Noni ( Morinda citrifolia L. ) had long been used to handle assorted conditions in folklore medical specialty. Soon, most surveies focused on antioxidant and anti-cancer activities, while it was claimed by the noni juice-consumers to bring forth felicity. Temper upsets e.g. anxiousness is a symptom reflected the upset of neurotransmitters, the most outstanding is the serotonergic system. Anxiety affects people throughout the universe, people have been seeking for intervention while the natural merchandises may relieve this unfavourable symptom. The present survey examined whether noni contained anxiolytic-like behaviour when tested with elevated plus-maze ( EPM ) , a standard trial for anxiousness and whether this consequence is related to alterations of monoamine senders in encephalon. Male Wistar rats were fed with either noni juice ( 1 ml/day, PO for 15 yearss ) or H2O, and the measuring of anxiousness was performed with EPM. The encephalon monoamines were so analysed with HPLC-electrochemical sensor. We found that noni treated-rats spent more clip in the opened-arm than control. Additionally, ratio of open- to closed-arm entry was higher in noni than control indicated the penchant to come in open- than protected closed-arm. The neurochemical analysis revealed important alterations of 5-HT and dopamine turnover in frontal cerebral mantle of noni group, and alterations of noradrenergic metamorphosis in substantia nigger of Valium rats. These findings indicated that the noni juice produced anxiolytic-like behaviours in rats by impacting neurochemical metamorphosiss in encephalon parts related to anxiousness.
Keywords: Anxiolytic, Elevated-plus labyrinth, Morinda citrifolia L. , Noni, 5-hydroxytryptamine, Dopastat, noradrenaline
Noni ( Morinda citrifolia L. ) is a little evergreen works that can be found from India through Southeast Asia to Eastern Polynesia. Its common names are noni, nonu, Indian mulberry, duppy prickly custard apple, cheese fruit, Ba Ji Tian, mergadu, and recognized as nhau ( yor ) in Southeast Asia. The noni has been used in assorted facets ; different parts of a works ( e.g. fruit, foliage, bark, root, flower and seed ) have long been employed in folklore medical specialty to handle wide scope of diseases including diabetes, high blood pressure, infections, colds and malignant neoplastic disease ( Wang et al. 2002 ) . There are more than 160 identified chemicals in noni, the major constituents are scopoletin, octanoic acid, terpene compounds, alkaloids, anthraquinones, b-sitosterol, provitamin A, vitamin A, vitamin C, K, flavone glycosides, linoleic acid, aminic acids, acubin, asperuloside, acubin, caproic acid, caprylic acid, ursolic acid, rutin, a putative proxeronine, glycosides, and a tri-saccharide fatty acid ester ( Liu et al. 2001 ; Sang et Al. 2001a, B ; Wang et Al. 2001, 2002 ) . Noni juice is an herbal drink produced from the riped fruit of noni. Recent surveies both in vivo and in vitro have shown that noni fruit infusion contained antioxidant, anti-cancer, anti-inflammatory and anti-angiogenic belongingss ( Liu et al. 2001 ; McKoy et Al. 2002 ; Wang et Al. 2002 ; Hornick et Al. 2003 ) . These groundss are non merely supported the cognition of noni as in folklore medical specialty, but besides supported some wellness benefits of the juice claimed by the consumer. Nowadays, noni juice is commercially market as a health-promoting drink and harmonizing to informations obtained in the USA for 2001, an mean figure of 46,603 people purchased a commercial noni juice per month ( Scientific commission on Food, 2002 ) . Interestingly, other than antioxidant or anticancer belongingss, one interesting claim from the consumer was experiencing of felicity and relaxing. While the cardinal consequence of noni was limited to the containing of ataractic consequence along with analgetic activity, and this consequence was from the root infusion ( Younos et al. 1990 ) . Unfortunately, the active substance was non resolved at that clip, and it is possible that noni juice may hold some similar good consequence on temper.
Anxiety is a signifier of temper upsets impacting people throughout the universe. It has been proposed that pull offing assorted neurotransmitters utilizing different medicines could relieve this upset. The use of either pharmacologically active substances or genetically modified animate beings had shown that anxiousness can be modified through monoaminergic activities including 5-hydroxytryptamine ( 5-HT ) , norepinephrine ( NE ) and Dopastat ( DA ) . The serotonergic system has been good accepted to play a cardinal modulatory function in cardinal nervous system processes that appeared to be dysregulated in psychiatric upsets including anxiousness. The uses of serotonin selective re-uptake inhibitor ( SSRI ) or assorted agonists or adversaries to 5-HT receptors had proven to bring on anxiolytic consequence both in animate being theoretical accounts and human topics ( for reviewed see Gingrich and Hen 2001 ) . Not merely the serotonergic system, but the noradrenergic system was besides suggested to be partly involved in the aggravation and fading of anxiousness. For illustration, the ?2-adrenergic receptor adversary was found to bring on anxiousness in rats tested with learned defensive burial and the modified force swim trial ( Tanaka et al. 2000 ) . Further, the dopaminergic system was besides take duty in modulating anxiousness, the Dopastat D3 receptor agonists was found to bring forth anxiolytic-like effects in behavioural trial theoretical accounts ( Rodgers et al 1996 ; Rogoz et Al. 2003 ) . Therefore, it is likely that anxiousness is non a merely consequence of one abnormalcy but so an integrating of different dysfunction/dysregulation of monoaminergic systems.
