Journal of Ethnopharmacology 166 (2015) 240–249

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Psychoneuropharmacological activities and chemical composition of essential oil of fresh fruits of Piper guineense (Piperaceae) in mice Idris Ajayi Oyemitan a,c,n, Omotola Aanuoluwa Olayera a, Akeeb Alabi a, Luqman Adewale Abass a, Christianah Abimbola Elusiyan b, Adebola Omowumi Oyedeji c, Moses Atanda Akanmu a a

Department of Pharmacology, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Osun State 220005, Nigeria Drug Research and Production Unit (DRPU), Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife, Osun State 220005, Nigeria c Department of Chemistry and Chemical Technology, Walter Sisulu University, Nelson Mandela Drive Campus, 5117 Mthatha, South Africa b

art ic l e i nf o

a b s t r a c t

Article history: Received 17 October 2014 Received in revised form 2 February 2015 Accepted 3 March 2015 Available online 11 March 2015

Ethnopharmacological relevance: Piper guineense Schum & Thonn (Piperaceae) is a medicinal plant used in the Southern States of Nigeria to treat fever, mental disorders and febrile convulsions. Aims of the study: This study aims at determining the chemical composition and the central nervous system (CNS) activities of the essential oil obtained from the plant's fresh fruits in order to rationalize its folkloric use. Materials and methods: Essential oil of P. guineense (EOPG) obtained by hydrodistillation was analysed by GC/MS. EOPG (50–200 mg/kg, i.p.) was evaluated for behavioural, hypothermic, sedative, muscle relaxant, anti-psychotic and anticonvulsant activities using standard procedures. Results and discussion: Analysis of the oil reveals 44 compounds of which 30 compounds constituting 84.7% were identified. The oil was characterized by sesquiterpenoids (64.4%) while only four monoterpeneoids (21.3%) were found present in the oil. Major compounds identified were βsesquiphellandrene (20.9%), linalool (6.1%), limonene (5.8%), Z-β-bisabolene (5.4%) and α-pinene (5.3%). The EOPG (50–200 mg/kg, i.p.) caused significant (p o0.01) inhibition on rearing {F(4,20) ¼ 43}, locomotor {F(4,20) ¼22} activity and decreased head dips in hole board {F(4,20) ¼7} indicating CNS depressant effect; decreased rectal temperature {F(4,20) ¼7–16}, signifying hypothermic activity; decreased ketamine-induced sleep latency {F(4,20) ¼ 7.8} and prolonged total sleeping time {F(4,20) ¼ 8.8}, indicating sedative effect; reduced muscular tone on the hind-limb grip test {F(4,20) ¼ 22}, inclined board {F(4,20) ¼4–49} and rota rod {F(4,20) ¼ 13–106}, implying muscle relaxant activity; induced catalepsy {F(4,20) ¼47–136}, inhibited apomorphine-induced climbing behaviour {F(4,20) ¼9} and inhibited apomorphine-induced locomotor {F(4,20) ¼ 16}, suggesting anti-psychotic effect; and protected mice against pentylenetetrazole-induced convulsions, indicating anticonvulsant potential. Conclusion: The most abundant component of the fresh fruits essential oil of P. guineense was βsesquiphellandrene (20.9%); and the oil possesses CNS depressant, hypothermic, sedative, muscle relaxant, antipsychotic and anticonvulsant activities, thus providing scientific basis for its ethnomedicinal applications. & 2015 Elsevier Ireland Ltd. All rights reserved.

Keywords: Piper guineense Volatile oil Behavioural Sedative Anti-psychotic Anticonvulsant β-sesquiphellandrene

1. Introduction Screening of natural products with the aim of identifying new compounds and activities is on the increase lately. Medicinal plants provide a veritable source for discovery of new biological molecules

n Corresponding author at: Department of Chemistry and Chemical Technology, Walter Sisulu University, Nelson Mandela Drive Campus, 5117 Mthatha, South Africa. Tel./fax: þ 234 80 38171360. E-mail addresses: [email protected], [email protected] (I.A. Oyemitan).

http://dx.doi.org/10.1016/j.jep.2015.03.004 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.

which also provide plausible rationale and justification for traditional uses in several regions of the world (Da Silva et al., 2014). Piper guineense Schum & Thonn (Piperaceae), commonly called West African Black Pepper, is an herbaceous climber commonly found in the African tropical forest zones, with hundreds species distributed in tropical regions of the world (Olonisakin et al., 2006). Locally in Nigeria, it is commonly known as Uziza and Iyere among the Igbos and Yorubas respectively. P. guineense has culinary, medicinal, cosmetic and insecticidal applications (Okwute, 1992; Arong et al., 2011). In folkmedicine, it is used in the treatment of rheumatism and bronchitis (Sofowora, 2008), cough, stomach disorder, intestinal diseases

I.A. Oyemitan et al. / Journal of Ethnopharmacology 166 (2015) 240–249

and gonorrhoea (Mensah et al., 2008; Sumathykutty et al., 1999), obstetrics and fertility enhancement in women (Mbongue et al., 2005; Udoh et al., 1999; Noumi et al., 1998), control of weight/obesity (Mba, 1994), the seeds as an aphrodisiac (Mbongue et al., 2005) and in the treatment of mental illness (Odugbemi, 2008). P. guineense found within the same species and geographical area has been reported to vary in their chemical composition, for example, Ekundayo et al. (1988) showed myristicin, safrole, sarisan and elemicin as main compounds; Oyedeji et al. (2005) reported β-pinene, α-pinene and germacrene-B as major components; Olonisakin et al. (2006) indicated β-pinene, D-Limonene, caryophyllene and car-3-ene as most abundant constituents, while Oboh et al. (2013) reported β-pinene, α-pinene, 1,8-cineole and γ-terpinene as the major components of the plant fruit essential oil from this Nigerian spp. The variations in chemical constituents of this plant species could contribute to its divergent biological activities and this therefore necessitate evaluating its chemical composition along with pharmacological activities. Biological studies attributed to the plant include antioxidant and anti-diabetic (Oboh et al., 2013; Etim et al., 2013), hypolipidemic and hypokalaemic (Nwaichi and Igbinobaro, 2012), insecticidal (Adewoyin et al., 2006), anti-microbial (Oyedeji et al., 2005), sedative activity (Tankam and Ito, 2013) and anticonvulsant activity of aqueous extract of its seed (Abila et al., 1993). The acute toxicity study of the oil conducted in our laboratory indicates the LD50 value of the oil to be 693 mg/kg, i.p. in mice (Unpublished data). Some of the traditional uses of P. guineense are closely associated with the central nervous system; hence it becomes imperative to further evaluate the essential oil of its fresh fruits for possible central activities. In the present work, the essential oil of P. guineense extracted by hydrodistillation was evaluated in terms of CNS activities in experimental animals using standard procedures to possibly validate some of its ethnomedicinal uses. Furthermore, the chemical composition of this particular essential oil species was carried out to determine its chemotype.

2. Materials and methods 2.1. Plant identification and authentication The fruits of P. guineense were authenticated by Mr. G. Ibhanesebor, of the herbarium unit, Department of Botany, Obafemi Awolowo University (OAU) Ile-Ife, Nigeria. Voucher specimen comprising the leaves and fruits was deposited as IFE 16772. 2.2. Plant collection and extraction of the essential oil Fresh fruits of P. guineense were purchased from the Central Market, Ondo, Ondo State in 2012. The fruits were commuted into smaller particles and subjected to hydrodistillation using a Clevenger-type apparatus (BP, 1988). The essential oil (EOPG) was dried over magnesium sulphate crystal and stored in an air tight bottle, and kept refrigerated until use. The oil was emulsified with Tween 80 prior to administration in all the tests with the final concentration of Tween being r5% v/v. Tween 80 (5% v/v) was used as the negative group for all tests. 2.3. GC–MS analysis of the essential oil Essential oil of P. guineense (EOPG) was analysed by GC/MS. The GC/MS analysis was carried out on the Hewlett–Packard Model 5971, GC/MS using a helium as gas carrier, 1 mL flow rate, 30 psi inlet pressure, and spilt ratio 1:30. Temperatures of the column were programmed from 35 to 180 1C at a rate of 4 1C/min, then heated at a rate of 10 1C/min from 180 to 250 1C/min. Mass spectra were

