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Neuropharmacological Activity of Teki Ladang (Cyperus Rotundus L.) Rhizome Fraction

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The content of flavonoids of Cyperus rotundus rhizomes is suspected to have activities on central nervous system involving GABAergik. This research was aimed to further investigate neuropharmacological properties of Cyperus rotundus rhizomes fraction by measuring hypnotic-siccative and anticonvulsant activities. The exploratory behavior test was performed using Hole Board method while motor coordination was assessed using Rotarod Test.

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The result showed that the 70% ethanol fraction of Cyperus rotundus rhizome in has significant (p <0,05) neuropharmacological activities. All three fractions tested (ethanol, ethyl acetate, and n-hexane), the ethanol fraction showed the best hypnotic-sedative and anticonvulsant activities. Ethanol Cyperus rotundus rhizome fraction could decrease the duration of HLE equal to sodium valproate and potentiated the phenobarbital-induced sleep by decreasing the onset and prolonging the duration of sleep. The highest dose of 70% ethanol fraction of Cyperus rotundus rhizome (356.7 mg/kg) showed the best anxiolytic activity comparable to diazepam (p> 0.05).

In conclusion, Teki ladang (Cyperus rotundus) rhizome appeared as potent neuropharmacological drug candidate with abilities comparable to synthetics drugs.

Drugs that work on the central nervous system (CNS) is the pharmacological agents that are most widely used ole public (Katzung and Trevor, 2015). These drugs have many therapeutic benefits like relieving pain, fever, pressing movement disorders, induce sleep or arousal, decrease appetite and relieve nausea. CNS drugs that work selectively can be used to overcome anxiety, depression, mania, or schizophrenia (Brunton et al., 2013). Cyperus rotundus has various benefits as medicine.

The research of Ahmad et al., (2014) shows that ethanol extract of Cyperus rotundus rhizome could inhibit inflammation, reduces gastric lesions, and cause sedation effects in mice. Other research also indicates the activity of this extract at a dose of 500 mg/kg rat as anticonvulsants (Biradar et al., 2010) This research was conducted on the fraction of 70% ethanol extracts of Cyperus rotundus Rhizome. The fraction used in this research was 70% ethanol, ethyl acetate, and n-hexane fraction. Phytochemical study on the rhizome of Cyperus rotundus showed a content of alkaloids, flavonoids, tannins, glycosides, saponins, starch, essential oil, and sesquiterpene (Yudistyawan, 2013). The dominant content of Cyperus rotundus Rhizome is quercetin (Krishna and Renu, 2013). This research aimed to test the effect of 70% ethanol, ethyl acetate and n-hexane Cyperus rotundus Rhizome fraction towards the central nervous system.

Materials Material used in the research were collected from Research Institute for Spices and Medicinal Plants (BALITTRO) Bogor and determined by Herbarium Bogoriense of Indonesian Institute of Sciences (LIPI). Healthy BALB/C male mice weighing 20-30 g and Sprague Dawley male rats weighing 200-250 g were used in this research. This study has been approved by the Committee of ethics of health research Faculty of Medicine University of Indonesia with the number approval: 699/UN2. F1/ETHICS/2017 and 726/UN2.F1/ETIK/2017. Methods Extraction and Fractionation Rhizome powder (2 kg) was soaked in ethanol 70% and left for 24 hours. This process was repeated 3 times with fresh solvent. The filtrate was evaporated using the Rotary Evaporator Vacuum. The extract then partitioned between ethanol, ethyl acetate, and n-hexane fraction. Maximal electroshock (MES) induced convulsion in rats The anticonvulsant property of the drug in this model was assessed by its ability to protect against MES induced convulsions. The rats were divided into 5 groups of 5 rats each group. Group 1 was given 1.54 mg/kg of valproic acid, group 2 received CMC-Na, group 3, 4, 5 were given 178.3 mg/kg 70% ethanol, ethyl acetate, and N-Hexane fraction of Cyperus rotundus rhizome. Maximal electroshock of 150mAmp and 50 hertz for 0.2 seconds was applied through ears electrodes to induce convulsion. The parameters observed were time for onset of HLE, duration of HLE and mortality of the animals over 24 hours duration. A preliminary screening test was done to include only those rats which had shown HLE in this experimental model. The experimental drugs were administered one hour before giving an electrical shock (Mudium and Kolasani, 2014; Biradar et al., 2010). The test will be considered positive if the animal exhibits tonic extensor seizure with rearward HLE more than 90o from the body and sustained for more than 3 s following 10 s after stimulation (Castel-Branco et al., 2009). Phenobarbital Induced Sleeping Test This method was done by observation of the onset of sleep and sleep duration on BALB/C mice (weight 25-30 grams, 2-3 months old).