In this survey, the consequence of the noni juice on cut downing anxiousness was tested utilizing an elevated plus-maze, a standard theoretical account particular for anxiousness. This trial is based on innate responses to a potentially unsafe environment, the combination of tallness, brightness and unfastened infinite is assumed to bring on fright or anxiousness in the gnawers. The grade of anxiousness is assessed by mensurating the clip spent on the open- and closed-arms, and the figure of entries made into each arm ( Pellow et al. 1985 ; Cruz et Al. 1994 ; Rodgers and Dalvi 1997 ) . Furthermore, in order to correlate the anxiolytic consequence of noni on neurotransmitter transition ; the alterations in monoaminergic activities were besides measured with high public presentation liquid chromatography-electrochemical sensor in assorted encephalon parts related to anxiousness ( i.e. frontal cerebral mantle, amygdaloid nucleus, hippocampus, etc. ) .
Materials and Methods
Male Wistar rats weighing 200-250 g at the beginning of the experiments were obtained from National Laboratory Animal Center, Mahidol University ( NLAC-MU ) , Thailand. All animate beings were housed in shoebox coop under 12h light/dark rhythm ( visible radiations on at 0700 hour ) at room temperature ( 25+2oC ) . Standard rat Zhou and H2O were supplied ad libitum. After 7-day version period, rats were assigned indiscriminately into 2 groups ( 6 rats per group ) : control and noni. The rats were fed with 1 milliliters of H2O or noni juices for 15 yearss. All processs herein were done under the blessing of Animal Used Committee, Faculty of Veterinary Science, Chulalongkorn University.
The juice of Morinda citrifolia L. ( noni ) was purchased commercially ( Siam Noni® , Suprederm International, Thailand ) . It composed of 99 % noni juice.
The behavioural experiments were performed utilizing an EPM, the standard trial to measure anxiety-like behaviours in rats ( Pellow et al. 1985 ; Cruz et Al. 1994 ; Rodgers and Dalvi1997 ) . The EPM was made of wood, elevated 50 centimeters above the floor, and consisted of four weaponries of equal dimension ( 10 x 50 centimeter ) in which two weaponries enclosed by high wall ( 30 centimeter ) and two weaponries opened. The last twenty-four hours of experiment, rats were placed in the centre of the EPM confronting a corner of the platform 15 min after having H2O or noni juice. The behavioural trial was conducted during the light stage of the rhythm, between 0900-1100 hour in a low natural visible radiation room. Each rat was allowed to research freely on the EPM for 5 min and recorded on VCR for subsequently analysis. The parametric quantities measured were clip spent in open-arm ( OT ) and closed-arm ( CT ) , including figure of entries into each arm ( open-arm entry, OE ; closed-arm entry, CE ) . Number of times of raising and training were besides recorded. An arm entry was defined as the arrangement of at least both forefeet into one arm. The labyrinth was carefully wiped with a moisture towel after each animate being. Harmonizing to Pellow et Al. ( 1985 ) , anxiousness is reflected in the innate antipathy to highs and unfastened infinites, the per centum of entries and clip spent in the unfastened weaponries provide steps of fear-induced suppression of explorative activity. This ratio is increased by minor tranquilizers and reduced by anxiogenic compounds.
