|Year : 2010 | Volume
| Issue : 2 | Page : 82-86
The antiepileptic effect of Centella asiatica on the activities of Na + /K + , Mg 2+ and Ca 2+ -ATPases in rat brain during pentylenetetrazol-induced epilepsy
G Visweswari1, K Siva Prasad1, V Lokanatha2, W Rajendra1
1 Department of Zoology, Division of Molecular Biology, Sri Venkateswara University, Tirupati, India
2 Department of Biotechnology, Dravidian University, Kuppam, Andhra Pradesh, India
|Date of Submission||29-Aug-2009|
|Date of Decision||28-Jan-2010|
|Date of Acceptance||14-Apr-2010|
|Date of Web Publication||23-Jun-2010|
Department of Zoology, Division of Molecular Biology, Sri Venkateswara University, Tirupati
Source of Support: University Grants Commission, Conflict of Interest: None
Background: To study the anticonvulsant effect of different extracts of Centella asiatica (CA) in male albino rats with reference to Na + /K + , Mg 2+ and Ca 2+ -ATPase activities.
Materials and Methods: Male Wistar rats (150±25 g b.w.) were divided into seven groups of six each i.e. (a) control rats treated with saline, (b) pentylenetetrazol (PTZ)-induced epileptic group (60 mg/kg, i.p.), (c) epileptic group pretreated with n-hexane extract (n-HE), (d) epileptic group pretreated with chloroform extract (CE), (e) epileptic group pretreated with ethyl acetate extract (EAE), (f) epileptic group pretreated with n-butanol extract (n-BE), and (g) epileptic group pretreated with aqueous extract (AE).
Results: The activities of three ATPases were decreased in different regions of brain during PTZ-induced epilepsy and were increased in epileptic rats pretreated with different extracts of CA except AE.
Conclusion: The extracts of C. asiatica, except AE, possess anticonvulsant and neuroprotective activity and thus can be used for effective management in treatment of epileptic seizures.
Keywords: Anticonvulsant effect, ATPases, Centella asiatica, epilepsy, pentylenetetrazol
|How to cite this article:|
Visweswari G, Siva Prasad K, Lokanatha V, Rajendra W. The antiepileptic effect of Centella asiatica on the activities of Na + /K + , Mg 2+ and Ca 2+ -ATPases in rat brain during pentylenetetrazol-induced epilepsy. Indian J Pharmacol 2010;42:82-6
|How to cite this URL:|
Visweswari G, Siva Prasad K, Lokanatha V, Rajendra W. The antiepileptic effect of Centella asiatica on the activities of Na + /K + , Mg 2+ and Ca 2+ -ATPases in rat brain during pentylenetetrazol-induced epilepsy. Indian J Pharmacol [serial online] 2010 [cited 2020 May 31];42:82-6. Available from: http://www.ijp-online.com/text.asp?2010/42/2/82/64504
| » Introduction|| |
Epilepsy, a common chronic nervous system disorder with repeated seizures affects 1-2% population worldwide.  There are likely to be multiple underlying cellular and molecular mechanisms responsible for various epileptiform phenomena, such as (a) imbalance between excitatory and inhibitory neurotransmission, (b) alterations in neurotransmitter expression and function, (c) channelopathies, and (d) aberrant neuronal synchronization. Purine nucleotides, in addition to serving as precursors of nucleic acids, play a critical role in diverse physiological functions such as energy metabolism, protein synthesis, regulation of enzyme activity, and signal transduction. 