The mice were divided into 5 groups: group 1 (normal control) was given CMC-Na, group 2 (negative control) were given Na-CMC and 41.1 mg/kg of phenobarbital, group 3,4,5 were given ethanol 70%, ethyl acetate, n-hexane Cyperus rotundus rhizome fraction at the same dose of 356.6 mg/kg.

All groups except normal controls were induced by phenobarbital (41.1 mg/kg i.m.) 45 minutes after oral administration of test substance. Sleeping time was considered to be the time interval between the disappearance and reappearance of the righting reflex. The prolongation of sleeping time was expressed as a percentage using the following ratio (Du et al., 2002; Chouksey, Upmanyu and Pawar, 2013): Prolongation of sleep time duration (%)= (Total time of treated animal)/(Total time of control animal) Г—100% Hole Board Test Hole Board test aimed to study the exploration behavior of the animal. Hole Board (Infrared Actimeter Orchid ScientificВ®) consisted of a box (28 Г— 28 Г— 20.5 cm) which had 16 equidistant holes of 3 cm in diameter in the floor and walls of clear Plexiglas. Infrared at the base of the hole detected the number of head dipping. At the beginning of the test, mice were allowed to freely explore the apparatus for 10 minutes. Soon after administering the test substance, the exploration activities were scored based on the number of head dipping during 60 minutes (Arenas et al., 2014; Doukkali et al., 2015; Ajao and Akindele, 2013).

Rotarod Test Effect on motor coordination is tested using Rotarod. Rotarod consisted of a base plant form and an iron rod of 3 cm diameter and 30 cm length, with a non-slippery surface. The animals were pre-selected in a training session 24 h before the test, based on their ability to remain on the bar (at 12 rpm) for 2 min. Mice were positioned on the bar (at 30 rpm) after given test substance. Previously, the mice were allowed to run on the Rotarod for 5 minutes so that the mice are used to it Time spent in the apparatus was observed for 30 minutes duration. Apparatus was cleaned thoroughly between trials with alcohol. All behavioral recordings were carried out with the observer blind to the treatment the mice had received (Doukkali et al., 2015). Data Analysis The data were analyzed statistically using one-way analysis of variance ANOVA, followed by the Tukey Kramer post hoc test for multiple comparisons. P < 0.05 was taken to be statistically significant. Results were presented as tables.

This research aimed to identify the activities of Cyperus rotundus Rhizome on the central nervous system. Methods used in this research were phenobarbital induced sleeping test, Maximal electroshock (MES) induced convulsion, hole board, and rotarod tests. Table I. Results of Anticonvulsants Activity of Cyperus rotundus 70% Fraction Group Onset of HLE (Second) Duration of HLE (Second) Valproic acid (1,54 mg/kg BW) 19,8 В± 2.58a 23,8 В± 2.28a CMC- Na 0 В± 0b 65,6 В± 5.54b CR 70% ethanol fraction (178,3 mg/kg BW) 15,2 В± 1.64ab 30,2 В± 3.03a CR Ethyl acetate fraction (178,3 mg/kg BW) 5,2 В± 1.30ab 35,0 В± 3.67ab CR n-hexane fraction (178,3 mg/kg BW) 3,4 В± 2.19ab 45,6 В± 4.44ab CR (Cyperus rotundus) a Significantly different from CMC-Na at the 0.05 level, b Significantly different from sodium valproate at the 0.05 level. Electrical seizures are induced by electroconvulsiometer, in which animals are subjected to non-lethal electric shock by means of electrodes applied to ear pinna or cornea (Mudium and Kolasani, 2014). The time for onset of tonic limb extension was prolonged by 70% ethanol, ethyl acetate and n-hexane Cyperus rotundus (CR) fraction significantly though incomparable to sodium valproate. There was also a significant reduction in the duration of HLE with both sodium valproate and CR fraction. However, only 70% ethanol CR fraction could decrease the duration of HLE equal to sodium valproate. There was no mortality among rats over 24 hours both for CR fractions and sodium valproate treated groups. MES-induced convulsion model was used for generalized tonic-clonic seizures. MES causes cellular damage by facilitating the entry of Ca2+ into the cells in large amounts, prolonging the duration of convulsions. also facilitate the entry of other positive ions like Na+, blockade of which, can prevent the MES-induced tonic extension (Venkatanarayana et al., 2013).