After the behavioural trial, rats were euthanized with isoflurane overdosage, encephalon was quickly removed, separately wrapped, freezed in liquid N and stored at -80oC for farther analysis. Assorted encephalon parts known to lend to anxiety were obtained from the frozen brains harmonizing to the direction of Heffner et Al. ( 1980 ) . Three encephalons from each group were indiscriminately selected for neurochemical analysis. These stray encephalons were sonicated in the iced-cold 0.1 M perchloric acid incorporating 3,4-dihydroxy-benzyl-amine hydrobromide ( DHBA ; Sigma, St. Louis, MO, USA ) , as an internal criterion. The samples were so centrifuged at 10,000x g for 30 min at 4 & A ; deg ; C. The supernatant was collected for farther analysis of monoamine neurotransmitters utilizing high-performance liquid chromatography with electrochemical sensor ( HPLC-EC ) . The HPLC-EC system composed of a glassy carbon-working electrode, an amperometric control ( sensitiveness 20 sodium and oxidative potency +0.70 V ; Bioanalytical systems, West Lafayette, IN, USA ) and a 15-cm Luna & A ; Ograve ; column ( Phenomenex, Torrance, CA, USA ) . The nomadic stage solution was 1 mM Heptane sulfonate, 100 millimeter Sodium dihydrogen phosphate, 1 millimeter Na2·EDTA and 5 % Methanol, adjusted to pH 4.1 with saturated citric acid. The supernatant ( 40 milliliter ) from the encephalon was injected into the HPLC-EC system to observe the monoamines including Dopastat ( DA ) and its metabolites 3,4-dihydroxyphenylacetic acid ( DOPAC ) and homovanillic acid ( HVA ) ; noradrenaline ( NE ) and its metabolite 3-methoxy-4-hydroxyphenylglycol ( MHPG ) ; and serotonin ( 5-HT ) and its metabolite 5-hydroxyindole acetic acid ( 5-HIAA ) . The HPLC extremums were integrated and analyzed with Delta 5.0 package ( Digital Solutions, Margate, QLD, Australia ) . The keeping clip and the concentration of neurotransmitters and its metabolites were calculated by shooting different concentrations of standard solutions into HPLC system. The concentrations of neurotransmitters and its metabolites were so converted and expressed as pmol/mg protein of encephalon tissue. Protein concentration of each encephalon parts was determined by the method of Lowry et Al. ( 1951 ) .
All informations were presented as agencies and standard mistakes of agencies ( S.E.M. ) . For comparings between two groups, odd Student ‘s t-tests were used. Differences were considered statistically important at P & A ; lt ; 0.05.
The anxiolytic consequence of noni juice
After having noni juice for 15 yearss, the noni treated-rat spent a significantly greater per centum of clip on the unfastened weaponries of the plus-maze ( Fig. 1A ; Table 1 ) than control rats. In add-on, the clip per entry into the unfastened weaponries ( Time/Entry ) was besides higher in noni rats ( Fig. 1B ; Table 1 ) than control rats. These informations indicated an anxiolytic belongings of the noni juice in the EPM. There were no important differences in the figure of entries into the unfastened weaponries ( Table 1 ) . The locomotor activity of animate beings, represented by the figure of closed arm and entire arm entries ( Table 1 ) , was non different between these two groups. Therefore, a lessening in anxiousness, as displayed by an addition in the clip spent on the unfastened weaponries, is independent of any alterations in locomotor activity. Further, the figure of times rise uping and preparing activity was recorded from the EPM was besides non different between groups ( Table 1 ) .
Brain neurochemistry and noni juice
The effects of noni juice on monoamines and their metabolite degrees in encephalon countries that contribute to anxiousness are shown in Tables 2. NE content in amygdaloid nucleus and hippocampus were significantly lower in noni group comparison to command ( Table 3 ) . The NE metabolite, MHPG in caudate putamen and substantia nigger of noni group were significantly higher than the control group. The NE turnover in the caudate putamen of noni group was higher than control. Furthermore, there was a tendency of higher NE turnover in the substantia nigger of noni group compared to command ( p = 0.0548 ) . In the hippocampus, the degree of DOPAC, the DA metabolite was lower in the noni group compared to command with a tendency of lower DOPAC ( 0.0652 ) and HVA ( 0.0561 ) in the substantia nigger of noni group. In add-on, the degrees of 5-HT and the 5-HT turnover in the amygdaloid nucleus were trended to be lower and higher in the noni compared to command ( p = 0.0976 and 0.0811, severally ) .
The neurochemical analyses showed that the monoaminergic degrees were, to different extents, affected by noni juice. In the frontal cerebral mantle, the noni group had important lowered 5-HT degree ( P = 0.0232 ; Table 2 ) , but had no effects on the 5-HIAA, DA, DOPAC, HVA, NE or MHPG. However, the alterations in metamorphosis of dopaminergic system were pronounced in noni group, the ratio of DOPAC/DA and HVA/DA were increased significantly ( DOPAC/DA, P = 0.0345 ; 0.42+0.08, 0.76+0.06 and 0.41+0.10 ; HVA/DA, P = 0.0212 ; 0.51+0.09, 1.83+0.43 and 0.57+0.18 ; control, noni, and Valium group, severally ) . In the amygdaloid nucleus, the degree of NE in the noni group was lower than the control but non important ( P = 0.0540 ) . The degrees of NE metabolite, MHPG was increased in the substantia nigger of the diazepam treated-group ( P = 0.0255, Table 2 ) and the ratio of MHPG/NE was besides increased in this group ( P = 0.0338, 56.68+4.69, 32.87+15.47 and 9.24+2.10 ; Valium, noni and control group, severally ) . Further, the alterations of NE, DA or 5-HT were besides affected by noni throughout the encephalon but non important ( Table 2, P & A ; gt ; 0.05 ) .
