NEUROPROTECTIVE PROPERITY OF CENTELLA ASIATICA AGAINST PENTYLENE- TETRAZOLE INDUCED EPILEPSY IN RAT BRAIN WITH PARTICULAR REFERENCE TO LIPID METABOLISM

NEUROPROTECTIVE PROPERITY OF CENTELLA ASIATICA AGAINST PENTYLENE- TETRAZOLE INDUCED EPILEPSY IN RAT BRAIN WITH PARTICULAR REFERENCE TO LIPID METABOLISM

Kanchi Siva Prasad

Department of Zoology, SVSSC Government Degree College, Sullurpet, SPSR Nellore - 524121, Andhra Pradesh, India.

ABSTRACT: This study evaluated the anticonvulsant effect of different extracts of Centella asiatica (CA) with particular reference to lipid metabolism in different regions of rat brain. The rats were randomly divided into 4 groups having 6 in each group: i.e. Control group received saline, PTZ-induced  epileptic group (60mg/kg  b.w op/ 1 day), epileptic  group pretreated with  chloroform extract  (CE),  epileptic group pretreated with aqueous (AE)  extract and  epileptic  group  pretreated  with  diazepam  (DP;  Reference  control)  (2 mg/kg  b.w/ip/ day).  The  CA extract  is  administered at the  dose  of 200  mg/kg  body  weight  orally  for  one week.  The experimental results were observed that the decreased content of phospholipids in the entire brain regions i.e. Cerebral cortex (CC), Cerebellum (CB), Hippocampus (HC) and Pons medulla (PM); total cholesterol, triglycerides and increased content of Lipid peroxidation in epileptic rats. The reversal changes were observed on pre­ treatment with the chloroform extract of CA and diazepam. Hence, it is evident that the different   bioactive factors   of CA offered protection against   PTZ-induced epilepsy.

Epilepsy, Anticonvulsant, Centella asiatica, Pentylenetetrazole, Lipid peroxidation

INTRODUCTION: Cholesterol is an essential component for neuronal physiology not only during development stage but also in the adult life. Cholesterol metabolism in brain is independent from that in peripheral tissues due to blood-brain barrier ¹. Lipids serve several functions  in the biological  systems  such as structural  components of the  membranes,  storage and  transport   forms of  metabolic  fuel,  protective coating  on  the surface concerned in cell recognition, species specificity  and  tissue  immunity ².  Epilepsy is a sudden surge electrical activity in the brain ³. It is well known that the epileptic seizures result from excessive discharge in a population of hyper excitable neurons.

Despite the multiple molecular  mechanisms  have been proposed in generating and spreading   epileptic   discharges, it has been well established that impaired GABAergic activity exaggerated glutama- tergic neurotransmission  primarily contribute to the various types of  epilepsies ⁴.  Glutamate   is  required  for normal  brain function; the  presence  of  excessive amounts  of  glutamate  can  lead  to  excitotoxic  cell death ⁵.

Through the inhibition of inhibitory neurotransmitter    (GABA),   the resting   membrane potential can  be  regulated   and  thus  can  reduce the probability of glutamate excitation ⁶.  Hopefully new antiepileptic drugs act on different neurotransmitter receptors or ion-channels will result  in  improved  control  of  seizures  and  drugs that are active on ion-channels have greater potential in restoring the function of epileptic neurons  to  normalcy ⁷. CA showed decrement in seizure score, improvement in learning deficits induced by PTZ and increased latencies in passive avoidance behavior ⁸.

It showed protection against electroshock induced convulsions, pentylenetetrazole and strychnine induced chemo convulsions ⁹. It also showed a reduction in lipid peroxidation, spontaneous motor activity, potentiation in diazepam withdrawal-induced hyperactivity, hypothermia and potentiation of pentobarbitone sleeping time ¹⁰. However, there are also anecdotal observations that multiple antiepileptic drugs regimens employed in ameliorating seizers generally met with partial success and suffer from substantial problems such as neurological disorders.

During the past few years, considerable progress has been made towards identifying active factors from indigenous medicinal plants for different human ailments including neurodegenerative disorders such as Alzheimer’s disease, parkin- sonism, epilepsy etc. Centella asiatica considerably increased the seizure threshold and reversed the neuro chemical abnormalities occurred in cholinergic system, monoamine neurotransmitter system and metabolism of glutamate and energy during PTZ- induced epilepsy. Keeping in view of this the present investigation is aimed at studying the modulations of  lipid  metabolism  during  PTZ-induced  epilepsy and antiepileptic treatment with Centella asiatica.