ATPases play an important role in the maintenance of ionic gradient by coupling ATP hydrolysis with energy processes.  ATP itself, as a neurotransmitter and neuromodulator, may influence the release of other neurotransmitters by acting through its own receptors or by altering the neurotransmitter receptors. , Na + , K + ATPase is a membrane-bound enzyme and inactivation of this enzyme is an important factor in epileptization of neurons.  Similarly it is demonstrated that inhibition of microsomal Mg 2+ /Ca 2+-ATPase may be associated with long-term plasticity changes associated with epileptogenesis.  Idiopathic epilepsies involve the Na + , K + , or Ca 2+ channels and the defects in the activities of Na + /K + or Ca 2+ -ATPases are responsible for maintaining ionic balance in the cell.  Several studies have reported significant inhibition of ATPase activity in the brain during different types of epileptic seizures. ,, Studies have also shown alterations in the activities of ATPases in different models of epilepsy and modulation of ATP hydrolysis by antiepileptic drug treatment. , Although the prognosis for seizure control is good in at least 60% patients, upto 40% individuals suffer from intractable, pharmacoresistant epilepsy. , Recently, focus on ethnopharmacology research has increased all over the world and a growing body of evidence has indicated immense potential of medicinal plants as alternative and complementary therapies for many human ailments.
Gotu kola, scientifically named as Centella asiatica, of the Apiaceae (Umbelliferae) family is an herbaceous annual plant native to the Asia. The most important bioactive components of C. asiatica leaf are triterpenoid glycosides (asiatic acid, medacassic acid), saponin glycosides (Brahmiside, Brahminoside), and flavonoids. , Gotu kola extract was reported to be beneficial in improving memory and also for the treatment of mental fatigue and anxiety. , CA reverses the effect of a GABA A antagonist, PTZ, and protects against PTZ-induced convulsions and ATPase inhibition. , Asiaticoside, an active constituent in methanol and ethylacetate extracts of C. asiatica, has anxiolytic activity.  Keeping in view of neuroprotective role of this medicinal plant, the present study has been taken up to examine the alterations in the activities of all three ATPases in different regions of brain during PTZ-induced epilepsy and on antiepliptic treatment with CA extracts.
| » Materials and Methods|| |
Collection of Plant Material
The leaves of C. asiatica used in this work were collected from Tirumala Hills, Andhra Pradesh, India, and authenticated by qualified botanist at Department of Botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India. The leaves were shade-dried and reduced to coarse powder.
The extraction was carried out as specified by Sowmyalakshmi  and Vattanajun.  The powdered plant material was soaked in methanol for 2 days at room temperature and the solvent was filtered. This was repeated three to four times until the extract gave no coloration. It was distilled and concentrated under reduced pressure in the Buchi rotavapour R-114 yielding a gum-like residue, which was then suspended in water and extracted with various organic solvents of increasing polarity (starting with the lipophilic solvent n-Hexane, ending with the more hydrophilic n-Butanol). The solvent from each extract was distilled and concentrated under reduced pressure in the Buchi rotavapor. Finally, the extracts were freeze dried and used for further studies.
Adult male Wistar rats (150±25g) were used for the present study. The experimental rats were housed in polypropylene cages under laboratory conditions of 28±2 0 C temperature with 75% relative humidity and photoperiod of 12 h light/dark cycle. The rats were given standard pellet diet (Gold Mohur Food and Feeds Ltd., Bangalore) and water ad libitum. The rats were maintained according to the ethical guidelines for animal protection and welfare bearing the CPCSEA 438/01/a/cpcsea/dt 17.07.2006 in its resolution No: 9/IAEC/SVU/2006/dt 04.03.2006.
Induction of Epilepsy
Convulsions were induced by an injection of pentylenetetrazol (60 mg/kg b.w., i.p.) in saline. 
Administration of Test Substance
Each fraction of CA extract (200 mg/kg b.w.) was dissolved in saline and given to the animals for 1 week prior to the injection of PTZ.  A gavage tube was used to deliver the substance by the oral route, which is the clinically expected route of administration of C. asiatica. The volume of administration was kept at 1 ml/kg b.w.
Drugs and Chemicals
Pentylenetetrazol was obtained from Sigma-Aldrich, St. Louis, MO, USA. All other chemicals were of analytical grade.
Isolation of Tissues
After stipulated duration, the animals were sacrificed by cervical dislocation and different brain regions such as cerebral cortex (CC), cerebellum (CB), pons medulla (PM), and hippocampus (HC) were immediately isolated, frozen in liquid nitrogen, and stored at -80 0 C until analysis.