The present survey indicated that noni juice when Federal for 15 yearss contained an anxiolytic activity in rats when tested with elevated plus maze ( EPM ) . The EPM is a valid behavioural trial for the selective anxiolytic and anxiogenic agent in the rat. The correlativity of behaviour, physiologically and pharmacologically have been stated ( Pellow et al. 1985 ) . Since the unfastened elevated country could arouse an innate fright, rats normally made fewer entries into the unfastened arm than into the closed arm, and spent less clip in unfastened arm. The agents that can increase the per centum of clip spent on the open-arm and the figure of entries into the open-arm is hence implied as an anxiolytic agent. Diazepam, a clinically effectual anxiolytic had been shown antecedently to increase both clip and figure of entry into open-arm ( Pellow et al. 1985 ; Rex et Al. 2002 ) . In this survey, the rats treated with individual unwritten dosage of diazepam spent more clip in the unfastened arm when compared to command, and this determination was in understanding with others. Similarly, we found that noni juice when given orally for 15 yearss could bring forth an consequence resembling to diazepam in that rat fed with noni juices spent more clip in the aversive opened country, while control rats avoid it and stayed in a protected closed-arm. Further, noni group prefer to come in the unfastened than the closed arm as shown by the ratio of unfastened to closed arm entry that higher than the control and Valium group. Because the motor activity of the rats, indicated by the entire figure of entries into both weaponries and the raising behaviour were non different between interventions, we can reason that the noni juice reduced anxiety-related behaviour in rats in the EPM.
It is known that cardinal neurotransmitters play an of import function in the control of anxiousness with the dysregulation of serotonergic system being the major concern, while noradrenergic and/or dopaminergic systems were besides believed to be partly involved. It is of involvement to see whether noni induced anxiolytic-like behaviours by modulated these neural systems. However, it is improbable that merely one neurotransmitter or merely one part is involved in the aggravation of anxiousness ; instead several neurotransmitters including noradrenaline, Dopastat and 5-hydroxytryptamine, and several encephalon parts are likely involved. In current survey, degrees of 5-HT in frontal cerebral mantle and NE in amygdaloid nucleus of noni group were lowered than control rat. Harmonizing to many surveies about the 5-HT hypothesis, anxiousness is normally associated with increased endogenous 5-HT, and anxiolytic tend to diminish endogenous 5-HT ( Briley et al. 1990 ; Barnes et Al. 1992 ; Handley 1995 ) . Therefore, the reduced 5-HT degrees in frontal cerebral mantle of noni group can explicate its anxiolytic actions. Furthermore, Tanaka et Al ( 2000 ) has revealed that NE release along with the increased of its metabolite, MHPG in amygdaloid nucleus and other encephalon parts was closely related with anxiogenic consequence produced by ?2-antagonist, yohimbine. Similarly in our survey, the reduced degrees of NE in amygdaloid nucleus were observed in noni group. It might be another tract by which noni Acts of the Apostless to diminish anxiety-like behaviours. In term of DA activity, antecedently Ge et Al. ( 1997 ) has shown that aversive state of affairs increased the degree of DA, DOPAC and HVA in encephalon parts related to anxiousness ( i.e. frontal cerebral mantle, amygdaloid nucleus, etc ) . However, contrary to Ge et Al. ( 1997 ) , we found the increased in DA turnover in frontal cerebral mantle but this rat has less anxiousness compared to command. This may explicate in portion that although the DA activity was likely to be increased but the DA degree along with its metabolites were lowered in this group although non different from control ( Table 2 ) . Interestingly, the NE and NE turnover was higher in substantia nigger of the Valium treated rats compared to others, this part was normally associated with locomotor activity and this may associate to the higher activity ( entire entry ) observed in EPM. Interestingly, although noni and Valium, both produced anxiolytic-like behaviour in the EPM, the neurochemical analysis of the Valium treated group was someway differed from those of noni treated group, proposing different mechanism in anxiolysis.
Conclusively, noni juice, a natural merchandise of Morinda citrifolia L. contained anxiolytic consequence in rat when tested with elevated plus-maze and this consequence is in portion mediated through the alterations of monoaminergic neurotransmitter degrees. Further surveies are required to decide the in deepness molecular mechanism of noni mediated anxiolytic consequence.
This work was supported in portion by The Grant for Development of New Faculty staffs and The Veterinary Research Fund of the Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.