MATERIALS AND METHODS:

Procurement and Maintenance of Experimental Animals: Male adult wistar rats weighing 150 ± 25 grams were used as the experimental animals in the present investigation. The rats were purchased from the Indian Institute of Science (IISc), Bangalore, maintained in the animal house of the department in polypropylene cages under laboratory conditions of 28 ± 2 ⁰C temperatures with photoperiod of 12 hours light and 12 hours dark and 75 % relative humidity. The rats were fed with standard pellet diet (Hindustan Lever Ltd., Mumbai) and water ad libitum.

Ethical Guidelines: 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:09/(i)/a/ CPCSCA/ IAEC/ SVU/ WR/KSP/Dt. 04.03.2006.

Selection of Drug: Pentylenetetrazole (PTZ), an anticonvulsant drug, was selected for the present study. It was obtained as commercial grade chemical from Sigma chemicals, USA.

Collection of the Plant Material: Centella asiatica (CA) plant was collected from Tirumala hills and indentified by a botanist, Department of Botany, S.V. University, Tirupati.  A voucher specimen was deposited in the herbarium of the Department of Botany, S. V. University, Tirupati (Voucher no. 1688).

Preparation of Plant Extracts: The active principles of the leaves of plant were extracted into Chloroform, since this solvent was predominantly used by several investigators for extracting anticonvulsant principle(s) from various plants ^{11, 12}. Powdered plant material was soaked in methanol for 2 days at room temperature and the solvent was filtered. This was repeated 3-4 times until the extract gave no coloration. The extract was distilled and concentrated under reduced pressure in the Buchi rotovapour R-114 yielding a gum-like residue, which was then suspended in water and extracted with chloroform.

Induction of Epilepsy: Convulsions were induced by an intraperitoneal (i.p.) injection of Pentylene- tetrazole (60 mg/Kg body weight) in saline ^{13, 14}.

Administration of Test Substance: Each fraction of CA extract (200 mg/Kg body weight) was dissolved in saline and given to the animals for one week prior to the injection of PTZ ¹⁵. A gavage tube was used to deliver the substance by the oral route, which is the clinically expected route of administration of CA ¹⁴. The volume of adminis- tration was kept at 1 ml/kg/ animal. Diazepam, an anticonvulsant drug, was dissolved in normal saline and given intraperitoneally (2 mg/Kg bw i.p.) for one week to the experimental animals (Reference control).

Drugs, Chemicals and Apparatus: All chemicals used in the present study were Analar grade (AR) and obtained from the following scientific companies:  Sigma,    Fisher (Pittsburg, PA, USA), Merck (Mumbai, India), Ranbaxy (New Delhi, India), Qualigens (Mumbai, India). Pentylene-tetrazole and diazepam were obtained from Sigma Aldrich (St. Louis, MO, USA). In the present investigation Barnstead Thermoline water purification plant for nanopure water, Kubota KR centrifuge and Hitachi U-2000 Spectrophotometer and other standard equipments were used for biochemical analyses.

Isolation of Tissues: 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 isolated, frozen in liquid nitrogen  and  were  stored    at  -80 ⁰C.

Experimental Design for Screening of Plant Extracts for Anticonvulsant Activity: The rats were randomly divided into 4 groups having 6 in each group: i.e. Control group received Saline, PTZ-induced epileptic group (60 mg/kg b.w./ i.p/ 1 day), Epileptic group pretreated with chloroform extract (CE)   and   epileptic group pretreated with Diazepam (DP; Reference control) (2 mg/kg b.w/i.p). The chloroform extract was administered at the dose of 200 mg/kg body weight orally for one week.

Biochemical Analysis: Phospholipids were estimated by the method of Zilversmidth and Davis ¹⁶. The total cholesterol and triglycerides contents were estimated by the method of Natelson ¹⁷. MDA levels were estimated by Ohkawa   et al., ¹⁸.

Statistical Treatment of Data: All assays were carried out with six separate replicates from each group.  The mean, standard error (SE) and Analysis of variance (ANOVA) were done using SPSS statistical software (11.5 ver.) for different parameters. Difference between control and experimental assays were considered as significant at P<0.05.

