CNS Activity of Myristica fragrans Houtt .-An Experimental Study

Objective: To evaluatethe role of Myristica fragrans Houtt. for its analgesic, sedative and anxiogenic activity. Method: Charles Foster rats were administered orally the ethanolic extract (EEMF) and fractions of extract in ethyl acetate (EAMF), chloroform (CMF), and n-hexane (HMF). They were screened for sedative activity using Pentobarbitone sleep potentiation test, analgesic activity using Eddy’s hot plate and Tail flick test; and anxiogenic or anxiolytic activity using Open field test. Result: In pentobarbitone induced sleep potentiation test, the ethanolic extract (EEMF) and ethyl acetate fraction (EAMF) showed significant decrease in latency of sleep and highly significant increase in duration of sleep. In Open field test, EEMF, EAMF and HMF showed highly significant decrease in locomotion parameters as shown by decrease in rearing, preening and ambulation. In Eddy’s hot plate test and Tail flick test, highly significant increase in reaction time was seen in EEMF, EAMF, HMF groups. Conclusion: The observed results suggest that Myristica fragrans has analgesic, anxiogenic and sedative activity.


Introduction:
Since long time, aromatic spices have been added as ingredients to improve the taste and flavor of food.These aromatic spices are also commonly used in phytotherapy and mostly related to various activities of their constituents.Nutmeg (Myristica fragrans Houtt.) is the seed kernel of the fruit while, mace is the lacy covering (aril) on the kernel 1 .Nutmeg has been studied to having antihelminthic, hepatoprotective, anti-oxidant 2 , aphrodisiac, and insecticidal properties and also used for treatment of rheumatism, diarrhea, asthma, atherosclerosis 3 .Sleep is a physiological process that is needed for recuperation.Sleep disorder has a relatively high prevalence and is a growing public health problem.On an average, more than 27% of population suffers from sleeping disorders with problem in initiating or maintaining sleep.It is estimated,that by the mid 21st century, approximately, 3-10% of all people will be frequent users of sleep medications 4, 5 .Chronic sleep disorder leads to poor memorizing, emotional disturbances, slower reactions and changes in the immune response 6 .Benzodiazepines are the most commonly used agents for sleep disorders.They are responsible for unpleasant adverse effects which include drug dependence, tolerance, rebound insomnia, amnesia, psychomotor impairment and potentiating other central depressant drugs 7 .For these reasons, newer hypnotic agents with better safety profile are needed.Anxiety is a pathological state of aggravated fear associated with motor tension, sympathetic overactivity,vigilance syndrome and apprehension leading to impairment of memory, intelligence and psychological function 8 .The anxiety disorder consists of spectrum of panic disorder, generalized anxiety disorder, phobias, separation anxiety disorder and post traumatic stress disorder 9 .One-DOI: http://dx.doi.org/10.3329/bjms.v17i1.35289eighth of the population in the world suffers from anxiety disorder 10 .It is more common in the female population as compared to males 11 .Recent trends have shown that one in every four Indians is affected by anxiety disorders 12 .Pain is one of the most common nonspecific manifestations of many diseases.Although nonsteroidal anti-inflammatory drugs (NSAIDs) and opiates forms the basis of treatment for most of the painful conditions, but they are associated with several adverse reactions like renal damage, respiratory depression, gastrointestinal disturbances, and possible dependence 13, 14 .So there has been an increasing trend these days to find analgesics with lesser side effect profile from natural sources and medicinal plants.The present study evaluates the analgesic, sleepprolonging and anxiogenic effect of Myristica fragrans.

Material and Methods:
The study protocol was approved by the Institutional Animal Ethics Committee (IAEC), Jawaharlal Nehru Medical College, A.M.U.  17 thus, toxicity study was done for three fractions of Myristica fragrans seeds on healthy adult female rats (100-150g)as per Organization for Economic Cooperation and Development (OECD) Guidelines 425.Each fraction of Myristica fragrans (MF) was administered per oral to five animals at a dose of 2gm/kg.All animals survived at the end of 14 days of observation.Accordingly, the LD 50 of each fraction is greater than 2000 mg/kg.

Pentobarbitone induced hypnosis potentiation test:
The animals were given (per oral) a single dose of extract and fractions.After 1 hr, pentobarbitone (30 mg/Kg, IP) was injected to induce sleep.The ratswere considered asleep if they stayed immobile and lost their righting reflex when positioned on their back.The time interval between pentobarbitone injection and onset of sleep was recorded as sleep latency and the total sleeping duration (the time between the loss and the recovery of the righting reflex) were determined for each rat.The rat was considered as being awake if it could right itself (return to upright position).Various groups (n=6) were divided as follows (Table1).

