolytic substances, mostly belonging to the benzodiazepine group, occupy a prominent position in the ranking of the drugs most utilized drugs by man (Uhlenhuth et al., 1999) to minimize stress, tension, and anxiety (Argyropoulos & Nutt, 1999). As a result of these effects, benzodiazepines are also able to treat insomnia (Schneider-Helmert, 1988). However, the anxiolytic drugs have an unfavorable risk/benefit ratio, as they produce anterograde amnesia, dependence, abstinence syndrome, paradoxical reaction in humans, and decay of psychomotor functions (Lader & Morton, 1991; Kan et al., 1997; Schweizer & Rickels, 1998). These symptoms can lead to an increased possibility of accidents while driving (Barbone et al., 1998; Pierfitte et al., 2001). As at present time, the etiologic factors responsible for anxiety and tension are not expected to decrease. There is a need for new anxiolytic drugs with less potential to induce adverse reactions. Since ancient times, some plants have been used for such purposes. Today, the use of their extracts is gaining increased acceptance by both the medical profession and patients. MN exhibited a weak anxiolytic activity in both animal models of anxiety, the EPM and the light/dark exploration test. Increase in occupancy by the animals in the open arm or decrease in the time spent in the enclosed arm indicates the anxiolytic activity of drug (Pellow et al., 1985). Many researchers have shown an inverted U-shaped doseresponse curve with anxiolytics (Weiss et al., 1981; Insel et al., 1984, Nutt & Glue, 1991), but this was not observed

with MN. Oral administration of an ethanol extract of MN bark dose-dependently increased the time spent in the open arm. The light/dark exploration test measures natural aversion of mice and rats to brightly lit places. Several researchers have used this model for evaluation of anxiolytic agents (Sanchez, 1995; Imaizumi et al., 1996; Bilkiei-Gorz et al., 1998). The observation that MN increased time spent in the lit box is in accord with these studies. The forced swimming test and tail suspension test have been employed widely to evaluate the effects of various agents on the CNS, such as CNS depressants, antidepressants, sedative-hypnotics, psychostimulants, euphorics, nootropics, adaptogens, and so forth. According to Porsolt et al. (1978), the immobility seen in rodents during swimming reflects behavioral despair as seen in human depression. The swimming test has also been used extensively to assess the antistress activity of plant drugs in mice and rats (Bhattacharya et al., 1987; Ramachandran et al., 1990; Oztu¨rk et al., 1996). The ethanol extract of MN significantly and dose-dependently increased the time of immobility in both models suggesting a depressive effect on the central nervous system. Therefore, on the basis of these experimental data, the profile relating to the effect of the ethanol extract of MN on central nervous system is not identical to that of the antidepressant drug used in these studies, but exhibited anxiolytic activity similar to diazepam. Further studies are required to identify the chemical moiety that is responsible for the anxiolytic and the depressive action.

References Argyropoulos SV, Nutt DJ (1999): The use of benzodiazepines in anxiety and other disorders. Eur Neuropsychopharmacol 9 (Suppl 6): S407–S412. Barbone F, McMahon AD, Davey PG, Morris AD, Reid IC, McDevitt, DG (1998): Association of road-traffic accidents with benzodiazepine use. Lancet 352: 1331–1336. Bhattacharya SK, Goel RK, Kaur R, Ghosal S (1987): Antistress activity of sitoinoside VII and VIII, new acylsterylglucosides from Withania somnifera. Phytother Res 1: 32–39. Bilkiei-Gorz OA, Gyertyan I, Levay G (1998): m-cpp-Induced anxiety in the light/dark box in rats a new method for screening anxiolytic activity. Psychopharmacology (Berlin) 136: 291–298. Chopra RN, Chopra IC, Nayer SL (1996): Glossary of Indian Medicinal Plants. New Delhi, CSIR Publications, pp. 171– 172. Crawley J, Goodwin FK (1980): Preliminary report of a simple animal behavior model for the anxiolytic effects of benzodiacepines. Pharmacol Biochem Behav 13: 167–170. Sun D, Zhao Z, Wong H, Foo LY. (1988): Tannins and other phenolics from Myrica esculenta bark. Phytochemistry 27: 579–583.