The Results of Hypnotic activity of Cyperus rotundus Rhizomes Fractions. Group the onset of sleep (minutes) Duration of sleep (minutes) Prolongation of sleep time duration (%) CMC- Na + phenobarbital 26 В± 0,89 200 В± 6,03 – CR 70% ethanol fraction (178,3 mg/kg BW) + phenobarbital 15 В± 3.52a 364 В± 8.22a 182 CR Ethyl acetate fraction (178,3 mg/kg BW) + phenobarbital 22 В± 1.89 226 В± 11,09a 113 CR n-hexane fraction (178,3 mg/kg BW) + phenobarbital 24 В± 2.52 214,8 В± 3,76a 107.4 CR (Cyperus rotundus Rhizome) a Significantly different from Na CMC at the 0.05 level All the fractions of Cyperus rotundus Rhizomes (CR) could prolonged duration of sleep incomparable to Na CMA (negative control). However, only 70% ethanol fraction of CR showed the significant faster onset of sleep (p<0.05). This fraction also had the highest percentage of prolonged sleep time, which increased by 182% at a dose of 178,3 mg/kg compared to negative control group (Table III). Phenobarbital exerts its pharmacological effect by binding to specific GABAA receptor subunits at CNS neuronal synapses facilitating GABA-mediated chloride ion channel opening duration, therefore, enhance membrane hyperpolarization (Katzung and Trevor, 2015). Phenobarbital-induced sleeping time was used to evaluate the possible sedative-hypnotic effects of Cyperus rotundus rhizomes fraction. The fraction that prolonged phenobarbital-induced sleeping time is considered as hypnotic agents. Ethanol fraction of CR potentiated the phenobarbital-induced sleep by decreasing the onset and prolonging the duration of sleep, which were considered to be the two aspects of a hypnotic effect.

Results of the Anxiolytic Activity Group The total number of Head Dip Time hangs on Rotarod without falling in the 30 minutes (seconds) Diazepam 0,82 mg/kg 111,6 В± 7,44b 359,20 В± 46,45b CR 70% ethanol fraction (89.16 mg/kg BW) 162.6 В± 6.31ab 670.93 В± 62.28ab CR 70% ethanol fraction (178,35 mg/kg BW) 147.0 В± 9.54ab 583.27 В± 54.93ab CR 70% ethanol fraction (356.7 mg/kg BW) 127,8 В± 6,10b 452,07 В± 47,10b NaCMC 197,8 В± 12,09a 825,62 В± 33,13a CR (Cyperus rotundus Rhizome) a different meaning with a positive control group (p < 0.05), b different meaning with the negative control group (p < 0.05) Hole Board test aimed to study the exploration behavior of the animal (Arenas et al., 2014). Placing the animal in a foreign environment could increase anxiety.

Therefore, anxiolytic activity could be determined by the decline of the behavior of exploration on testing animal. The parameters observed as exploratory behavior was the head dip (test animals entering his head in the hole). Anxiety in animals was characterized by an increase in the number of head dip (Brown and Nemes, 2008). This research showed that FECR in all doses could cause a calming effect and can reduce anxiety indicated by a significant decrease in the number of head dip (Table I). Study of ethanol extract of the rhizome teki (Cyperus rotundus) at doses of 500 mg/kg mice have been shown to have neuropharmacology activity, demonstrated by a decrease in the activity of the central nervous system. But the decline in exploratory activity at this dose is not yet comparable to diazepam (Ahmad et al., 2014). Rotarod Test aimed to evaluate peripheral neuromuscular blockade and motor coordination (Doukkali et al., 2015).

Administering FECR could cause sedation effect, therefore, lowering motor activity significantly compared to a negative control (Na CMC). The results showed that 356.7 mg/kg FECR had the best anxiolytic activity and comparable to diazepam (Table III). The results of this study indicated the fraction of 70% ethanol Cyperus rotundus rhizome at dose 356.7 mg/kg had higher potential as anxiolytic the extract at dose 500 mg/kg (Ahmad et al., 2014). Traditional medicine has been proven clinically in reducing anxiety and depression through the mechanism of inhibition of reuptake of monoamine such as noradrenaline, serotonin, and dopamine, also GABAergic effects (Saki, Bahmani, and Rafieian-Kopaei, 2014). The results of phytochemicals study showed that the fraction of 70% ethanol Cyperus rotundus Rhizome contained flavonoids, alkaloids, saponins and tannins (Table II). Flavonoids of Cyperus rotundus Rhizome have been examined consists of quercetins and myricetins (Krishna and Renu, 2013). Quercetin of other plants such as Tilia Americana has been investigated to have the mechanism of GABAergic inhibition. Mechanisms of flavonoids from each plant may be different, depending on the chemical structure and pharmacology activity, but anxiolytic and sedative activities typically involve a system of GABAergic and serotonergic (Aguirre-HernГЎndez et al., 2016). In addition, based on (Du et al., 2002) research, flavonoids activity as anticonvulsants involving GABA receptor agonist

The ethanol fraction of Cyperus rotundus rhizome showed the highest hypnotic-sedative and anticonvulsant activities, comparable to positive control. Ethanol fraction of Cyperus rotundus rhizome could decrease the duration of HLE equal to sodium valproate and potentiated the phenobarbital-induced sleep by decreasing the onset and prolonging the duration of sleep. The highest dose of 70% ethanol fraction of Cyperus rotundus rhizome (356.7 mg/kg) showed the best anxiolytic activity comparable to diazepam (p> 0.05).

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