ATPases (E.C. 184.108.40.206):(ATP Phosphohydrolase)
The activities of ATPases were assayed by the method of Fritz and Hamrick  as reported by Desaiah and Ho  with slight modifications and the inorganic phosphate liberated was estimated by the method of Lowry and Lopez  as modified by Phillips and Hayes. 
Tissue homogenates were prepared in ice cold 0.32 M sucrose containing 1.0 mM EDTA and 10 mM imidazole (pH 7.5). The homogenates were centrifuged at 1000 Χ g and the supernatant obtained was used as an enzyme source.
Na + , K + -ATPase
The reaction mixture in a total volume of 3.0 ml contained 3 mM ATP, 3 mM MgCl 2 , 100 mM NaCl, 20 mM KCl, 135 mM imidazole-HCl buffer (pH 7.5) and 10 mg of tissue. The reaction mixture was incubated at 37 0 C for 30 min and stopped by the addition of 0.1 ml of 50% TCA. Samples were then assayed for inorganic phosphate by the method of Lowry and Lopez  as modified by Phillips and Hayes.  The color intensity was read at 800 nm in a spectrophotometer (Hitachi). Mg 2+ - ATPase activity was measured in the presence of 1 mM Ouabain, a specific inhibitor of Na + , K + - ATPase. Ouabain-sensitive Na 2+ , K + -ATPase activity was obtained by the difference between total ATPase and Mg 2+ -ATPase activity. The enzyme activity was expressed as ΅moles of inorganic phosphate formed/mg protein/hr.
Ca 2+ -ATPase activity
The reaction mixture in a total volume of 3 ml contained 135 mM imidazole - HCl buffer (pH 7.5), 5 mM MgCl 2 , 0.05 mM CaCl 2 , 4 mM ATP, and 30 mg of tissue. The mixture was incubated at 37 0 C for 30 min and stopped by the addition of 0.1 ml of 50% TCA. The inorganic phosphate formed was estimated by the method of Lowry and Lopez  as modified by Phillips and Hayes.  Mg 2+ - ATPase activity was measured in the presence of 0.5 mM EGTA and this value was subtracted from total ATPase activity to get Ca 2+ -ATPase activity. Enzyme activity was expressed as ΅moles of inorganic phosphate formed/mg protein/h.
Mean, standard deviation (SD), and analyses of variance (ANOVA) for the data were calculated by using the SPSS (Statistical Package for Social Sciences) software.
| » Results|| |
The activity levels of Na + , K + -ATPase, Mg 2+ -ATPase, and Ca 2+ -ATPase were studied in different regions of brain during PTZ-induced epilepsy and on pre-treatment with different extracts of C. asiatica. In general, all the three ATPases showed conspicuous inhibition during PTZ-induced epilepsy in all the brain regions (CC, CB, PM, and HC) when compared to respective saline controls. On pre-treatment with different CA extracts, the activity levels, in general, were increased in all the brain regions except for the treatment with aqueous extract (AE) when compared to the respective PTZ-induced epileptic group [Table 1],[Table 2],[Table 3].
| » Discussion|| |
Mitochondrial dysfunction and consequent energy depletion are the major causes of oxidative stress and alterations in the ionic homeostasis in the central nervous system which causes loss of cellular integrity and cell death. Mitochondrial dysfunction, excitotoxicity, and generation of reactive oxygen species have been associated with various CNS disorders such as seizures, Parkinsonism More Details, Huntington, and Alzheimer's diseases. ,
In the present investigation, a decrease in Na + , K + -ATPase, Mg 2+ -ATPase, and Ca 2+ -ATPase activities was observed in all the brain areas during PTZ-induced epilepsy, which is in agreement with the earlier reports. , Inhibition of Na + , K + -ATPase in monkey and human epileptic brain and inhibition of Ca 2+ -ATPase in seizure prone mice have also been reported.  Similar inhibition of Na + , K + -ATPase activity was also recorded in hippocampus and cortex during kainic acid-induced epilepsy.  Since ATPases play a pivotal role in maintenance of cellular ionic gradient, their inhibition probably enhance neuronal excitability and facilitates the appearance of excitatory activity and convulsions.  Accordingly, ATPase inhibition leads to uncontrolled dendrite discharge in purkinje cells of rat cerebellum and causes electrographically recorded seizures in mice.  Since the Na + , K + and Ca 2+ ions are important in the development and conduction of action potential, the decrease in the activities of respective ATPases may alter the rate of influx and efflux of cations correlating with altered membrane permeability properties. Further, the decrement in the activities of ATPases reflects the decreased turnover of ATP, presumably due to inhibition of the oxidoreductase system and uncoupling of oxidative phosphorylation.