241

recorded from 30 to 450 m/z. Individual components were identified by matching their 70 eV mass spectra with those of the spectrometer database using Wiley L-built library as well as by comparison of the fragmentation pattern with those reported in the literature. 2.4. Pharmacological experiments 2.4.1. Materials and equipment Metler Toledo balance (Switzerland), Plexiglas observation cage (25  25  30 cm3), digital thermometer, suspended iron rod (30 cm long, 0.3 cm diameter and 36 cm above the table), adjustable rectangular wooden inclined board (30  60 cm2, 301, 651), and Rota-Rod machine (Ugo Basile Rota-Rod, Model 7650). 2.4.2. Drugs Ketamine HCL (Alpha Pharm. Nig.), diazepam (Valium(R) Roche, Switzerland), pentylenetetrazole (Sigma, USA), strychnine (Sigma, Switzerland, MSDS), fluoxetin (FLUTEXs Medbios, India), normal saline (Unique Pharm. Nig. Ltd.), haloperidol HCl, Apomorphine HCl, normal saline (Unique Pharm. Nig. Ltd.), and other reagents were of analytical grade. 2.4.3. Laboratory animals Adult male and female albino mice (VOM strain) 18–25 g were obtained from the Animal house, Department of Pharmacology, Faculty of Pharmacy, OAU, Ile-Ife. The animals were maintained on standard animal pellets and water ad libitum. The ethical clearance for this research was obtained through the Faculty Postgraduate Committee and all animal experiment was carried out in strict compliance with the National Institute of Health (NIH, 1985) as being implemented by the OAU Research Committee. 2.4.4. General experimental design Animals were randomly selected into 5 groups (n ¼ 5). Group I serves as the negative control which received the vehicle (5% Tween 80, 10 ml/kg) only. Test groups II–IV were treated with the EOPG at doses of 50, 100 and 200 mg/kg respectively, while the positive control group received the appropriate standard drug. All treatments were by intraperitoneal (i.p.) route. 2.4.5. Effect of the EOPG on novelty-induced behaviours (NIB) in mice The novelty induced behavioural effects scores of rearing and locomotion were performed according to Onigbogi et al. (2000) with minor modification. Each mouse was placed inside Plexiglas's cage and observed for rearing (20 min) and locomotive activity (20 min) after 30 min of pre-treatment. The floor of the cage was divided into 16 equal squares and the number of squares crossed with all the fore and hind limbs was counted as locomotion, while rearing was the number of times the animal places its fore paws against the wall of the cage or in the air. The positive control group V received diazepam (1 mg/kg, i.p.). Head-dips performed by the mouse were also counted for a period of 5 min (Takeda et al., 1998). Diazepam (1 mg/kg, i.p.) also serves as the standard treatment. 2.4.6. Effect of the EOPG on rectal temperature The effect of the EOPG on body temperature of mice was determined using a thermo probe digital thermometer. The rectal temperature of each mouse was taken by inserting the probe 2 cm deep into the anus at time 0, 30, 60 and 120 min posttreatment. Mean7SEM were calculated for each treated group (Al-Naggar et al., 2003). 2.4.7. Effect of the EOPG on ketamine induced-hypnosis The effect of the EOPG on ketamine-induced sleeping time was measured as described by Mimura et al. (1990). The animals were

242

I.A. Oyemitan et al. / Journal of Ethnopharmacology 166 (2015) 240–249

pre-treated 30 min prior to administration of ketamine (100 mg/ kg, i.p.). The interval between the administration of ketamine until loss of righting reflex was recorded as onset of sleep or sleep latency (SL), while the time from the loss, to the regaining of the righting reflex as duration of sleep or total sleeping time (TST) (Bastidas-Ramirez et al., 1998). Diazepam (1 mg/kg, i.p.) served as the standard drug. 2.4.8. Effect of the EOPG on skeletal muscle 2.4.8.1. Hind-limb grip test. The grip strength was used to assess the effects of the EOPG on muscular incoordination of mice. Modified (Oyemitan et al. 2008) method of Asuzu et al. (1998) was used. Diazepam (2 mg/kg, i.p.) was used as the positive control. The apparatus consisted of an iron rod, about 0.5 cm thick and 30 cm long, suspended on two perpendicular retort stands (40 cm) high. All the mice used were first pretested before the start of the experiment by suspending them on the rod with their fore paws. Only mice that were able to pull-up within 15 s were selected for the test. Thirty minutes after the animals were administered the vehicle, the oil or diazepam, each animal was then suspended on the rod with its forepaws and the pull-up time for each mouse was scored as follows (Oyemitan et al., 2008): Assessment

Scores

(i) Able to pull-up within 15 s (ii) Pull-up after 20 s (iii) Unable to pull-up after 20 s but hold with forepaws before falling (iv) Unable to hold the rod with forepaws/fall instantly

0 1 2 3

The scores were recorded at 30, 60, 90 and 120 min posttreatment. 2.4.8.2. Inclined board test. The method originally used by Randall et al. (1961) was used with minor modification (inclined board was set at 651). Mice after treatment were placed on the inclined plane screen individually 20 cm from the top of the plane and the time to slide-down 30, 60, 90 and 120 min post-treatment was recorded. The mice were left on the screen to observe whether the paralysant effect was severe enough to cause the mice to slide-down the screen. Cut-off time was fixed at 60 s. 2.4.8.3. Rota rod test. Naive mice were placed on a horizontal wooden rod (32 mm diameter) rotating at a speed of 17 rpm on the rota rod machine. Animals remaining on the rod for up to 180 s or more on three successive trials were selected for the experiment. Positive control group was administered diazepam (2 mg/kg, i.p.). The animals were placed on the rod after 30 min of pretreatment and the time taken for the mice to fall-off from the rotating rod was noted and recorded (Sravani et al., 2012). The cutoff point was fixed at 180 s. 2.4.9. Effect of the EOPG on experimental psychosis 2.4.9.1. Catalepsy test. Each mouse (after 30 min of pre-treatment) was placed in the observation cage with its fore paws over a 3.5 cm bar and watched for the time it takes to remove its fore paws from the bar. This procedure was repeated at 30, 60 and 120 min post-treatment. The intensity of catalepsy was measured by the duration of time the animal took to remove both fore limbs from the bar to the floor of the observation cage (Navarro et al., 1998). Haloperidol (5 mg/kg, i.p.) was the standard drug.

2.4.9.2. Effect of EOPG on apomorphine-induced climbing test. The method described by Davis et al. (1986) was adopted with minor modification. The following scoring system was employed: 0 ¼all paws on cage floor; 1 ¼two paws placed on the side of the cage; 2¼ all paws off floor; 3 animal climbed and remain on the wall. The scores achieved by individual animals were summed so that each animal obtained a final score between 0 and 6. Climbing behaviour assessment was for 2 min after 10, 20 and 30 min postapomorphine injection (2 mg/kg, i.p.). 2.4.9.3. Swimming-induced grooming behaviour test. Grooming behaviour was induced in mice by a short period of swimming as described by Chesher and Jackson (1981). Prior to short swimming in a chamber (8 cm high) filled with water (30 1C) for 1 min, the animals were pre-treated following the standard protocol. Afterwards, the animals were towel-dried and immediately placed inside the observation cage and scored as follows: Presence of grooming¼1; absence of grooming¼ 0, for every 2 min and up to total time of 20 min. The maximum score possible is 10 points. 2.4.9.4. Apomorphine-induced locomotor activity test. Locomotor activity was assessed as described by Eilam and Szechtman, 1967. Standard treatments were administered 30 min prior to apomorphine (2 mg/kg, i.p.). Spontaneous locomotor activity was measured immediately after mice were placed in the observation cage and counted the number of line cross for 15 min. Haloperidol (5 mg/kg, i.p.) was used as the positive control. 2.4.9.5. Apomorphine-induced hypothermia test. Hypothermia was induced by apomorphine (2 mg/kg, i.p.) and was recorded in all the groups. Treatments followed the standard procedure. The rectal temperature of each mouse was taken with a digital thermometer inserted 2 cm deep into their anus prior to apomorphine injection and at 30, 60 and 90 min post-apomorphine administration (Puech et al., 1981). 2.4.10. Effect of the EOPG on pentylenetetrazole (PTZ)-induced convulsion Pentylenetetrazole (85 mg/kg, i.p.) was used to induce tonic–clonic convulsions in mice (Swinyard et al., 1989). The standard treatments were administered 30 min prior to PTZ (85 mg/kg, i.p.) and diazepam (1 mg/kg, i.p.) was used as the positive control. Each animal was observed for tonic–clonic convulsion. Animals that survived beyond 30 min were regarded as protected (Pourgholami et al., 1999). 2.4.11. Statistical analysis The results were expressed as mean7SEM. All parametric tests were analysed using one-way analysis of variance (ANOVA) followed by Dunnett's posthoc test for comparison between the treated groups and control; while all non-parametric tests involving scoring such as in hind-limb grip test (muscle relaxant), swimming-induced grooming and apomorphine-induced climbing behaviour (antipsychotic) were analysed with Kruskal–Wallis followed by Dunn's multiple comparison test. The level of significance was set at 95% confidence interval at po0.05. The statistical softwares used were GraphPad Instat3.0 and GraphPad Prism 5 (Copyright (c) 2007 GraphPad Software Inc.).