RESULTS:

Phospholipids: The phospholipids content was decreased significantly in all the brain regions in induced epileptic rats (PTZ), with highest decrease noted in the hippocampus (HC). Pretreatment with CA extract i.e. CE and diazepam (Reference control) were resulted in significantly increased phospholipids content in all the brain regions and highest increment is noted in HC (Table 1).

Total Cholesterol: When compared with saline control, PTZ-induced animals had significantly decreased the total cholesterol in all the brain regions, with highest decrease noted in the hippocampus (HC). Pre-treatment with CA extract i.e. CE and diazepam (Reference control) were resulted in significantly increased total cholesterol content in all the brain regions. (Table 2)

Triglycerides: The Triglycerides content was decreased significantly in all the brain regions in induced epileptic rats (PTZ), with highest decrease noted in the hippocampus (HC). Pre-treatment with CA extract i.e. CE, and were resulted in significantly increased Triglycerides content in all the brain regions and highest increment was noted in HC (Table 3).

Lipid Peroxidation: The malondialdehyde content was significantly increased in all the areas of brain during PTZ- induced epilepsy, the highest elevation was noted in hippocampus (HC). Meanwhile pre-treatment with the extract of CA, showed significant decrease malondialdehyde content in all the brain regions, the highest decrement was noted in HC (Table 4).

TABLE 1: CHANGES IN THE PHOSPHOLIPIDS CONTENT IN DIFFERENT REGIONS OF RAT BRAIN DURING PTZ- INDUCED  EPILEPSY  AND  PRE-TREATMENT  WITH  DIFFERENT  EXTRACTS  OF  CENTELLA  ASIATICA

All  the  values  are  mean,  ± SEM   of  six  individual  observations. Values  in  '(  )'parentheses  are  %  change  over  saline  control. *Values  are  significant  at  P  <  0.05  in  Scheffe  test. (Values  are  expressed  in  mg  of  phospholipids/g  wet  wt  of  the  tissue)

TABLE  2:  CHANGES  IN  THE  TOTAL  CHOLESTEROL  CONTENT  IN  DIFFERENT  REGIONS    OF  RAT  BRAIN  DURING PTZ-INDUCED  EPILEPSY  AND  PRE-TREATMENT  WITH  DIFFERENT  EXTRACTS  OF  CENTELLA  ASIATICA

All  the  values  are  mean,  ± SEM  of  six  individual  observations. Values  in  '(  )'parentheses  are  %  change  over  saline control. *Values  are  significant  at  P  <  0.05  in  Scheffe  test. (Values  are  expressed  in  mg  of  phospholipids/g  wet  wt  of  the  tissue)

TABLE  3:  CHANGES  IN  THE  TRIGLYCERIDES  CONTENT  IN  DIFFERENT  REGIONS  OF  RAT  BRAIN  DURING  PTZ- INDUCED  EPILEPSY  AND  PRE-TREATMENT  WITH  DIFFERENT  EXTRACTS  OF  CENTELLA  ASIATICA

All  the  values  are  mean,  ± SEM  of  six  individual  observations. Values  in  '(  )'parentheses  are  %  change  over  saline  control. *Values  are  significant  at  P  <  0.05  in  Scheffe  test. (Values  are  expressed  in  mg  of  triglycerides  /  g  wet  wt  of  the  tissue)

TABLE  4:  CHANGES  IN  THE  LIPID  PEROXIDATION  CONTENT  IN  DIFFERENT  REGIONS  OF  RAT  BRAIN  DURING PTZ-INDUCED  EPILEPSY  AND  PRE-TREATMENT  WITH  DIFFERENT  EXTRACTS  OF  CENTELLA  ASIATICA

All  the  values  are  mean,  ± SEM  of  six  individual  observations. Values  in  '(  )' parentheses  are  %  change  over  saline  control. *Values  are  significant  at  P  <  0.05  in  Scheffe  test. (Values  are  expressed  in  µ  moles  of  malondialdehyde  formed  /  gram  wet  wt  of  the  tissue).