All the three ATPases were elevated in different regions of brain during pre-treatment with CA extracts in PTZ-induced epileptic animals except for the treatment with AE. Although not related to the present studies, it has been shown that Curcumin plays neuroprotective role by elevating Na + , K + -ATPase activity in all the brain regions of rat.  Studies with Bacoside A isolated from Bacopa monniera showed that administration of Bacoside A inhibited lipid peroxidation, improved the activities of Na + , K + -ATPase, Ca 2+ -ATPase, and Mg 2+ -ATPase, and maintained the ionic equilibrium.  The present findings coupled with earlier reports suggest that the bioactive factors present in selected extracts of CA offer neuroprotection by directly or indirectly modulating the activities of ATPases and thus may be helpful in the treatment of seizure disorders.
| » Acknowledgments|| |
We are highly thankful to University Grants Commission for the financial assistance. Project was sanctioned to corresponding author Prof. W. Rajendra, UGC-MRP/32-504/2006(SR).
| » References|| |
|1.||Loscher W. Animal models of epilepsy for the development of antiepileptic and disease modifying drugs: A comparison of the pharmacology of kindling and post-status epilepticus models of temporal lobe epilepsy. Epilepsy Res 2002;50:105-23. |
|2.||Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper's Illustrated Biochemistry. 28 th ed. New York: McGraw Hill Medical; 2009. P. 285-91. |
|3.||Kodama T. Thermodynamic analysis of muscle ATPase mechanisms. Physiol Rev 1985;65:467-551. [PUBMED] [FULLTEXT] |
|4.||Gendron FP, Benrezzak O, Krugh BW, Kong Q, Weisman GA, Beaudoin AR. Purine signaling and potential new therapeutic approach: Possible outcomes of NTPDase inhibition. Curr Drug Targets 2002;3:229-45. [PUBMED] [FULLTEXT] |
|5.||Littleton JT, Bellen HJ. Synaptotagmin controls and modulates synaptic-vesicle fusion in a calcium-dependent way. Trends Neurosci 1995;18:18-24. |
|6.||Kryzhanovskii GN, Tverdislova IL, Karpova MN, Agatova OL, Glebov RN. Effect of metrazol-induced epileptic activity on transport Ca-ATPase activity in rat brain synaptic membranes. Bull Exp Biol Mede 1987;104:895-8. |
|7.||Parsons TJ, Churn SB, Kochan LD, DeLorenzo RJ. Pilocarpine-induced status epilepticus causes N-Methyl-D-aspartate receptor-dependent inhibition of microsomal Mg 2+ /Ca 2+ ATPase-mediated Ca 2+ uptake. J Neurochem 2001;75:1209-18. |
|8.||Arundhati K, Mohan Das S, Padma T. Reduced activity of red Cell Na + K + ATPase and Ca 2+ - ATPase in patients with idiopathic generalized epilepsy. Int J Hum Genet 2003;3:59-63. |
|9.||Kinjo ER, Arida RM, De Oliveira DM, De Silva Fernandes MJ. The Na + /K + -ATPase activity is increased in the hippocampus after multiple status epilepticus induced by pilocarpine in developing rats. Brain research 2006;1138:203-07. |
|10.||Streck EL, Feier G, Bϊrigo M, Franzon R, Dal-Pizzol F, Quevedo J, et al. Effects of electroconvulsive seizures on Na(+),K(+)-ATPase activity in the rat hippocampus. Neurosci Lett 2006;404:254-7. |