3. Result 3.1. Chemical composition of the EOPG EOPG was obtained as colourless oil with characteristic aromatic smell. Its relative density was 833 mg/ml. GC/MS analysis indicates that the EOPG contained 45 compounds out of which 30 constituents made-up approximately 85% of the oil's constituents

I.A. Oyemitan et al. / Journal of Ethnopharmacology 166 (2015) 240–249

243

Table 1 Chemical composition of the EOPG.

H

ß-sesquiphellandrene Linalool

Limonene

Z-β-Bisabolene

Fig. 1. Some major compounds identified in the EOPG fresh fruits.

(Fig. 1 and Table 1). The sesquiterpene β-sesquiphellandrene accounted for 20.9% of the total constituents. Other prominent compounds identified in the oil are linalool (6.1%), limonene (5.8%), Z-β-bisabolene (5.4%) and α-pinene (5.3%). Although this report is on the fresh oil, the chemical profile is quite different from those in literatures which therefore suggest that this particular species may be of a different chemotype from those previously reported. 3.2. Effects of the EOPG on novelty-induced behaviours in mice The EOPG significantly decreased rearing [p o0.05–p o0.01; F(4,20) ¼43] and spontaneous locomotive activity [p o0.01; F(4,20) ¼22] compared to vehicle. Diazepam, also demonstrated similar effect (Fig. 2). The EOPG dose dependently suppressed exploratory head dipping behaviour significantly [p o0.01; F(4,20) ¼7] in mice (Fig. 3).

Peak no.

Retention time (min)

RI

Relative abundance (%)

Identified compound

CAS identification numbera,b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20– 22 23 24 25 26 27 28– 29 30 31

4.363 5.444 6.617 8.740 16.785 17.563 18.267 18.708 19.085 19.801 20.081 20.527 20.991 21.174 21.286 21.471 21.826 21.975 22.501 23.262– 23.331 23.428 23.480 23.646 23.886 24.023 24.498– 27.727 24.842 24.968

946 986 1021 1058 1379 1389 1418 1421 1445 1447 1455 1460

5.3 4.1 5.8 6.1 0.5 0.4 0.3 3.3 0.5 0.5 1.7 4.6 0.3 1.3 3.1 4.5 1.1 4.2 5.4 20.9

α-pinene β-pinene Limonene Linalool α-copaene β-elemene α-cedrene β-caryophyllene Cubeb-11-ene cis-caryophyllene α-humulene (Z,Z)-α-farnesene Unidentified γ-muurolene germacrene D ar-curcumeme β-himachalene α-zingiberene Z-β-bisabolene βsesquiphellandrene E-γ-bisabolene Unidentified Unidentified Elemol Unidentified E-nerolidol

80-56-8 127-91-3 5989-27-5 78-70-8 3856-25-5 515-13-9 469-61-4 87-44-5 13744-15-5c 118-65-0 6753-98-6 28973-99-1c

77171-55-2 1139-30-6

32 33 34 35 36 37 38 39

25.328 25.626 25.866 26.003 26.198 26.341 26.507 26.638– 26.730

Spathulenol caryophyllene oxide Unidentified Rosifoliol Humulene-epoxide Cedrol T-cadinol T-murrolol Unidentified Cedrenol

Total a b

3.3. Effect of the EOPG on rectal temperature The EOPG showed both significant dose-and time-dependent effect on the normal rectal temperature of the treated mice compared to vehicle and diazepam (Table 2). EOPG at 100 mg/kg and DZM (1 mg/kg) caused significant (p o0.05) reduction in rectal temperature at 60 min; however only EOPG at 200 mg/kg caused significant [p o0.01; F(4,20) ¼ 16, 15 and 7.2] reduction in rectal temperature at 30, 60 and 120 min respectively. 3.4. Effect of the EOPG on ketamine-induced hypnosis The EOPG (50, 100 F(4,20) ¼7.8] reduced SL 200 mg/kg significantly to vehicle. The EOPG at to DZM (1 mg/kg, i.p.).

and 200 mg/kg) significantly [p o0.05; (Fig. 4A) and prolonged TST at 100 and [p o0.01; F(4,20) ¼8.8] (Fig. 4B) compared 200 mg/kg induced higher TST compared

3.5. Effect of the EOPG on the skeletal muscle 3.5.1. Effect of the EOPG on hind-limb grip test The EOPG at 50 mg/kg did not cause significant muscle relaxant effect compared to control throughout the observation period. However, at 100 mg/kg it caused significant muscle relaxation

c

1476 1478 1479 1480 1489 1503 1520 1521

1560

2.6 1.4 0.4 0.4 0.4 2.4

1576 1586

0.6 0.7

1549

1590 1595 1596 1640 1642 1658

1.2 0.3 0.6 0.4 1.6 0.3 1.2 2.2

30021-74-0 23986-74-5 644-30-4 94297-35-5c 495-60-3 495-61-4c 20307-83-9c 53585-13-0c

639-99-6 716-66-3

63891-61-2 19888-34-7 77-53-2 23178-88-3 19912-62-0 2831-03-0

90.6

Adams (1989). ESO (2000, 1990). Cross reference with the literature (Joulain and Koenig, 1998).

(*p o0.05) at 30, 60 and 90 min. The oil at 200 mg/kg also caused significant (po 0.01) muscle relaxation at 30, 60 and 90 min posttreatment. Diazepam (2 mg/kg) induced muscle relaxation compared to vehicle but non-significantly (Table 3). 3.5.2. Effect of the EOPG on the inclined board (70º) The EOPG at 50 or 100 mg/kg did not produce significant (p 40.05) reduction in retention time (60 s) compared to vehicle. EOPG caused significant (po 0.01) reduction in retention time throughout the test period (120 min), while DZM (2 mg/kg) caused significant reduction in retention time on the inclined board at 30 (p o0.01) and (p o0.05) 60 min only (Fig. 5A). 3.5.3. Effect of the EOPG on rota rod test The EOPG at 50 mg/kg did not produce significant (p 40.05) reduction in retention time on the rota rod compared to vehicle, as the mice remained on the rotating rod beyond 180 s. The EOPG at 100 mg/kg caused a significant (p o0.05) reduction in retention time on the rod at 30 min only, but at 200 mg/kg, it caused significant (po 0.01) reduction in endurance time on the rod.

244

I.A. Oyemitan et al. / Journal of Ethnopharmacology 166 (2015) 240–249 80

Number oh head dips (5 min)

70 60 50

**

40 30

**

20 10 0 VEH

50 100 200 _____________________________ EOPG

DZM 1

Treatment (mg/kg,i.p.)

Fig. 3. Effect of EOPG on the head-dips behaviour in mice. VEH, EOPG and DZM represent vehicle, essential oil of P. guineense and diazepam respectively. N ¼5. Each bar represents mean7 SEM. **p o 0.01 statistically lower than VEH (ANOVA, Dunnett's test).

Table 2 Effect of EOPG on rectal temperature of mice. Treatment mg/kg, i.p., n¼ 5 Variation (1C) in rectal temperature posttreatment at:

Vehicle EOPG 50 EOPG 100 EOPG 200 Diazepam 1

30 min

120 min

60 min

 0.2 7 0.03  0.3 7 0.04nn  0.6 7 0.07nn  0.9 7 0.10nn  0.6 7 0.08nn

 0.2 7 0.02  0.3 7 0.01  1.17 0.02n  2.4 7 0.04nn  1.0 7 0.02n

0.4 7 0.01 0.3 7 0.01  0.8 7 0.02  1.3 7 0.50nn 0.2 7 0.02

Each value is the mean variation in rectal temperature (1C) from the basal value at time zero. EOPG represents the essential oil of P. guineense. n

p o 0.05; statistically lower than vehicle (ANOVA, Dunnett's test). p o 0.01; statistically lower than vehicle (ANOVA, Dunnett's test).

nn

Fig. 2. Effect of EOPG on rearing (A) and locomotor activity (B). VEH, EOPG and DZM represent vehicle, essential oil of P. guineense and diazepam respectively. Each bar represents mean 7SEM. N ¼ 5. *p o0.05 statistically lower than VEH; **po 0.01 statistically lower than VEH (ANOVA, Dunnett's test).