DISCUSSION: The treatment with extracts of Centella asiatica and diazepam restored the levels of cholesterol in different regions of brain of epileptic rats. The membrane micro domains are rich in cholesterol; the alterations in cerebral cholesterol in induced epilepsy could alter the cellular signaling pathways which possibly play a pivotal role in the neuro degeneration process ¹⁹. Increase in cholesterol levels were reported in rats fed both CA  extract  and  powder  during  H₂O₂   induced oxidative stress ²⁰. The decreased levels of triglycerides in different regions of brain during PTZ-induced epilepsy might be due to enhanced lipolysis  through  lipase  activity.  It  is  well established that glutamate excitotoxicity and oxidative stress contribute to neuronal degeneration in acute conditions such as stroke, epilepsy, trauma, hypoxia and hypoglycemia and chronic neuro-degenerative diseases such as Parkinson’s disease, Alzheimer’s and Huntington’s disease ²¹.

Since the bioactive factors of CA significantly attenuate the glutamate induced excitation and oxidative stress, it is possible that the CA extract possibly ameliorate the deregulated lipid metabolism in general and cholesterol metabolism in particular, thus protecting the progressive cell damage that occurs in induced epilepsy ²². Lipid peroxidation is a complex process generating reactive radicals, which is regarded as an etiologic or pathogenic factor in several diseases of central nervous system including epilepsy.

The cell membranes, enriched with polyunsaturated fatty acids (PUFAs), are more prone to free radical mediated lipid peroxidation. Lipid peroxidation of cell membranes causes  a  loss  of  the  fluid  properties  of  the membrane  as  well  as  increase  in  membrane permeability ²³. Lipid peroxidation products are constantly     involved in some of  the pathophysiological  effects   associated   with oxidative stress in cell and tissues. Unlike reactive free radicals, aldehydes can produce lipid peroxidation products, which are rather long lived and can therefore diffuse from the site of their origin, reaching and attacking intracellular and extra cellular targets  ²⁴.  They disrupt  various important structural and protective functions associated with bio-membranes in various in-vivo pathologic events and are implicated as a result of this oxidation ²⁵.

The key functions of nerve cells, such as creation and maintenance of transmembrane potential, reception and subsequent transmission of signal, synthesis and regulation of signal transducers, and uptake and secretion of neurotransmitters are highly susceptible to excessive accumulation of endogenous products of lipid peroxidation in neuronal membrane structures.

Hence, lipid peroxidation is regarded as an etiologic or pathological factor in myriad number of neurological disorders such as Parkinson’s disease, Downs’s syndrome, schizophrenia, epilepsy etc. oxidative damage induced by lipid peroxidation has been recognized as key factor for the occurrence of many human diseases ²⁶. It has been hypothesized that COx enzyme induction leads to an increase in various prostaglandins, particularly PGE2 which  facilitates  the  massive  release  of glutamate  from  nerve  terminals  and  astrocytes and subsequently increase the free radical production leading to oxidative stress followed by apoptosis of GABAergic neurons ending in epileptic discharges ²⁷.

Similar increases in MDA, XO and NO levels have also been recorded in the brains of mice treated with PTZ ²⁸.  Liu et al., ²⁹ have indicated that the made cassoside, the active constituent of CA, decreased nitric oxide (NO) levels and malanoldehyde (MDA) content in the burn skin tissue. Decreased MDA content and an increase in glutathione and catalase activities have been reported in rats treated with aqueous extract of CA in intra cerebroventricular streptozotocan model of Alzheimer ’s  disease  in  rats.  Decreased  lipid peroxidation and increased enzymatic and non- enzymatic antioxidants have been elucidated by the asiaticoside derived from CA ³⁰.

CONCLUSION: The present findings in conjugation with the earlier reports it is speculated that the bioactive factors of CA has the propensity to a modulate excitotoxic glutamate induced oxidative impairments in the brain and may be efficiently employed as a neuroprotective adjuvant to abrogate  the  oxidative  stress  that  occur  during induced epilepsy. However, further in depth studies are required to understand the physiological mechanism of different bioactive compounds present in the CA extracts and to suggest that the therapeutic modality of these compounds with particular reference of anticonvulsant and  neuroprotective  activity.

ACKNOWLEDGEMENT: The corresponding author would like to thank for the support of the Department of Zoology, Sri Venkateswara University, Tirupathi.

CONFLICTS OF INTEREST: The authors have no conflict of interests to declare regarding the publication of this paper.

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    How to cite this article:

    Prasad KS: Neuroprotective properity of Centella asiatica against pentylene - tetrazole induced epilepsy in rat brain with particular   reference to lipid metabolism. Int J Pharm Sci Res 2018; 9(2): 665-71.doi: 10.13040/IJPSR.0975-8232.9(2).665-71.

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