Noni (Morinda citrifolia L.) had long been used to treat various conditions in folklore medicine. Presently, most studies focused on antioxidant and anti-cancer activities, while it was claimed by the noni juice-consumers to produce happiness. Mood disorders e.g. anxiety is a symptom reflected the disorder of neurotransmitters, the most prominent is the serotonergic system. Anxiety affects people throughout the world, people have been seeking for treatment while the natural products may alleviate this unfavorable symptom. The present study examined whether noni contained anxiolytic-like behavior when tested with elevated plus-maze (EPM), a standard test for anxiety and whether this effect is related to changes of monoamine transmitters in brain. Male Wistar rats were fed with either noni juice (1 ml/day, PO for 15 days) or water, and the measurement of anxiety was performed with EPM. The brain monoamines were then analysed with HPLC-electrochemical detector. We found that noni treated-rats spent more time in the opened-arm than control. Additionally, ratio of open- to closed-arm entry was higher in noni than control indicated the preference to enter open- than protected closed-arm. The neurochemical analysis revealed significant changes of 5-HT and dopamine turnover in frontal cortex of noni group, and changes of noradrenergic metabolism in substantia nigra of diazepam rats. These findings indicated that the noni juice produced anxiolytic-like behaviors in rats by affecting neurochemical metabolisms in brain regions related to anxiety.
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Keywords: Anxiolytic, Elevated-plus maze, Morinda citrifolia L., Noni, serotonin, dopamine, norepinephrine
Noni (Morinda citrifolia L.) is a small evergreen plant that can be found from India through Southeast Asia to Eastern Polynesia. Its common names are noni, nonu, Indian mulberry, duppy soursop, cheese fruit, Ba Ji Tian, mergadu, and recognized as nhau (yor) in Southeast Asia. The noni has been used in various aspects; different parts of a plant (e.g. fruit, leaf, bark, root, flower and seed) have long been employed in folklore medicine to treat broad range of diseases including diabetes, hypertension, infections, colds and cancer (Wang et al. 2002). There are more than 160 identified chemicals in noni, the major components are scopoletin, octanoic acid, terpene compounds, alkaloids, anthraquinones, b-sitosterol, carotene, vitamin A, vitamin C, potassium, flavone glycosides, linoleic acid, amino 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 studies both in vivo and in vitro have shown that noni fruit extract contained antioxidant, anti-cancer, anti-inflammatory and anti-angiogenic properties (Liu et al. 2001; McKoy et al. 2002; Wang et al. 2002; Hornick et al. 2003). These evidences are not only supported the knowledge of noni as in folklore medicine, but also supported some health benefits of the juice claimed by the consumer. Nowadays, noni juice is commercially market as a health-promoting beverage and according to data obtained in the USA for 2001, an average number of 46,603 people purchased a commercial noni juice per month (Scientific committee on Food, 2002). Interestingly, other than antioxidant or anticancer properties, one interesting claim from the consumer was feeling of happiness and relaxing. While the central effect of noni was limited to the containing of sedative effect along with analgesic activity, and this effect was from the root extract (Younos et al. 1990). Unfortunately, the active substance was not resolved at that time, and it is possible that noni juice may have some similar beneficial effect on mood.
Anxiety is a form of mood disorders affecting people throughout the world. It has been proposed that managing various neurotransmitters using different medications could alleviate this disorder. The utilization of either pharmacologically active substances or genetically modified animals had shown that anxiety can be modified through monoaminergic activities including serotonin (5-HT), norepinephrine (NE) and dopamine (DA). The serotonergic system has been well accepted to play a key modulatory role in central nervous system processes that appeared to be dysregulated in psychiatric disorders including anxiety. The usages of serotonin selective reuptake inhibitor (SSRI) or various agonists or antagonists to 5-HT receptors had proven to induce anxiolytic effect both in animal models and human subjects (for reviewed see Gingrich and Hen 2001). Not only the serotonergic system, but the noradrenergic system was also suggested to be partially involved in the provocation and attenuation of anxiety. For example, the α2-adrenergic receptor antagonist was found to induce anxiety in rats tested with conditioned defensive burying and the modified force swim test (Tanaka et al. 2000). Further, the dopaminergic system was also take responsibility in regulating anxiety, the dopamine D3 receptor agonists was found to produce anxiolytic-like effects in behavioral test models (Rodgers et al 1996; Rogoz et al. 2003). Therefore, it is likely that anxiety is not a simply result of one abnormality but indeed an integration of different dysfunction/dysregulation of monoaminergic systems.