Diazepam (2 mg/kg) also significantly (p o0.01) reduced retention time on the rotating rod at 30 and 60 min only (Fig. 5B).

3.6.3. Effect of the EOPG on swimming-induced grooming test in mice The EOPG at 50–200 mg/kg inhibited non-significantly (p40.05) swimming-induced grooming behaviour when compared to vehicle but haloperidol (5 mg/kg, i.p.) caused significant (po0.001) reduction in swimming-induced grooming behaviour compared to vehicle (Table 6).

3.6. Effect of the EOPG on experimental psychosis 3.6.1. Effect of the EOPG on catalepsy test in mice The oil (50 and 100 mg/kg) and vehicle did not show significant (p40.05) delay in the reaction time of the mice in this model. The EOPG at 200 mg/kg extended significantly [po0.01; F(4,20) ¼47, 136 and 88] the mean latency time it took the mice to remove their fore limbs from the bar at 30, 60 and 120 min post-treatment respectively which were comparable to haloperidol, 5 mg/kg, i.p. (Table 4).

3.6.4. The effect of the EOPG on apomorphine-induced locomotion in mice The result of the effect of the oil (50, 100 and 200 mg/kg, i.p.) on apomorphine-induced locomotion indicates that the oil and haloperidol (5 mg/kg, i.p.) significantly [p o0.01; F(4,20) ¼25], reduced the apomorphine-induced locomotion in mice (Fig. 6).

3.6.2. Effect of the EOPG on apomorphine-induced climbing behaviour test in mice The EOPG (50–200 mg/kg) and vehicle did not reverse apomorphine induced climbing behaviour in this test; however haloperidol reversed apomorphine-induced climbing behaviour but non-significantly (Table 5).

3.6.5. The effect of the EOPG on apomorphine-induced hypothermia in mice The EOPG at all doses tested and haloperidol (5 mg/kg) did not cause significant (p 40.05) reversal of the apomorphine-induced hypothermia compared to vehicle throughout the test period (result not shown).

I.A. Oyemitan et al. / Journal of Ethnopharmacology 166 (2015) 240–249

245

100% protection against the PTZ-induced convulsion similarly to diazepam (1 mg/kg), a standard anticonvulsant drug (Table 7).

120 100

4. Discussion and conclusion *

Sleep latency (s)

80

** ** **

60 40 20 0 VEH

50 100 200 _________________________ EOPG

DZM 1

Treatment (mg/kg, i.p.)

4000

##

##

3500

##

Total sleeping time (s)

3000 2500 2000 1500 1000 500 0 VEH

50 100 200 __________________________ EOPG

DZM 1

Treatment (mg/kg, i.p.) Fig. 4. Effect of EOPG on ketamine-induced hypnosis in mice. Panel A is sleep latency and panel B is total sleeping time. VEH, EOPG and DZM represent vehicle, essential oil of P. guineense and diazepam respectively. N ¼ 5. Each bar represents mean 7 SEM. *p o 0.05; **p o0.01 statistically lower than VEH (Panel A). ## p o 0.01; statistically higher than VEH (Panel B), (ANOVA, Dunnett's test).

Table 3 Effect of the EOPG on the hind-limb grip test in mice. Treatment mg/kg, i.p. (n¼ 5) Muscle relaxant scores (mean 7 SEM) assessed after:

Control (5% Tween 80) EOPG 50 EOPG 100 EOPG 200 Diazepam 2

30 min

60 min

90 min

0.0 7 0 0.0 7 0 3.0 7 0.0n 3.0 7 0.0n 2.6 7 0.3

0.0 7 0 0.0 7 0 2.8 7 0.2n 3.0 7 0.0nn 1.8 7 0.4

0.0 7 0 0.0 7 0 2.4 7 0.3n 37 0.0nn 1.0 7 0.3

EOPG is essential oil of P. guineense. n

po 0.05; significantly different from vehicle (Kruskal–Wallis, Dunn's). p o0.01; significantly different from vehicle (Kruskal–Wallis, Dunn's).

nn

3.7. Effect of the EOPG on PTZ-induced convulsion in mice The EOPG (50 mg/kg) and vehicle did not protect mice against PTZ-induced convulsion. However, at 100 mg/kg it offered a 40% protection while the animals that survived convulsed but at 200 mg/kg, EOPG prevented episodes of convulsion and offered

4.1. Discussion This study evaluated the psychoneuropharmacological effects of the essential oil of P. guineense (EOPG) for behavioural, hypothermic, hypnotic, muscle relaxant, antipsychotic and anticonvulsant activities in mice. The result of this study showed that P. guineense essential oil produced mainly depressant effects on the central nervous system (CNS). In the behavioural tests the oil caused significant (po0.05) inhibition of rearing and spontaneous locomotor behaviour when compared to the vehicle (Fig. 2), indicating central depressant activity (Amos et al., 2004). It has been confirmed that CNS depressant lowers the level of exploration or inquisitiveness of animals (HellionIbarrola et al., 1999) hence this result showed that EOPG may possess inhibitory effects on the CNS. It can be suggested that the inhibitory effect of the EOPG on novelty induced behaviour may be mediated through augmentation of GABA neurotransmission in the CNS (Rang et al., 2007). The novelty-induced behavioural responses are known to be regulated by multiple neurotransmitter systems including: gamma amino butyric acid (GABA), acetylcholine, dopamine, opioid, serotonin (5-HT), and many others yet to be ascertained. Increased rearing behaviours are mainly modulated by enhanced dopamine neurotransmission (Ayhan and Randrup, 1973) or cholinergic stimulation (Jones et al., 1981). Furthermore, it has been observed that most behavioural effects of dopamine transmission in the CNS are directly modulated by 5-HT transmission (Scalzitti et al., 1999). In the hole-board, nose-poking has been shown to indicate curiosity in rodents. It has been shown that anxiolytic drugs increase exploration of the holes by rodents (da Silva and Elisabetsky, 2001). The EOPG (50–100 mg/kg, i.p.) and DZM (1 mg/ kg) decreased the exploratory head dipping behaviour of mice in this study (Fig. 3) therefore suggesting anxiogenic-like behaviour due to the administration of the oil. Increase in head-dipping has been associated with anxiolytic-like effect while decreased headdipping is related to anxiogenic-like effect (Takeda et al., 1998; Walf and Frye, 2007) while benzodiazepines tend to suppress nosepocking at relatively low doses (Vogel, 2002). However, decreased head-dipping may be due to sedative effect at higher doses, implying that EOPG and diazepam at the doses used in this study manifested significant sedative effect thereby inhibiting the headdips in this study (McNamara et al., 1989; Rabbani et al., 2003). The EOPG at all doses used dose-dependently caused a significant (po0.05) decrease in rectal temperature compared to vehicle (Table 2). The oil at 50 mg/kg caused significant (po0.05) decrease in the rectal temperature at 30 min only while at dose level of 100 mg/kg, it significantly (po0.01) decreased the rectal temperature at 30 and 60 min. However, at 200 mg/kg, i.p., it significantly (po0.01) decreased the rectal temperature up to 90 min posttreatment signifying hypothermic effect (Devi et al., 2003). Diazepam also caused a significant (po0.05) decrease in the rectal temperature similarly to the EOPG at 100 mg/kg, i.p., thus confirming the earlier report of benzodiazepines hypothermic effect in animals (Jackson and Nutt, 1990). Changes in body temperature have been variously linked with several endogenous neurotransmitters in the hypothalamus including but not limited to dopamine, ACh, GABA, norepinephrine and opioid (Rang et al., 2007). Ketamine-induced hypnosis test is used normally to investigate the sedative effect of an agent (Mimura et al., 1990). The EOPG at the test doses (50, 100, and 200 mg/kg) significantly (po0.01) decreased SL compared to the vehicle-treated animals and significantly (po0.05)

246

I.A. Oyemitan et al. / Journal of Ethnopharmacology 166 (2015) 240–249

VEH EOPG 50

60

EOPG 100

*

EOPG 200

**

40

DZM 2

** **

20

90

60

0

30

Time able to remain on the sliding board (s)

80

Time of assessment after treatment (min)

Endurance time on the rota rod (s)

200

VEH EOPG 50

150

EOPG 100 EOPG 200

100

DZM 2

**

* 50

**

** ** ** 90

30

60

0 Time of assessment after treatment (min) Fig. 5. Effect of EOPG on the time spent on the incline board (A) and endurance time on the rota rod (B) by the mice. Each bar represent score (mean 7 SEM). VEH, EOPG and DZM represent vehicle (5% Tween 80), essential oil of P. guineense and diazepam respectively. *p o 0.05; **p o0.01; significantly different from vehicle (ANOVA, Dunnett's test).