|11.||Guillaume D, Grisar T, Delgado-escueta AV, Bureau-heeren MJ, Laschet J. Phosphyorylation of brain (Na + , K + ) -ATPase alpha catalytic subunits in normal and epileptic cerebral cortex: The audiogenic mice and the cat with a freeze lesion. J Neurosci Res 2003;29:207-17. |
|12.||Ludwig M, Pittman QP. Talking back: Dendritic neurotransmitter release. Trends Neurosci 2003;26:5. |
|13.||Cognato Gde P, Bruno AN, da Silva RS, Bogo MR, Sarkis JJ, Bonan CD. Antiepileptic drugs prevent changes induced by pilocarpine model of epilepsy in brain Ecto-Nucleotidases. Neurochem Res 2007;32:1046. [PUBMED] [FULLTEXT] |
|14.||Tanganelli P, Regesta G. Clinical aspects and biological bases of drug-resistant epilepsies. Epilepsy Res 1999;34:109-22. |
|15.||Kwan P, Brodie MJ. Early identification of refractory epilepsy. N Engl J Med 2000;342:314-9. [PUBMED] [FULLTEXT] |
|16.||Inamdar PK, Yeole RD, Srivastava de Souza NJ. Stability study of the active constituents in the Centella asiatica extract formulations. Drug Dev Indust Pharm 1996;22:211-6. |
|17.||Maquart FX, Chastang F, Simeon A, Birembaut P, Gillery P, Wegrowski Y. Triterpenes from Centella asiatica stimulate extracellular matrix accumulation in rat experimental wounds. Eur J Dermatol 1999;9:289-96. [PUBMED] [FULLTEXT] |
|18.||Hamid AA, Shah Z, Md Musa R, Mohammad S. Characterization of antioxidative activities of various extracts of Centella asiatica (L) Urban. Food Chem 2002;77:465-9. |
|19.||Snyder SH, Jaffrey SR, Zakhary R. Nitric oxide and carbon monoxide: Parallel roles as neural messengers. Brain Res Rev 1998;26:2-3. |
|20.||Oliveira MS, Furian AF, Royes FF, Fighera MF, de Carvalho Myskiw J, Fiorenza NG, et al. Ascorbate modulates pentylenetetrazol-induced convulsions biphasically. Neuroscience 2004;128:721-8. |
|21.||Fighera MR, Royes LFF, Furian AF, Oliveira MS, Fiorenza NG, Frussa-Filho R, et al. GM1 ganglioside prevents seizures, Na + , K + -ATPase activity inhibition and oxidative stress induced by glutaric acid and pentylenetetrazole. Neurobiol Dis 2006;22:611-23. |
|22.||Wijeweera P, Arnason JT, Koszyck D, Merali Z. Evaluation of anxiolytic properties of Gotukola-(Centella asiatica) extracts and asiaticoside in rat behavioral models. Phytomedicine 2006;13:668-76. |
|23.||Sowmyalakshmi S, Nur-e-Alam M, Akbarsha MA, Thirugnanam S, Jurgen Rohr, Chendil D. Investigation on semecarpus lehyam-a siddha medicine for breast cancer. Planta 2005;220:910-8. |
|24.||Vattanajun A, Wattanabe H, Tantisira MH, Tantisira T. Isobolographically additive anticonvulsant activity between Centella asiatica's Ethyl Acetate fraction and some antiepileptic drugs. J Med Assoc Thai 2005;88:131-40. |
|25.||Ray SK, Poddar MK. Effect of pentylenetetrazol on carbaryl-induced changes in striatal catecholamines. Biochem Pharmacol 1985;34:553-7. [PUBMED] [FULLTEXT] |
|26.||Saxena G, Flora SJ. Changes in brain biogenic amines and haem biosynthesis and their response to combined administration of succimers and Centella asiatica in lead poisoned rats. J Pharm Pharmacol 2006;58:547-59. [PUBMED] [FULLTEXT] |
|27.||Fritz PJ, Hamrick ME. Enzymatic analysis of adenosine triphosphatase. Enzymologia 1966;30:57-64. [PUBMED] |
