J Wood Sci DOI 10.1007/s10086-012-1277-3 ORIGINAL ARTICLE Neuropharmacological activities of fruit essential oil from Litsea cubeba Persoon Chi-Jung Chen • Yen-Hsueh Tseng • Fang-Hua Chu • Tin-Ya Wen • Wei-Wen Cheng • Yu-Ting Chen • Nai-Wen Tsao • Sheng-Yang Wang Received: 21 March 2012 / Accepted: 18 June 2012 Ó The Japan Wood Research Society 2012 Abstract Litsea cubeba (Lauraceae) is woody plant Keywords Litsea cubeba Essential oil endemic to Taiwan that is traditionally used as a spice. In Neuropharmacology activitiesÁ Á the current study, several behavioral analyses were per- formed to evaluate the neuropharmacological activity of the Introduction essential fruit oil of L. cubeba in ICR mice. Oral adminis- tration of 100, 300 and 500 mg/kg of L. cubeba fruit oil The World Health Organization (WHO) reported that in 2003 significantly prolonged pentobarbitone-induced mouse more than 450 million people suffered from mental or sleeping time by 20.0, 110.8, and 159.6 %, respectively. In behavioral disorders [1]. Thus, discovery of new neurophar- addition, after administration of L. cubeba oil, mice sig- macologically active phytocompounds as therapeutic alter- nificantly increased the time spent in the open arms and natives for such disorders is of interest. The local name for number of entries into the open arms of an elevated plus Litsea cubeba (Lour.) Persoon (Lauraceae) is mountain pep- maze compared to saline-treated mice suggesting that per. Indigenous people in Taiwan traditionally use this plant to L. cubeba oil has anxiolytic activity. A tail-flick test con- treat inflammation, headache, and intoxication [2]. Many ducted after treatment of mice with 500 mg/kg L. cubeba studies have shown that essential oils of L. cubeba exhibit a fruit oil also suggested that this oil has potent analgetic range of bioactivities such as antitermite [3], antioxidant [4], activity. According to GC/MS analyses, the essential fruit larvicidal [5], and cytotoxic [6] activities. However, reports oil of L. cubeba oil consists of 23 compounds. The main on the neuropharmacological activity of the essential oil from components are geranial (37.16 %), neral (28.29 %), and L. cubeba are rare. In this study, several animal behavioral d-limonene (22.90 %). We conclude that L. cubeba oil has a analyses were performed to evaluate the neuropharmacolog- potent effect on the central nervous system of mice. ical activities of the oil from the fruit of L. cubeba to under- stand its effect on the central nervous system. The composition of the essential oil was also characterized. C.-J. Chen Y.-H. Tseng T.-Y. Wen W.-W. Cheng Materials and methods Y.-T. ChenÁ N.-W. Tsao Á S.-Y. WangÁ (&) Á DepartmentÁ of Forestry/AgriculturalÁ Biotechnology Center, National Chung-Hsing University, Taichung, Taiwan Plant materials e-mail: taiwanfi[email protected] Mature fruit of 15-year-old L. cubeba were collected from C.-J. Chen Y.-H. Tseng T.-Y. Wen W.-W. Cheng Y.-T. ChenÁ N.-W. Tsao Á Á Á Huisun Experimental Forest, Nantou, Taiwan in June 2008. National Chung-HsingÁ University, Prof. Yen-Hsueh Tseng from the Department of Forestry, No. 250 Kuo-Kuang Road, Taichung 402, Taiwan National Chung-Hsing University, confirmed taxonomic identification, and voucher specimens were deposited at the F.-H. Chu School of Forestry and Resource Conservation, Herbarium of the Department of Forestry, National Chung- National Taiwan University, Taipei 106, Taiwan Hsing University, Taiwan. 123 J Wood Sci Fruit essential oil preparation Open-field test Fruits of L. cubeba (800 g) underwent water distillation for The open-field test was used to evaluate the exploratory 6 h in a Clevenger-type apparatus, and oil content (ml/kg) activity of the animals [8]. Open-field activity was mea- was determined based on the dry weight of the fruit. The sured in a wooden cage (76 cm 9 76 cm 9 40 cm) divi- essential oil was stored in sample vials after deoxygenation ded into 25 squares of equal area. The mice were divided with nitrogen prior to analysis by gas chromatography into four groups (n = 10) and oral administered L. cubeba (GC) and GC–mass spectrometry (GC/MS). fruit oil, which were diluted with corn oil (100, 300, and 500 mg/kg/d) using a gastric feeding tube or corn oil orally GC/MS analyses of essential oil of L. cubeba for 8 days. On day 8, 1 h after L. cubeba fruit oil admin- istration, each mouse was placed in the center of the open- The compositions of the fruit essential oils were analyzed field arena. The distance of movement, mean speed and the by GC/MS (HP G1800A; Hewlett Packard, USA), equipped time spent immobile during a 5-min observation period with a DB-5 ms column (30 m 9 0.25 mm i.d., 0.25 lm were recorded to indicate the exploratory activity of mice. film thickness; J & W Scientific). The temperature program was as follows: 40 °C for 1 min, then increased by 4 °C/min Pentobarbital-induced sleeping time to 260 °C and held for 4 min. The other parameters were as follows: injection temperature, 270 °C; ion source tem- The mice were divided into 5 groups (n = 8). The control perature, 280 °C; EI, 70 eV; carrier gas, He at 1 ml/min; group was injected with pentobarbitone sodium [45 mg/kg; injection volume, 1 ll; spilt ratio, 1:50; and mass range, m/z intraperitoneally (i.p.)] only. The other groups were injected 45–425. Quantification was obtained from percentage peak with pentobarbitone sodium (45 mg/kg; i.p.) sixty minutes areas from the gas chromatogram. A Wiley/NBS Registry after oral administration of L. cubeba fruit oil (100, 300 and of Mass Spectral Data search and authentic reference 500 mg/kg/d), or zolpidem hemitartrate (0.3 mg/kg/d), compounds were used for substance identification. The respectively. The time elapsing between loss and recovery Kovats retention index (KI), which is a parameter calcu- of the righting reflex was considered as sleeping time and lated in reference to n-alkanes that converts retention times recorded for control and treated animals [9]. into system-independent constants, was also confirmed [7]. Chromatography results expressed as area percentages were Elevated plus-maze test calculated with a response factor of 1.0. The elevated plus maze is a widely used behavioral assay Animals for rodents and it has been validated to assess the anti- anxiety effects of pharmacological agents [10]. The ele- Male ICR mice (4 weeks old, 25–28 g) were purchased vated plus maze for mice consists of two opposing open from BioLasco (Taiwan). Mice were allowed 1 week to arms (32 cm 9 6 cm) perpendicular to two opposing acclimatize before testing. They were housed under con- closed arms (32 cm 9 6 cm) with walls (15 cm). The plus ditions of controlled temperature (25 ± 2 °C), relative maze was elevated 50 cm above the floor. The control humidity (55 ± 5 %), and lighting (light period 06:00– group was treated with saline only, and a positive control 18:00), with food and water ad libitum. The animals were group was treated with trazodone hydrochloride (10 mg/kg), transferred to the laboratory at least 1 h before the start of a well-known psychoactive compound with sedative and each experiment. All animal experiments were conducted anti-depressant properties used clinically for relief of an in accordance with the Guide for the Care and Use of anxiety disorder. The other groups were fed orally with L. Laboratory Animals, and Taiwan laws relating to the pro- cubeba fruit oil (100, 300 and 500 mg/kg/d) for 7 days, tection of animals, and were approved by the local ethics respectively. On day 7, mice were individually placed on committee. the center of the maze. The number of entries and the time spent in the open arms of the apparatus were recorded Behavioral analysis during a 5-min observation period. During behavioral assays, all the activities of test mice Tail-flick test were recorded for visual and automated quantitative anal- ysis using a DSP CCD camera (Model: KMS-63F4) con- The tail-flick test measures pain response in animals. It is nected to a computer installed with Noldus software used in basic pain research and to measure the effective- (Ethovision version 4.0, Noldus Information Technology, ness of analgesics by observation of animal reaction to Wageningen, the Netherlands) for data acquisition. intense heat [11]. Mice that withdrew their tails from hot 123 J Wood Sci water (52 °C) in 3.0 ± 1.0 s were pre-selected 24 h before Table 1 Compositions of fruit essential oil from Litsea cubeba the start of the experiment. The test mice were divided into Compound Concentration KI Identificationa five groups (n = 10). The control group was treated with (%) saline only, the positive control group was treated with acetaminophen (60 mg/kg) and the experimental groups a-Pinene 0.98 929 MS, KI, ST were fed L. cubeba fruit oil (100, 300, and 500 mg/kg/d) Camphene 0.05 953 MS, KI, ST orally for 9 days. On day 9, the tail of each test mouse was Sabinene 0.16 967 MS, KI, ST placed in hot water (52 °C) 0, 30, 60, and 120 min after b-Pinene 0.87 969 MS, KI, ST administration of L. cubeba fruit oil, acetaminophen or 6-Methyl-5-hepten-2-one 0.31 983 MS, KI saline. Withdrawal of the tail from the hot water was taken b-Myrcene 2.06 987 MS, KI, ST as the end point. A cut-off time of 10 s was adopted to p-Cymene 0.03 1016 MS, KI, ST prevent damage to mouse tails [12].