Table 4 Effect of the EOPG on catalepsy test in mice. Treatment (mg/kg, i.p.) n ¼5

VEH EOPG 50 EOPG 100 EOPG 200 Haloperidol 5

Table 5 Effect of the EOPG on apomorphine-induced climbing behaviour in mice.

Reaction time (mean 7SEM) in second at: 30 min

60 min

120 min

1.0 7 0.0 1.0 7 0.0 1.0 7 0.0 7.4 7 0.7nn 12.2 7 1.5nn

1.0 7 0.0 1.0 7 0.0 1.0 7 0.0 30.0 7 3.5nn 70.67 4.7nn

1.0 7 0.0 1.0 7 0.0 1.0 7 0.0 53.0 7 5.4nn 49.07 3.7nn

VEH and EOPG represent vehicle (5% Tween 80) and the essential oil of Piper guineense respectively. Reaction time is the time the mouse takes to remove its forepaws from the bar. nn

Treatment (mg/kg), i.p. n¼ 5 Climbing scores (mean7 SEM) after apomorphine injection at:

VEH EOPG 50 EOPG 100 EOPG 200 Haloperidol 5

10 min

20 min

30 min

5.4 7 0.7 5.2 7 0.5 5.2 7 0.5 4.4 7 0.4 4.0 7 1.3

5.4 7 0.7 4.4 7 0.4 4.4 7 0.4 4.4 7 0.4 2.4 7 1.0

5.4 70.7 4.4 70.4 3.6 71.0 1.6 71.0 0.0 70.0

VEH and EOPG represent vehicle (5% Tween 80) and the essential oil of P. guineense respectively.

p o0.01; statistically higher than vehicle (ANOVA, Dunnett's test.

prolonged TST compared to vehicle (Fig. 4). Diazepam similarly caused significant (po0.05) decrease in the SL and prolonged TST compared to vehicle. These results suggest that EOPG possess significant sedative activity in this model (Haque et al., 2001; Rabbani et al., 2008). The mechanism of action of the EOPG in mediating this sedative activity may be through augmentation of GABA neurotransmission in the brain (Dhawan et al., 2003), enhancing blockade of N-methyl-Daspartate (NMDA) subtype of glutamate receptor by ketamine (Orser et al., 1997; Sleigha et al., 2014) or both. Further study is imperative to

determine the specific mechanism of hypnotic action. This sedative result also provides scientific basis for the ethnomedicinal uses of the plant in the management of some CNS-related ailments. The EOPG dose-dependently produced reduction in muscle tone compared to both the control and the standard drug, diazepam (Table 3 and Fig. 5). In unaffected animal, the hind limbs normally recline upward and grip the rod to support the fore limbs from losing its grip (Abid et al., 2006), but failure to do this within specific timelimit indicates muscle relaxant effect (Oyemitan et al., 2008). The EOPG (100 and 200 mg/kg) significantly (po0.01) caused muscle

I.A. Oyemitan et al. / Journal of Ethnopharmacology 166 (2015) 240–249

Table 6 Effect EOPG on swimming-induced grooming in mice. Treatment mg/kg, i.p. n ¼5

Number of grooms (mean 7SEM) in 20 min

VEH EOPG 50 EOPG 100 EOPG 200 Haloperidol 5

9.4 7 0.4 7.8 7 0.7 5.8 7 0.6 6.6 7 0.4 2.8 7 0.4nnn

EOPG is essential oil of P. guineense. nnn

p o 0.01; significantly different from vehicle (Kruskal–Wallis, Dunn's). VEH

EOPG 50

APO 5

EOPG 200

EOPG 100

Number of apomorphine-induced locomotion/15 min

300

200

* **

**

100

** 0

Treatment (mg/kg, i.p., n=5)

Fig. 6. Effect of EOPG on apomorphine-induced locomotor activity in mice. VEH, EOPG and APO represent vehicle (5% Tween 80), the essential oil of P. guineense and apomorphine respectively. *p o0.05; **p o 0.01, statistically less than vehicle (ANOVA, Dunnett's test).

Table 7 Effect of the EOPG on PTZ-induced convulsion in mice. Treatment (mg/kg, i.p.) n ¼5

Incidence of convulsions

% Mortality

% Protection

VEH EOPG 50 EOPG 100 EOPG 200 Diazepam 1

þ þ þ  

100 100 60 0 0

0 0 40 100 100

VEH and EOPG represent vehicle (5% Tween 80) and the essential oil of Piper guineense respectively. (  ) and (þ ) represent incidence and absence of seizures respectively.

relaxation after 30, 60 and 90 min post-treatment with the mice unable to hold the rod or fall-off from the rod instantly (Fig. 5). The muscle relaxant effect of the EOPG on the 651 inclined board showed that EOPG significantly (po0.05) induced muscle weakness at 100– 200 mg/kg by causing the mice to slide-off the inclined board faster than vehicle-treated mice (Fig. 5A), indicating muscle relaxant effect (Shalam et al., 2007). The rota rod test was performed to determine if the EOPG acts via the neuro-muscular junction or centrally (Chindo et al., 2014), and it assessed the ability of the mice to hold on to the rotating horizontal rod, mice that fall off from the rod within the observation period indicate positive muscle relaxant effect (Sravani et al., 2012). The EOPG at 100 and 200 mg/kg induced significant (po0.05–0.01) muscular weakness (Fig. 5B), suggesting that the mechanisms involved may be central as well as peripheral (Vongtau et al., 2005). Furthermore, EOPG can also be suggested to contain constituent(s) that cause blockage of neurotransmission at the neuromuscular junction similarly to tubocurarine, a non-depolarizing neuromuscular blocking drug which acts by interacting with nicotinic receptor and prevent the binding of acetylcholine at the neuronal synapses (Karpen and Hess, 1986), thus, preventing the depolarization of the muscle cell membrane and inhibiting muscular