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In this study, the effect of the noni juice on reducing anxiety was tested using an elevated plus-maze, a standard model specific for anxiety. This test is based on unconditioned responses to a potentially dangerous environment, the combination of height, luminosity and open space is assumed to induce fear or anxiety in the rodents. The degree of anxiety is assessed by measuring the time spent on the open- and closed-arms, and the number 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 effect of noni on neurotransmitter modulation; the changes in monoaminergic activities were also measured with high performance liquid chromatography-electrochemical detector in various brain regions related to anxiety (i.e. frontal cortex, amygdala, 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 animals were housed in shoebox cage under 12h light/dark cycle (lights on at 0700 hr) at room temperature (25+2oC). Standard rat chow and water were supplied ad libitum. After 7-day adaptation period, rats were assigned randomly into 2 groups (6 rats per group): control and noni. The rats were fed with 1 ml of water or noni juices for 15 days. All procedures herein were done under the approval 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 behavioral experiments were performed using an EPM, the standard test to assess anxiety-like behaviors in rats (Pellow et al. 1985; Cruz et al. 1994; Rodgers and Dalvi1997). The EPM was made of wood, elevated 50 cm above the floor, and consisted of four arms of equal dimension (10 x 50 cm) in which two arms enclosed by high wall (30 cm) and two arms opened. The last day of experiment, rats were placed in the center of the EPM facing a corner of the platform 15 min after receiving water or noni juice. The behavioral test was conducted during the light phase of the cycle, between 0900-1100 hr in a low natural light room. Each rat was allowed to explore freely on the EPM for 5 min and recorded on VCR for later analysis. The parameters measured were time spent in open-arm (OT) and closed-arm (CT), including number of entries into each arm (open-arm entry, OE; closed-arm entry, CE). Number of times of rearing and grooming were also recorded. An arm entry was defined as the placement of at least both forefeet into one arm. The maze was carefully wiped with a wet towel after each animal. According to Pellow et al. (1985), anxiety is reflected in the unconditioned aversion to heights and open spaces, the percentage of entries and time spent in the open arms provide measures of fear-induced inhibition of exploratory activity. This ratio is increased by anxiolytics and reduced by anxiogenic compounds.
After the behavioral test, rats were euthanized with isoflurane overdosage, brain was rapidly removed, individually wrapped, freezed in liquid nitrogen and stored at -80oC for further analysis. Various brain regions known to contribute to anxiety were obtained from the frozen brains according to the instruction of Heffner et al. (1980). Three brains from each group were randomly selected for neurochemical analysis. These isolated brains were sonicated in the iced-cold 0.1 M perchloric acid containing 3,4-dihydroxy-benzyl-amine hydrobromide (DHBA; Sigma, St. Louis, MO, USA), as an internal standard. The samples were then centrifuged at 10,000x g for 30 min at 4°C. The supernatant was collected for further analysis of monoamine neurotransmitters using high-performance liquid chromatography with electrochemical detector (HPLC-EC). The HPLC-EC system composed of a glassy carbon-working electrode, an amperometric control (sensitivity 20 nA and oxidative potential +0.70 V; Bioanalytical systems, West Lafayette, IN, USA) and a 15-cm LunaÒ column (Phenomenex, Torrance, CA, USA). The mobile phase solution was 1 mM Heptane sulfonate, 100 mM Sodium dihydrogen phosphate, 1 mM Na2·EDTA and 5% Methanol, adjusted to pH 4.1 with saturated citric acid. The supernatant (40 ml) from the brain was injected into the HPLC-EC system to detect the monoamines including dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA); norepinephrine (NE) and its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG); and serotonin (5-HT) and its metabolite 5-hydroxyindole acetic acid (5-HIAA). The HPLC peaks were integrated and analyzed with Delta 5.0 software (Digital Solutions, Margate, QLD, Australia). The retention time and the concentration of neurotransmitters and its metabolites were calculated by injecting different concentrations of standard solutions into HPLC system. The concentrations of neurotransmitters and its metabolites were then converted and expressed as pmol/mg protein of brain tissue. Protein concentration of each brain regions was determined by the method of Lowry et al. (1951).
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All data were presented as means and standard errors of means (S.E.M.). For comparisons between two groups, unpaired Student's t-tests were used. Differences were considered statistically significant at p < 0.05.
The anxiolytic effect of noni juice
After receiving noni juice for 15 days, the noni treated-rat spent a significantly greater percentage of time on the open arms of the plus-maze (Fig. 1A; Table 1) than control rats. In addition, the time per entry into the open arms (Time/Entry) was also higher in noni rats (Fig. 1B; Table 1) than control rats. These data indicated an anxiolytic property of the noni juice in the EPM. There were no significant differences in the number of entries into the open arms (Table 1). The locomotor activity of animals, represented by the number of closed arm and total arm entries (Table 1), was not different between these two groups. Therefore, a decrease in anxiety, as displayed by an increase in the time spent on the open arms, is independent of any changes in locomotor activity. Further, the number of times rearing and grooming activity was recorded from the EPM was also not different between groups (Table 1).
Brain neurochemistry and noni juice
The effects of noni juice on monoamines and their metabolite levels in brain areas that contribute to anxiety are shown in Tables 2. NE content in amygdala and hippocampus were significantly lower in noni group compare to control (Table 3). The NE metabolite, MHPG in caudate putamen and substantia nigra of noni group were significantly higher than the control group. The NE turnover in the caudate putamen of noni group was higher than control. Moreover, there was a trend of higher NE turnover in the substantia nigra of noni group compared to control (p = 0.0548). In the hippocampus, the level of DOPAC, the DA metabolite was lower in the noni group compared to control with a trend of lower DOPAC (0.0652) and HVA (0.0561) in the substantia nigra of noni group. In addition, the levels of 5-HT and the 5-HT turnover in the amygdala were trended to be lower and higher in the noni compared to control (p = 0.0976 and 0.0811, respectively).