EEMF-Ethanolic extract Myristica fragrans, EAMF-Ethyl acetate fraction of Myristica fragrans, CMF-Chloroform fraction of Myristica fragrans, HMF-n-Hexane fraction of Myristica fragrans. Open Field Behaviour test:
The open field apparatus was made of plywood and consisted of sixteen 18 x 18 cm squares with a central square (18 cm x 18 cm) in the middle.The rats in each group were first trained for few days 18 .One hour after test drug administration, the rats were placed individually in the open-field arena three times after a gap of 3 minutes each and observed for a period of 3 minutes using modified methodology by Rahman SZ et al. 16 .The parameters recorded in the open field were Ambulation, Rearing, Preening, Defecation count.The Test was done in 9 groups of 6 animals each as shown in Table 2.The method is based upon the reaction of rats to heat stimulus applied to their tail.It was performed by using the analgesiometer (Orchid Scientifics, India).Rats of either sex (150-200 gm) were placed in restraining holder so that the tail between the hole and tail tip or single point 3-5 cm from the tip of tail were directly kept over heated nichrome wire.
The time taken by the rats to withdraw the tail was recorded 20 .Heat intensity was adjusted such that the average withdrawn latency is 3-6 sec and a maximum cut-off time of 15 sec adopted to prevent undue tissue damage.Tail flick latency was tested at 30 min interval for 4 hours.The test was done in 10 groups (n=6) as shown in Table 4. of Ambulation, Preening and Rearing.n-Hexane fraction (400mg) also showed significant increase in defecation count (Table 6).Ethyl acetate fraction of Myristica fragrans showed significant decrease in mean score of Ambulation only (Fig 3).

Tail flick Test:
The rats in the control group responded within cutoff time of 6 seconds in all time periods (Table8).The standard pentazocine group showed significant (p<0.001)increase in reaction time (seconds) as5.21, 6.10, 9.10, 8.10, 7.54, 7.23, 5.50, at interval (minutes) of 30, 60, 90, 120,150, 180, 210 respectively.Ethanolic extract, HMF, EAMF showed highly significant increase in reaction time (seconds) at the interval (minutes) of 60, 90, 120,150 and 180 respectively.It was further noticed that mean response time was higher in the high dose group.The peak effect in both groups was seen at 90 mins.

Discussion:
Pentobarbitone enhances GABA receptor function or GABAergic neurotransmission in various brain regions 21 (Collingridge et al., 1984).The connection between GABA receptors activation and hypnotic response is still not known.Moreover, it is also not known in which region the GABA receptors that mediate the hypnotic response are present.One of the hypothesis associated with sleep generation is that, the stimulation of GAB Aergic ventrolateral preoptic nucleus (VLPO) neurons during sleep inhibits nuclei involved in arousal such as the histaminergic neurons of tuberomammillary nucleus (TMN) or serotonergic neurons of the dorsal raphe nucleus (DRN) 22 .The effect of pentobarbitone is reported to be enhanced by additional sedative medicine.Serotonin has long been implicated in the regulation of sleep-wake states and it plays an important role in the initiation and maintenance of sleep 23,24 .Although it is still not clear where and how serotonin exerts itseffect,as later it was found that serotonergic neurons play a role in inhibiting sleep 25 .
Su-Ying et al., suggested that the potentiating effect of diltiazem on pentobarbital-induced sleep may be related to 5-HT 1A and 5-HT 2A/2C receptors, and DRN Our result obtained from present study showed significant analgesic effect in both hot plate and tail flick tests suggesting enrichment in components (primarily non-lipid and/or aromatic compounds) that might activate a spinally-mediated analgesic pathway.Further lack of various constituents in chloroform fraction showed no statistically significant effects.Further mechanistic studies are needed to better understand the analgesic action of nutmeg.