Downloaded By: [INFLIBNET India Order] At: 08:32 17 November 2009

Anxiolytic effect of Myrica nagi Dawson GR, Tricklebank MD (1995): Use of the elevated plusmaze in the search for novel anxiolytic agents. Trends Pharmacol Sci 16: 33–36. De Smet, PAGM (1997): The role of plant-derived drugs and herbal medicines in healthcare. Drugs 54: 801–840. Dhawan K, Kumar S, Sharma A (2001): Anti-anxiety studies on extracts of Passiflora incarnata Linneaus. J Ethnopharmacol 78: 165–170. Harborne JB (1973): Phytochemical Methods. London, Chapman and Hall, Ltd., pp. 49–188. Imaizumi M, Miyazaki S, Onodera K (1996): Effects of theophylline in p-chlorophenylalanine treated mice in light:dark test. Exp Clin Pharmacol 18: 513–520. Insel TR, Ninan PT, Aloi J, Jimerson DC, Skolnick P, Paul SM (1984): A benzodiazepine receptor mediated model of anxiety studies in nonhuman primate and clinical implications. Arch Gen Psychiatry 41: 741–750. Kan CC, Breteler MHM, Zitman FG (1997): High prevalence of benzodiazepine dependence in out-patient users, based on the DSM-III-R and ICD-10 criteria. Acta Psychiatr Scand 96: 85–93. Kiritikar B (1975): Indian Medicinal Plants, Vol. II & III. New Delhi, Periodical Experts, pp. 2350–2351. Lader M, Morton S (1991): Benzodiazepine problems. Br J Addict 86: 823–828. Lister RG (1987): The use of plus maze to measure anxiety in mouse. Psychopharmacology 92: 180–185. Malterud KE, Diep OH, Sund RB (1996): C-Methylated dihydrochalcones from Myrica gale L: Effects as antioxidants and as scavengers of 1,1-diphenyl-2-picryl hydrazyl. Pharmacol, Toxicol 78: 111–116. Mathiesen L, Malterud KE, Sund BR (1997): Hydrogen bond formation as basis for radical scavenging activity, a structureactivity of C-methylated dihydrochalcones from Myrica gale and structurally related acetophenone. Free Radic Biol Med 22: 307–311. Nadkarni KM (1954): Indian Materia Medica, Vol. I. Bombay, Popular Book Depot, pp. 828–829. Nutt DJ, Glue P (1991): Psychopharmacology of Anxiolytic and Depressant. File SE, ed. New York, Pergamon, pp. 1–28.

761

` HC (1996): Effects of exOzturkY, Aydin S, Ozturk N, BasAer tracts from certain Sideritis species on swimming performance in mice. Phytother Res 10: 70–73. Pellow S, Chopin P, File SE, Briley M (1985): Validation of open:closed arm entries in an elevated plus maze as a measure of anxiety in rats. J Neurosci Methods 14: 149–167. Pierfitte C, Macouillard G, Thico´y¨pe M, Chaslerie A, Pehourcq F, A´y¨ssou M (2001): Benzodiazepines and hip fractures in elderly people, casecontrol study. Br Med J 322: 704–708. Porsolt RD, Anton G, Blavet N, Jalfre M (1978): Behaviour despair in rats, a new model sensitive to antidepressant treatments. Eur J Pharmacol 47: 379–391. Ramachandran U, Divekar HM, Grover SK, Srivastava KK (1990): New experimental model for the evaluation of adaptogenic products. J Ethnopharmacol 29: 275–281. Rastogi RP, Mehrotra BN (1995): Compendium of Indian Medicinal Plants. New Delhi, CSIR Publication, 4:490–492. Sanchez C (1995): Serotonergic mechanism involved in the exploratory behavior of mice in a fully automated two compartment black and white test box. Pharmacol Toxicol 77: 71–80. Schneider-Helmert, D (1988): Why low-dose benzodiazepinedependent insomniacs can’t escape their sleeping pills. Acta Psychiatr Scand 78: 706–711. Schweizer E, Rickels K (1998): Benzodiazepine dependence and withdrawal: A review of the syndrome and its clinical management. Acta Psychiatr Scand 98 (Suppl 393): 95–101. Steru L, Chermat R, Thierry D, Simon P (1985): Tail suspension test: A new model for screening antidepressant in mice. Psychopharmacology 85: 367–370. Trease GE, Evans WC (2002): Pharmacognsy. 15th ed. Saunders Publishers, London, pp. 139–146. Uhlenhuth EH, Balter MB, Ban TA, Yang K (1999): Trends in recommendations for the pharmacotherapy of anxiety disorders by an international expert panel, 1992–1997. Eur Neuropsychopharmacol 9 (Suppl 6): S393–398. Weiss JM, Goodman PA, Losito BG, Corrigan S, Charry JM, Biley WH (1981): Behavioral depression produced by an uncontrollable stressor: Relationship to norepinephrine, dopamine and serotonin level in various regions of rat brain. Brain Res Rev 3: 167–205.