|28.||Desaiah D, Ho IK. Effect of acute and continuous morphine administration on catecholamine-sensitive adenosine triphosphatase in mouse brain. J Pharmacol Exp Ther 1979;208:80-5. [PUBMED] [FULLTEXT] |
|29.||Lowry OH, Lopez JA. The determination of inorganic phosphate in the presence of labile phosphate esters. J Biol Chem 1946;162:421-8. |
|30.||Phillips TD, Hayes AW. Effects of patulin on ATPase in mouse. Toxicol Appl Pharmacol 1977;42:175-88. [PUBMED] |
|31.||Poon HF, Frasier M, Shreve N, Calabrese V, Wolozin B, Butterfield DA. Mitochondrial associated metabolic proteins are selectively oxidized in A30P A-synuclein transgenic mice: A model of familial Parkinson's disease. Neurobiol Dis 2005;18:492-8. [PUBMED] [FULLTEXT] |
|32.||Cristina Rego A, Oliveira R. Mitochondrial dysfunction and reactive oxygen species in excitotoxicity and apoptosis: Implications for the pathogenesis of neurodegenerative diseases. Neurochem Res 2003;28:1563-74. |
|33.||Vitezic, D, Pelcic JM, Zupan G, Vitezic M, Ijubicic D, Simonic A. Na+, K+- ATPase activity in the brain of the rats with kainic acid-induced seizures: Influence of lamotrigine. Psychiatr Danub 2008;20:269-76. |
|34.||Vasilets LA, Schwartz W. Structure-function relationships of cation binding in the Na + /K + ATPase. Biochem Biophys Acta 1993;1154:201-22. |
|35.||Jamme I, Petit E, Divoux D. Modulation of mouse cerebral Na + , K + -ATPase activity by oxygen free radicals. Neuro Rep 1995;7:333-7. |
|36.||Vajragupta O, Boonchoong P, Watanabe H, Tohda M, Kummasud N, Sumanont Y. Manganese complexes of curcumin and its derivatives: Evaluation for the radical scavenging ability and neuroprotective activity. Free Radic Biol Med 2003;35:1632-44. [PUBMED] [FULLTEXT] |
|37.||Anbarasi K, Vani G, Balakrishna K, Shyamala Devi CS. Effect of Bacoside A on membrane-bound ATPases in the brain of rats exposed to cigarette smoke. J Biochem Mol Toxicol 2000;19:59-65. |
[Table 1], [Table 2], [Table 3]
|This article has been cited by|
||Induction of root colonization by Piriformospora indica leads to enhanced asiaticoside production in Centella asiatica
| ||Satheesan, J., Narayanan, A.K., Sakunthala, M. |
| ||Mycorrhiza. 2012; 22(3): 195-202 |
||Centella asiatica (L.) Urban: From Traditional Medicine to Modern Medicine with Neuroprotective Potential
| ||Ilkay Erdogan Orhan |
| ||Evidence-Based Complementary and Alternative Medicine. 2012; 2012: 1 |
|[Pubmed] | [DOI]|
||Centella asiatica (L.) Urban: From traditional medicine to modern medicine with neuroprotective potential
| ||Orhan, I.E. |
| ||Evidence-based Complementary and Alternative Medicine. 2012; art(946259) |
||Centella asiatica modulates neuron cell survival by altering caspase-9 pathway
| ||Omar, N.S., Zakaria, Z.A.C., Mian, T.S., Ngah, W.Z.W., Mazlan, M. |
| || Journal of Medicinal Plant Research. 2011; 5(11): 2201-2209 |
||Anticonvulsant activity of ethanolic extract of fruits of Terminalia chebula on experimental animals
| ||Debnath, J., Sharma, U.R., Kumar, B., Chauhan, N.S. |
| || International Journal of Drug Development and Research. 2010; 2(4): 764-768 |