247

contraction. The effect of diazepam (2 mg/kg, i.p.) in this study was lower than that of EOPG (200 mg/kg, i.p.) implying that the mechanism(s) involved may not only be by increasing presynaptic inhibition in the spinal cord (Mycek et al., 1997). The muscle relaxant results obtained in this study corroborated earlier report in which methanolic extract of the plant demonstrated muscle relaxant effect in rat and frog in vitro (Udoh et al., 1999). The results may be used to justify the ethno-medicinal uses of the fruits of the plant particularly in the management of pain and arthritis among other uses. The antipsychotic effect of the EOPG was evaluated on 5 different animal models; catalepsy, apomorphine-induced climbing, swimminginduced grooming, apomorphine-induced hypothermia and apomorphine-induced locomotion in mice and the results indicate that the EOPG showed positive anti-psychotic effect. The result of the catalepsy test shows that the oil (200 mg/kg, i.p.) caused prolongation in the reaction time for the mice to remove their fore paws from the bar compared to the vehicle group at up-to 120 min posttreatment (Table 5), signifying positive antipsychotic effect. Haloperidol also showed positive effect in this model that was higher than that of the oil at 200 mg/kg. The catalepsy test has frequently been used to detect antipsychotic effect of several agents and may be suitable for screening of essential oil of the plant. Table 3 shows EOPG (200 mg/kg, i.p.) significantly (po0.01) inhibited apomorphine-induced climbing behaviour compared to vehicle and comparable to haloperidol at 30 min post-treatment suggesting possible antipsychotic activity which could be mediated through central depressant effects (Ogren et al., 1984) or dopamine D1 receptor inhibition (Vasse et al., 1988). The result of swimming-induced grooming (Fig. 6A) shows that the oil (100 and 200 mg/kg, i.p.) significantly (po0.01) reduced the episodes of apomorphine-induced grooming in mice compared to vehicle. The oil at 100 and 200 mg/kg, i.p., significantly (po0.01) reduced grooming behaviour. This result indicates that EOPG tested positive for antipsychotic activity in this model which may also be mediated through selective blockade of D1 receptor (Van Wimersma Greidanus et al., 1989). The EOPG at all doses used and haloperidol significantly (po0.01) reduced the number of lines crossed (locomotion) by mice compared when compared to vehicle-treated mice (Fig. 6), indicating antipsychotic activity. The result obtained for apomorphine-induced hypothermia shows that neither the EOPG nor haloperidol decreases rectal compared to vehicle. The oil (200 mg/kg, i.p.) and haloperidol (5 mg/kg, i.p.) inhibited but not reversed the apomorphine-induced hypothermia in this model, indicating no activity. It has been reported that haloperidol did not antagonize hypothermia induced by apomorphine because the initial effect on the receptor showed that hypothermia is associated with D2 dopamine receptor stimulation (Colboc and Costentin, 1980). Supporting evidence for this view is that neuroleptics antagonize apomorophine-induced hypothermia through their affinity for the D3 receptor (Millan et al., 1994); hence, the mechanism of action of the EOPG in this model cannot be predicted based on the available data. In summary, the results obtained from all the models of antipsychotic tests strongly suggest that EOPG may exhibit important antipsychotic property thus providing the scientific and pharmacological basis for the use of the plant in ethnomedicine in the treatment of some neurological disorders. Drugs with significant activity against seizures due to PTZ may be useful in controlling myoclonic and absence seizures in humans (Löscher and Schmidt, 1988). PTZ has been reported to inhibit chloride conductance by binding to picrotoxin sites of GABAA receptor complex (MacDonald and Kelly, 1995). The EOPG significantly (po0.05) decreased mortality dose dependently (Table 7). The EOPG at 100 and 200 mg/kg, i.p., offered 40% and 100% protections respectively, against the PTZ-induced convulsion similar to diazepam (1 mg/kg, i.p.) of 100% protection indicating potential anti-convulsant activity which may be effective in the management of generalized convulsion such as tonic, clonic, aclonic and myoclonic (McNamara, 1989). The mechanism of action of the anticonvulsant effect of the

248

I.A. Oyemitan et al. / Journal of Ethnopharmacology 166 (2015) 240–249

EOPG may be via the augmentation of GABA neurotransmission in the CNS like diazepam, a standard anticonvulsant (Nicoll, 2007). This result also provides plausible rationale for the ethnomedicinal uses of the plant in the management of epilepsy and related ailments. Concerning the specific compound(s) responsible for the various CNS activities of the EOPG or the general mode of action of this oil, it is impossible to point to a particular constituent of the oil as it is well known that essential oils have very complex mechanisms (Djilani and Dicko, 2012); and it will be necessary to evaluate some of the major compounds using the standard reference compounds especially β-sesquiphellandrene with a relative abundance of 20.9%. Other prominent constituents of EOPG are linalool (6.1%), limonene (5.8%), Z-β-bisabolene (5.4%) and α-pinene (5.3%). There is paucity of the literature on the biological activity of βsesquiphellandrene but it has been reported to serve as insect sex attractant by the stink bug (Piezodorus guildinii) as sexual pheromone (Moraes et al., 2008). Recent studies on limonene showed that it inhibits methamphetamine-induced locomotor activity possibly through regulation of 5-HT and dopamine neurotransmission (Yun, 2014), possesses anxiolytic (Lima et al., 2012) and gastroprotective activities (Moraes et al., 2009). S-(þ)-linalool isolated from Lippia alba has been reported to possess both sedative and anaesthetic properties (Heldwein et al., 2014), sedative effect through inhalation (Lincka et al., 2009), anti-inflammatory activity (Peana et al., 2002) and anticonvulsant activity (Elisabetsky et al., 2001). The overall effects observed in this study may be addictive or synergistic as phyto-constituents act mostly through this mechanism (Williamson, 2001). These compounds have been reported to demonstrate several biological activities both in vitro and in vivo and can be suggested here that one or more of these compounds have contributed considerably to the overall CNS activities of the oil in this study. The chemical profile of the fresh fruit essential oil reported here is quite different from those in literatures; we therefore report that this particular species is a β-sesquiphellandrene chemotype.

5. Conclusion The essential oil of the fresh fruit of P. guineense was found to be β-sesquiphellandrene chemotype and it demonstrated depressant effect on the CNS; and possesses significant hypothermic, sedative, muscle relaxant, anti-psychotic and anticonvulsant effects in mice. The CNS activities may be mediated through augmentation of GABA at the GABAA–benzodiazepine receptor complex pathway, or inhibition of dopamine neurotransmission at D1/D2 receptors. The results obtained in this study provide (scientific) pharmacological evidence for the contribution of the essential oil to the various CNS activities reported for the plant.

Author contributions 1. Author IAO initiated, designed and supervised the work, analysed the results, wrote the original draft and coordinated the preparation of the manuscript. 2. Author OAO carried out the experiment on behaviour, hypothermia, hypnosis and wrote preliminary report. 3. Author AA carried out the experiment on antipsychotic tests and wrote preliminary report. 4. Author LAA carried out the experiment on muscle-relaxant activities and wrote preliminary report. 5. Author CAE is a research collaborator of author IAO. She carried out the extraction of the essential oil, processed the GC/MS analysis, reviewed and edited the manuscript.

6. Author AOO is the Post-doc Supervisor of author IAO. She analysed the GC/MS results, identified the chemical constituents of the essential oil, reviewed and edited the manuscript. 7. Author MAA is the Head of Department where the experiment was conducted; he provided materials for the work, reviewed and edited the manuscript.