Conclusion:
In our study, Myristica fragrans has significantly potentiated the pentobarbitone induced sleep, it has also significantly increased the latency of reaction in Hot plate and Tail flick test and finally in the Open field arena it decreased the locomotor activity significantly.Hence, in conclusion we can suggest that Myristica fragrans has a complex CNS activity and acts as a sedative has analgesic and anxiogenic activity.These activities can further be explored to find a new plant based therapy helpful in sleep disorder and in painful conditions.Its anxiogenic activity can be used to screen anxiolytic agents in experimental model.
, Aligarh (Ref.no.8662/ CAH dated 21.03.2016.).All animal experiments were carried out as per the rules and regulations framed by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Table 2 : Experimental design (Open field Behaviour test)
19MF-Ethanolic extract of Myristica fragrans, EAMF-Ethyl acetate fraction of Myristica fragrans, CMF-Chloroform fraction of Myristica fragrans, HMF-n-Hexane fraction of Myristica fragrans Analgesic activity by Eddy's Hot Plate method:This is one of the most common and reliable method used for screening of centrally acting analgesic agents.The present study was performed by using the Eddy's Hot Plate Analgesiometer (Orchid Scientifics, India).The hot plate is an electrically heated aluminum plate with a temperature ranging between 55˚ to 56˚C19.ascontrolwhereas Pentazocine 30 mg/kg i.p. was administered as standard drug.The cut-off time for response reaction was 30 seconds.The plate was wiped clean every time with saline if urination/ defecation is found.The test was done in 10 groups as shown in Table3.

Table 4 : Experimental design (Analgesic activity by Rat Tail flick test)
oStatistical analysis:All the values were expressed as Mean ± SEM.Duration of loss of righting reflex was significantly increased at both the doses of EAMF.The chloroform and n-hexane fractions of M.f were unable to produce any significant changes in the latency or duration of righting reflex.

Table 5 : Effect of Myristica fragrans on Pentobarbitone Induced Hypnosis test
The rats in the test group were treated with the extract and fractions of M.f. at dose of 200 and 400 mg/kg p.o., while the rats in the control group were given the same volume of Propylene glycol in the same manner.One hour later, all the animals were tested thrice after a gap of three minutes each for three minutes in the open field arena.As shown in Table6, in the control group mean score of ambulation, rearing, preening and defecation count were 49.50, 17.67, 4.67 and 1.67 respectively (Table6).Significant results were shown by the Ethanolic extract, Ethyl acetate fraction and n-Hexane fraction of Myristica fragrans.Ethanolic extract and n-hexane fraction of Myristica fragrans at dose of 400 mg/kg p.o. showed maximum and almost similar decrease in mean score

Table 8 : Effect of Myristica fragrans seed on reaction time in rats using Tail flick test.
36,37,38 (n= 6) sec.*P<0.05,**P<0.01,***P<0.001ascompared to normal control.maybeinvolved26.In our study, MF was found to potentiate pentobarbitalinduced sleep.These observations points to the sedative effect of MF.The sedative effect may be due to the presence of various compounds in Myristica fragrans.Earlier study suggested that Myristica fragrans might be increasing the serotonergic activity in brain27.In the open-field test, we noticed a decrease in ambulation, rearing and grooming behavior in the HMF, EAMF and EEMF groups.These findings suggest that M. fragrans possess anxiogenic activity and are consistent with previous study by Sonavane et al., who also noted decrease in locomotion and rearing by both Hexane extract of Myristica fragrans and xrimyristin, thus possessing anxiogenic activity, whereas the anxiolytic agents increased both rearing as well as locomotion32.Decreased locomotion is an indicator of diminished dopaminergic transmission, which may be due to the rise in 5-HT level caused by anxiogenic agents33.Leiter et al. also suggested that myristicin does not reduce anxiety by GABA receptor modulation but may promote anxiogenesis.They concluded that myristicin antagonize the anxiolytic effects of midazolam, increase anxiety, and affect motor movements34.Muchtaridi et al. reported that myristicin, 4-terpineole and safrole were associated with inhibition of locomotor activity in mice.They suggested that this inhibition by nutmeg seed essential oil is due to the direct pharmacological action of one or more of its constituents35.More investigations are needed to identify the anxiogenic principles of nutmeg and to know their mechanism of anxiogenesis as it can act as an experimental tool in the screening of anxiolytic agents.In our study EEMF, EAMF and HMF showed significant increase in reaction time in both hot plate and tail flick test, suggesting towards the analgesic activity of Myristica fragrans Human and animal data in previous studies, with pure nutmeg compounds also noted CNS depressant activity36,37,38.Earlier researcher, Joyce OO et al., (2012) concluded that analgesic effect of ethanolic extract of Myristica fragrans is both peripherally and centrally significant 39 .Similarly, Olajide et al., 1999 reported that oral administration of the Myristica fragrans possessed a potent analgesic effect against acetic acid-induced writhing in mice 40 .Lopes et al., 2009 reported that both responses of hot plate test (paw licking and jumping) integrate at supraspinal structures with the C and Aδ type I and II sensitive fibers participating in this model 41 .