The neurochemical analyses showed that the monoaminergic levels were, to different extents, affected by noni juice. In the frontal cortex, the noni group had significant lowered 5-HT level (P = 0.0232; Table 2), but had no effects on the 5-HIAA, DA, DOPAC, HVA, NE or MHPG. However, the changes in metabolism 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 diazepam group, respectively). In the amygdala, the level of NE in the noni group was lower than the control but not significant (P = 0.0540). The levels of NE metabolite, MHPG was increased in the substantia nigra of the diazepam treated-group (P = 0.0255, Table 2) and the ratio of MHPG/NE was also increased in this group (P = 0.0338, 56.68+4.69, 32.87+15.47 and 9.24+2.10; diazepam, noni and control group, respectively). Further, the changes of NE, DA or 5-HT were also affected by noni throughout the brain but not significant (Table 2, P>0.05).
The present study indicated that noni juice when fed for 15 days contained an anxiolytic activity in rats when tested with elevated plus maze (EPM). The EPM is a valid behavioral test for the selective anxiolytic and anxiogenic agent in the rat. The correlation of behavior, physiologically and pharmacologically have been stated (Pellow et al. 1985). Since the open elevated area could evoke an unconditioned fear, rats usually made fewer entries into the open arm than into the closed arm, and spent less time in open arm. The agents that can increase the percentage of time spent on the open-arm and the number of entries into the open-arm is therefore implied as an anxiolytic agent. Diazepam, a clinically effective anxiolytic had been shown previously to increase both time and number of entry into open-arm (Pellow et al. 1985; Rex et al. 2002). In this study, the rats treated with single oral dose of diazepam spent more time in the open arm when compared to control, and this finding was in agreement with others. Similarly, we found that noni juice when given orally for 15 days could produce an effect resembling to diazepam in that rat fed with noni juices spent more time in the aversive opened area, while control rats avoid it and stayed in a protected closed-arm. Further, noni group prefer to enter the open than the closed arm as shown by the ratio of open to closed arm entry that higher than the control and diazepam group. Because the motor activity of the rats, indicated by the total number of entries into both arms and the rearing behavior were not different between treatments, we can conclude that the noni juice reduced anxiety-related behavior in rats in the EPM.
It is known that central neurotransmitters play an important role in the control of anxiety with the dysregulation of serotonergic system being the major concern, while noradrenergic and/or dopaminergic systems were also believed to be partially involved. It is of interest to see whether noni induced anxiolytic-like behaviors by modulated these neuronal systems. However, it is unlikely that only one neurotransmitter or only one region is involved in the provocation of anxiety; rather several neurotransmitters including norepinephrine, dopamine and serotonin, and several brain regions are probably involved. In current study, levels of 5-HT in frontal cortex and NE in amygdala of noni group were lowered than control rat. According to many studies about the 5-HT hypothesis, anxiety is usually associated with increased endogenous 5-HT, and anxiolytic tend to decrease endogenous 5-HT (Briley et al. 1990; Barnes et al. 1992; Handley 1995). Thus, the decreased 5-HT levels in frontal cortex of noni group can explain its anxiolytic actions. Furthermore, Tanaka et al (2000) has revealed that NE release along with the increased of its metabolite, MHPG in amygdala and other brain regions was closely related with anxiogenic effect produced by α2-antagonist, yohimbine. Similarly in our study, the decreased levels of NE in amygdala were observed in noni group. It might be another pathway by which noni acts to decrease anxiety-like behaviors. In term of DA activity, previously Ge et al. (1997) has shown that aversive situation increased the level of DA, DOPAC and HVA in brain regions related to anxiety (i.e. frontal cortex, amygdala, etc). However, contrary to Ge et al. (1997), we found the increased in DA turnover in frontal cortex but this rat has less anxiety compared to control. This may explain in part that although the DA activity was likely to be increased but the DA level along with its metabolites were lowered in this group although not different from control (Table 2). Interestingly, the NE and NE turnover was higher in substantia nigra of the diazepam treated rats compared to others, this region was usually associated with locomotor activity and this may related to the higher activity (total entry) observed in EPM. Interestingly, although noni and diazepam, both produced anxiolytic-like behavior in the EPM, the neurochemical analysis of the diazepam treated group was somehow differed from those of noni treated group, suggesting different mechanism in anxiolysis.
Conclusively, noni juice, a natural product of Morinda citrifolia L. contained anxiolytic effect in rat when tested with elevated plus-maze and this effect is in part mediated through the changes of monoaminergic neurotransmitter levels. Further studies are required to resolve the in depth molecular mechanism of noni mediated anxiolytic effect.
This work was supported in part by The Grant for Development of New Faculty staffs and The Veterinary Research Fund of the Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.