References Abid, M., Hrishikeshavan, H.J., Asad, M., 2006. Pharmacological evaluation of Pachyrrhizus erosus (L) seeds for central nervous system depressant activity. Indian J. Physiol. Pharmacol. 50 (2), 143–151. Abila, B., Richens, A., Davies, J.A., 1993. Anticonvulsant effects of extracts of the West African black pepper, Piper guineense. J. Ethnopharmacol. 39 (2), 113–117. Adams, R.P., 1989. Identification of Essential Oil Components by Ion Trap Mass Spectroscopy. Academic Press, New York, U.S.A. Adewoyin, F.B., Odaibo, A.B., Adewunmi, C.O., 2006. Mosquito Repellent Activity of Piper guineense and Xylopia Aethiopica fruits oils on Aedes Aegypti. Afr. J. Tradit. Complement. Altern. Med. 3 (2), 79–83. Ayhan, I., Randrup, A., 1973. Behavioural and pharmacological studies on morphine-induced excitation of rats. Possible relation to brain catecholamines. Psychopharmacology 29, 317–328. Al-Naggar, T.B., Gómez-Serranillos, M.P., Carretero, M.E., 2003. Neuropharmacological activity of Nigella sativa L. extracts. J. Ethnopharmacol. 88 (1), 63–68. Amos, S., Akah, P.A., Emwerem, N., Chindo, B.A., Hussaini, I.M., Wambebe, C., Gamaliel, K., 2004. Behavioural effect of Pavetta crassipes. Pharmacol. Biochem. Behav. 77, 751–759. Arong, G.A., Oku, E.E., Obhiokhenan, A.A., Adetunji, B.A., Mowang, D.A., 2011. Protectant ability of Xylopia aethiopica and Piper guineense leaves against the cowpea bruchid Callosobruchus maculatus (Fab.) (Coleoptera: Bruchidae). World J. Sci. Technol. 1 (7), 14–19. Asuzu, I.U., Ezejiofor, S., Njoku, C.J., 1998. The pharmacological activities of Olax viridis root bark extract on central nervous system. Fitoterapia 69 (3), 265–268. Bastidas-Ramirez, B.E., Navarro, R.N., Quezada Arellano, J.D., Ruiz, M.B., Villanueva Michel, M.T., Garzon, P., 1998. Anticonvulsant effects of Magnolia grandiflora L. in the rat. J. Ethnopharmacol. 61, 143–152. British Pharmacopoeia, 1988. Her Majesty Stationary Office london, vol. 2; , pp. A137–A138. Chesher, G.B., Jackson, D.M., 1981. Swim-induced grooming in mice is mediated by a dopaminergic substrate. J. Neural Transm. 50, 47–55. Chindo, B.A., Ya’U, J., Danjuma, N.M., Okhale, S.E., Gamaniel, K.S., Becker, A., 2014. Behavioural and anticonvulsant effects of the standardized extract of Ficus platyphylla stem bark. J. Ethnopharmacol. 154, 351–360. Colboc, O., Costentin, J., 1980. Evidence for thermoregulatory dopaminergic receptors located in the preopticus medialis nucleus of the rat hypothalamus. J. Pharm. Pharmacol. 32, 624–629. Da Silva, A.O., Alves, A.D., de Almeida, D.A.T., Balogun, S.O., de Oliveira, R.G., Aguiar, A.A., Soares, I.M., Marson-Ascêncio, P.G., Ascêncio, S.D., Martins, D.T.D., 2014. Evaluation of anti-inflammatory and mechanism of action of Macrosiphonia longiflora (Desf.) Müll. Arg. J. Ethnopharmacol. 154, 319–329. Davis, A.S., Jenner, P., Marsden, C.D., 1986. A comparison of motor behaviours in groups of rats distinguished by their climbing response to apomorphine. Br. J. Pharmacol. 87, 129–137. Devi, B.P., Boominathan, R., Mandal, S.C., 2003. Evaluation of antipyretic potential of Cleome viscosa Linn. (Capparidaceae) extract in rats. J. Ethnopharmacol. 87 (1), 11–13. Dhawan, K., Kumar, S., Sharma, A., 2003. Evaluation of central nervous system effects of Passiflora incarnata in experimental animals. Pharm. Biol. 41 (2), 87–91. Djilani, A., Dicko, A., 2012. In: Jouad, B. (Ed.), The Therapeutic Benefits of Essential Oils, Nutrition, Well-Being and Health. In Teck Europe, Rijeka, Croatia, ISBN: 978-953-51-0125-3. Eilam, D., Szechtman, H., 1967. Biphasic effect of the D2 agonist quinpirole on locomotion and movements. Eur. J. Pharmacol. 161, 151–323. Elisabetsky, E., Silva Brum, L.F., Souza, D.O., 2001. Anticonvulsant properties of linalool in glutamate-related seizure models. Phytomedicine 6 (2), 107–113. Ekundayo, O., Laasko, I., Adegbola, R.M., Oguntimein, B., Sofowora, A., Hiltunen, R., 1988. Essential oil constituents of Ashanti Pepper (Piper guineense) fruits (Berries). J. Agric. Food Chem. 36, 880–882. ESO, 2000. The Complete Database of Essential Oils B.A.C.I.S. The Netherlands, 1999. Etim, O.E., Egbuna, C.F., Odo, C.E., Udo, N.M., Awah, F.M., 2013. In vitro antioxidant and nitric oxide scavenging activities of Piper guineense seeds. Glob. J. Res. Med. Plants Indig. Med. 2 (7), 485–494. Haque, S., Choudhui, M.S.K., Islam, M.N., Hannan, J.M.A., Shahriar, M., 2001. Pharmacological study of Sri mahalaxmibilas (Rasayan). Hamdard Med. 44, 56–60. Heldwein, C.G., Silva, L.D.L., Gai, E.Z., Roman, C., Parodi, T.V., Bürger, M.E., Baldisserotto, B., Flores, É.M.D.M., Heinzmann, B.M., 2014. S-( þ )-Linalool from Lippia alba: sedative and anesthetic for silver catfish (Rhamdia quelen). Vet. Anaesth. Analg. 41 (6), 621–629.

I.A. Oyemitan et al. / Journal of Ethnopharmacology 166 (2015) 240–249

Hellion-Ibarrola, D.A., Montalbetti, Y., Villalba, D., Heinichen, O., Ferro, E.A., 1999. Acute toxicity and general pharmacological effect on central nervous system of the crude rhizome extract of Kyllinga brevifolia Rottb. J. Ethnopharmacol. 66, 271–276. Jackson, H.C., Nutt, D.J., 1990. Body temperature discriminates full and partial benzodiazepine receptors agonist. Eur. J. Pharmcol. 185, 243–246. Jones, D.L., Mogenson, G.J., Wu, M., 1981. Injections of dopaminergic, cholinergic, serotonergic and GABAergic drugs into the nucleus accumbens: effects on locomotor activity in the rat. Neuropharmacology 20, 29–37. Joulain, D., Koenig, W.A., 1998. The Atlas of Spectral Data of Sesquiterpenes Hydrocarbons. E.B-Verlag Hamburg, Germany. Karpen, J.W., Hess, G.P., 1986. Acetylcholine receptor inhibition by d-tubocurarine involves both a competitive and a noncompetitive binding site as determined by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles. Biochemistry 5 (7), 1786–1792. Lima, N.G., De Sousa, D.P., Pimenta, F.C., Alves, M.F., De Souza, F.S., Macedo, R.O., Cardoso, R.B., de Morais, L.C., Melo, D.M.D., de Almeida, R.N., 2012. Anxiolyticlike activity and GC–MS analysis of (R)-( þ )-limonene fragrance, a natural compound found in foods and plants. Pharmacol. Biochem. Behav. 103 (3), 450–454. Lincka, V.D.M., da Silva, A.L., Figueiróa, M., Piatoa, A.L., Herrmanna, A.P., Bircka, F.D., Caramãod, E.B., Nunese, D.S., Morenof, P.R.H., Elisabetsky, E., 2009. Inhaled linalool-induced sedation in mice. Phytomedicine 16 (4), 303–307. Löscher, W., Schmidt, D., 1988. Which animal models should be used in the search for new antiepileptic drugs? A proposal based on experimental and clinical considerations. Epilepsy Res. 2, 145–181. MacDonald, R.L., Kelly, K.M., 1995. Antiepileptic drug mechanisms of action. Epilepsia 36, S2–S12. Mba, M.A., 1994. Effect of dietary intake of Piper guineense on growth and indices of fitness in Rattus rattus, isc. Innoa 4, 383–388. Mbongue, F.G.Y., Kamtchouing, P., Essame, O.J.L., Yewah, P.M., Dimo, T., Lontsi, D., 2005. Effect of the aqueous extract of dry fruits of Piper guineense on the reproductive function of adult male rats. Indian J. Pharmacol. 7, 30–32. McNamara, J.O., 1989. Development of new pharmacological agents for epilepsy: lessons from the kindling model. Epilepsia 30, S13–S18. Mensah, J.K., Okoli, R.I., Ohaju-Obodo, J.O., Eifediyi, K., 2008. Phytochemical, nutritional and medical properties of some leafy vegetables consumed by Edo people of Nigeria. Afr. J. Biotechnol. 7 (14), 2304–2309. Mimura, M., Namiki, A., Kishi, R., Ikeda, T., Miyake, H., 1990. Antagonistic effect of physostigmine on ketamine-induced anaesthesia. Psychopharmacology 102, 399–403. Millan, M.J., Audinot, V., Rivet, J.M., Gobert, A., Vian, J., Prost, J.F., Spedding, M., Peglion, J.L., 1994. ( þ )-S 14297, a novel selective ligand at cloned human dopamine D3 receptors, blocks ( þ )-7-OH-DPAT-induced hypothermia in rats. Eur. J. Pharmacol. 260, R3–R5. Moraes, M.C.B., Pareja, M., Laumann, R.A., Borges, M., 2008. The chemical volatiles (Semiochemicals) produced by neotropical stink bugs (Hemiptera: Pentatomidae). Neotrop. Entomol. 37 (5), 489–505. http://dx.doi.org/10.1590/S1519566X2008000500001. Moraes, T.M., Kushima, H., Moleiro, F.C., Santos, R.C., Rocha, L.R., Marques, M.O., Vilegas, W., Hiruma-Lima, C.A., 2009. Effects of limonene and essential oil from Citrus aurantium on gastric mucosa: role of prostaglandins and gastric mucus secretion. Chem. Biol. Interact. 180 (3), 499–505. Mycek, M.J., Harvey, R.A., Champe, P.C., 1997. Lippincott’s illustrated reviews. In: Pharmacology. vol. 2, pp. 51–91. Navarro, J.F., Pedraza, C., Martin, M., Manzaneque, J.M., Davila, G., Enrique Maldonado, E., 1998. Tiapride-induced catalepsy is potentiated by gammahydroxybutyric acid administration. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 22, 835–844. National Institute of Health (NIH-1985), 1996. Guide for the Care and Use of Laboratory Animals, National Research Council. National Academy Press, Washington, DC. Nicoll, R.A., 2007. Introduction to the pharmacology of CNS drugs. In: Katzung, B.G. (Ed.), Basic and Clinical Pharmacology. McGraw-Hill Medical, San Francisco, pp. 233–239. Noumi, E., Amvam, Z.P.H., Lontsi, D., 1998. Aphrodisiac plants used in Cameroon. Fitoter 69, 5–34. Nwaichi, E.O., Igbinobaro, O., 2012. Effects of some selected spices on some biochemical profile of wister albino, rats. Am. J. Environ. Eng. 2 (1), 8–11. Oboh, G., Ademosun, A.O., Odubanjo, O.V., Akinbola, I.A., 2013. Antioxidative properties and inhibition of key enzymes relevant to type-2 diabetes and hypertension by essential oils from black pepper. Adv. Pharmacol. Sci. 2013, http://dx.doi.org/10.1155/2013/926047. Odugbemi, T., 2008. A Textbook of Medicinal Plants from Nigeria. University of Lagos Press, Lagos, Nigeria p. 594. Ogren, S.O., Hall, H., Kohler, C., Magnusson, O., Lindbom, L.O., Angeby, K., Florvall, L., 1984. Remoxipride, a new potential antipsychotic compound with selective antidopaminergic actions in the rat brain. Eur. J. Pharmacol. 20 (102), 459–474. Okwute, S.K., 1992. Plant derived pesticidal and antimicrobial agents for use in agriculture. A review of phytochemical and biological studies on some Nigerian plants. J. Agric. Sci. Technol. 2 (1), 62–70. Olonisakin, A., Oladimeji, M.O., Lajide, L., 2006. Chemical composition and antibacterial activity of steam distilled oil of Ashanti pepper (Piper guineense) fruits (Berries). Electron. J. Environ. Agric. Food Chem. 5 (5), 1531–1535.

249

Onigbogi, O., Ajayi, A.A., Ukponwan, O.E., 2000. Mechanism of chloroquine induced body scratching behaviour in rats, evidence of involvement of endogenous opioid peptides. Pharmacol. Biochem. Behav. 65, 333–337. Orser, B.A., Pennefather, P.S., MacDonald, J.F., 1997. Multiple mechanisms of ketamine blockade of N-methyl-D-aspartate receptors. Anesthesiology 86 (4), 903–917. Oyedeji, O.A., Adeniyi, B.A., Ajayi, O., König, W.A., 2005. Essential oil composition of Piper guineense and its antimicrobial activity. Another chemotype from Nigeria. Phytother. Res. 19 (4), 362–364. Oyemitan, I.A., Iwalewa, E.O., Akanmu, M.A., Olugbade, T.A., 2008. Sedative, hypothermic and muscle relaxant effects of the essential oil of Dennettia tripetala G. Baker (Annonaceae) and its mechanisms in mice. Ife J. Sci. 10 (1), 1–9. Peana, A.T., D’Aquila, P.S., Panin, F., Serra, G., Pippia, P., Moretti, M.D., 2002. Antiinflammatory activity of linalool and linalyl acetate constituents of essential oils. Phytomedicine 9 (8), 721–726. Pourgholami, M.H., Kamalinejad, M., Javadi, M., Majzoob, S., Sayyah, M., 1999. Evaluation of the anticonvulsant activity of the essential oil of Eugenia caryophllata, in male mice. J. Ethnopharmacol. 64, 167–171. Puech, A.J., Rioux, P., Poncelet, M., Brochet, D., Chrmat, R., Simon, P., 1981. Pharmacological properties of new antipsychotic agents: use of animal models. Neuropharmacology 20, 1279–1284. Rabbani, M., Sajjad, S.E., Zareitt, H.R., 2003. Anxiolytic effects of Stachys lavandufolia Vahl on the elevated plus-maze model of anxiety in mice. J. Ethnopharmacol. 89, 271–276. Rabbani, M., Sajjadi, S.E., Mohammadi, A., 2008. Evaluation of the anxiolytic effect of Nepeta persica Boiss in mice. Evid. Based Complement. Altern. Med. 5, 181–186. Randall, L.O., Heise, G.A., Schallek, W., Bagdon, R.E., Banzinger, R., Boris, A., Moe, R. A., Abrams, W.B., 1961. Pharmacological and clinical studies on Valium(TM) a new psychotherapeutic agent of the benzodiazepine class. Curr. Ther. Res. 3, 405–425. Rang, H.P., Dale, M.M., Ritter, J.M., Flower, R.J., 2007. Rang & Dale's Pharmacology, 6th ed. Churchill Livingstone, London. Scalzitti, J.M., Carvera, L.S., Hensler, J.G., 1999. Serotonin 2A receptor modulation of D1 dopamine receptor-mediated grooming behaviour. Pharmacol. Biochem. Behav. 23, 385–389. Shalam, M., Shantakumar, S.M., Narasu, M.L., 2007. Neuropharmacological profile of trans-01, A polyherbal formulation in mice. Pharmacologyonline 1, 146–151. da Silva, A.L., Elisabetsky, E., 2001. Interference of propylene glycol with the hole board test. Braz. J. Med. Biol. Res. 34, 545–547. Sleigha, J., Harvey, M., Vossa, L., Denny, B., 2014. Ketamine—more mechanisms of action than just NMDA blockade. Trends Anaesth. Crit. Care 4 (2–3), 76–81. Sofowora, A., 2008. Medicinal Plants and Traditional Medicine in Africa, 3rd edition Spectrum Books Limited, Ibadan, pp. 181–207. Sravani, K.M.R., Kumar, C.K.A., Lakshmi, S.M., Rani, G.S., 2012. Psychopharmacological profiles of Pergularia daemia (Forsk.) Chiov. Asian J. Pharm. Clin. Res. 5 (2), 112–114. Sumathykutty, M.A., Rao, J.M., Padmakumari, K.P., Narayanan, C.S., 1999. Essential oil constituents of some piper species. Flavors Fragr. J. 14, 279–282. Swinyard, E.A., Woodhead, J.H., White, H.S., Franklin, M.R., 1989. Experimental selection, quantification and evaluation of anticonvulsants. In: Levy, R., Mattson, R., Meldrum, B.S., Penry, J.K., Dreifuss, F.E. (Eds.), Antiepileptic Drugs, 3rd ed. Raven press, New York, pp. 85–102. Takeda, H., Tsuji, M., Matsumiya, T., 1998. Changes in head-dipping behaviour in the hole-board test reflect the anxiogenic and/or anxiolytic state in mice. Eur. J. Pharmacol. 350 (1), 21–29. Tankam, J.M., Ito, M., 2013. Inhalation of the essential oil of Piper guineense from Cameroon shows sedative and anxiolytic-like effects in mice. Biol. Pharm. Bull. 36 (10), 1608–1614. Udoh, F.V., Lot, T.Y., Braide, V.B., 1999. Effect of extracts of seed and leaf of Piper guineense on skeletal muscle activity in rat and frog. Phytother. Res. 13, 106–110. Van Wimersma Greidanus, T.B., Maigret, C., Torn, M., Ronner, E., Van der Kracht, S., Van der Wee, N.J.A., Versteeg, D.H., 1989. Dopamine D1 and D2 receptor agonists and antagonists and neuropeptide-induced excessive grooming. Eur. J. Pharmacol. 173, 227–231. Vasse, M., Chagraoui, A., Protais, P., 1988. Climbing and stereotyped behaviours in mice require the stimulation of D1 dopamine receptors. Eur. J. Pharmacol. 148, 221–229. Vogel, H.G., 2002. Drug Discovery and Evaluation, 2nd ed. Springer, N.Y., pp. 390–500. Vongtau, H.O., Abbah, J., Chindo, B.A., Mosugu, O., Salawu, A.O., Kwanashie, H.O., Gamaniel, K.S., 2005. Central inhibitory effect of the methanol extract of Neorautanenia mitis root in rats and mice. Pharm. Biol. 43, 113–120. Walf, A.A., Frye, C.A., 2007. The use of the elevated plus maze as an assay of anxietyrelated behavior in rodents. Nat. Protoc. 2 (2), 322–328. Williamson, E.M., 2001. Synergy and other interactions in phytomedicines. Phytomedicine 8 (5), 401–409. Yun, J., 2014. Limonene inhibits methamphetamine-induced locomotor activity via regulation of 5-HT neuronal function and dopamine release. Phytomedicine 21, 883–887.