INTRODUCTION

Reactive oxygen species (ROS) has been reported to play an important role in ageing process. Increase in ROS production and imbalance in antioxidant defense and repair mechanisms, leads to cell death during ageing and age related neurological disorders (Bodis-Wollner et al., 1991). Parkinsons disease (PD) is one of the widespread neurodegerative diseases, caused by the loss of dopamin-ergic neurons in the substantia nigra of basal ganglia. PD is characterized by resting tremor, rigidity and bradyki-nesia. Even though the exact mechanism of neurodegen-eration in substantia nigra is elusive, alterations of sev-eral biochemical pathways appear to be involved in the cascade of events leading to cellular dysfunction in sub-stantia nigra. Mitochondrial dysfunction and increased production of ROS are known to induce oxidative neuro-

degeneration in PD (Subathra et al., 2005). Though lev-odopa is the treatment of choice in PD, there are motor complications and oxidative neuronal damage reported with continuous therapy (Lan and Jiang, 1997; Savitt et al., 2006). 1-methyl-4-phenyl-1, 2, 3, 6 - tetrahydropyrid-ine (MPTP), when injected will be converted to the most prominent mitochondrial complex I inhibitors, and cause striatal dopamine depletion and loss of tyrosine hydroxy-lase immunoreactivity to produce Parkinsons like symp-toms (Bashkatova et al., 2004; Lane et al., 2008).

Plants having medicinal properties have gained impor-WDQFHQRZDGD\VEHFDXVHRIWKHLUEHQHFLDOHIIHFWRQhuman health care. Centella asiatica (Umbelliferae) syn Hydrocotyl asiatica, belongs to the family of apiaceaeand widely found all over the world. The extract of this plant has been used in Indian system of medicine for dif-ferent ailments like asthma, wound healing, skin disor-

Neuroprotective effect of Centella asiatica extract (CAE) on experimentally induced parkinsonism in aged

Sprague-Dawley ratsNagaraja Haleagrahara1 and Kumar Ponnusamy2

1Division of Human Biology, Faculty of Medicine and Health, International Medical University, No. 126, Jalan 19/155B, Bukit Jalil, 57000 Kuala Lumpur, Malaysia

2St. Matthew's University School of Medicine, Grand Cayman Islands British West Indies

(Received August 9, 2009; Accepted October 29, 2009)

ABSTRACT Reactive oxygen species (ROS) play an important role in ageing and age-related neuro-degenerative changes including Parkinsons disease (PD). PD is characterized by signs of major oxidative stress and mitochondrial damage in the pars compacta of the substantia nigra. Present study was designed to investigate whether the Centella asiatica extract (CAE) would prevent 1-methyl-4-phenyl-1,2,3,6 - tet-rahydropyridine (MPTP) - induced neurotoxicity in aged Sprague-Dawley rats. Adult, male Sprague-daw-ley rats of 300-350 g were divided into control, C. asiatica alone, MPTP alone (20 mg/kg, for 21 days) and MPTP with C. asiatica (300 mg/kg for 21 days) groups. Effect of aqueous extract of C. asiatica on oxidative biomarker levels in corpus striatum and hippocampus homogenate was examined. MPTP-chal-OHQJHGUDWVHOLFLWHGDVLJQLFDQWLQFUHDVHLQOLSLGK\GURSHUR[LGHV/32SSURWHLQFDUERQ\OFRQ-WHQW3&&SDQG[DQWKLQHR[LGDVH;2SZKHQFRPSDUHGZLWKFRQWUROUDWV7KHUHZDVDVLJQLFDQWGHFUHDVHLQWRWDODQWLR[LGDQWV7$SVXSHUR[LGHGLVPXWDVH62'SJOXWDWKLRQHSHUR[LGDVH*3[SDQGFDWDODVH&$7SOHYHOVZLWK0373WUHDWPHQW6XS-SOHPHQWDWLRQRI&$(UHGXFHG/32DQG3&&DQGVLJQLFDQWO\LQFUHDVHGS7$DQGDQWLR[LGDQWHQ]\PHOHYHOVSLQFRUSXVVWULDWXPDQGKLSSRFDPSXV7KHVHUHVXOWVVKRZWKDWDGPLQLVWUDWLRQRI&asiatica was effective in protecting the brain against neurodegenerative disorders such as Parkinsonism.

Key words: Parkinsonism, Centella asiatica, Neurotoxicity, Oxidative stress, Antioxidants

Correspondence: Nagaraja Haleagrahara (E-mail: hsnagaraja@gmail.com)

Original Article

The Journal of Toxicological Sciences (J. Toxicol. Sci.)Vol.35, No.1, 41-47, 2010

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ders, ulcers and body ache (Sahu et al., 1989; Babu et al., 1995; Suguna et al., 1996; Zainol et al., 2003; Kumar and Gupta, 2002). The plant extract also showed cen-tral nervous system depressant activity and inhibition on biosynthetic activity of fibroblasts (Del Vecchio et al., 1984; Babu et al., 1995). In the recent reports, it has been proved that C. asiatica has anti-lipid peroxidative and free radical scavenging activities (Jayashree et al., 2003; Gnanapragasam et al., 2004). The major active constitu-ents present in the extract of C. asiatica extract are trit-erpenes and polyphenols (Inamdar et al., 1996; Zainol et al., 2003). Triterpenes like asiatic acid and high polyphe-nolic contents are reported to contribute to the antioxida-tive activities of this plant extract (Gnanapragasam et al., 2004).

The most important challenge in the research on PD is to develop a neuroprotective therapy that can be supple-mented with levodopa or other dopaminergic agents, in the early course of disease to slow the progression of dis-ease. The present study was aimed to investigate wheth-er C.asiatica extract when administered orally would pre-vent MPTP induced neurotoxicity in the corpus striatum of Sprague Dawley rats and our hypothesis was, C. asi-atica extract offers neuroprotection in experimentally induced PD.

MATERIALS AND METHODS

C. asiatica extractFresh whole plant of C. asiatica was procured from the

commercial market and samples of the plant were iden-WLHGDQGDXWKHQWLFDWHGIRUWKHLUFRUUHFWERWDQLFDOLGHQWL-ty by a Botanist. The whole plants were cleaned, air-dried and were powdered. The powder was soaked in dou-ble distilled H2O:C2H5OH (1:1) in shaking incubator at &IRUGD\V7KHH[WUDFWHGVROXWLRQZDVOWHUHGWKURXJK:KDWPDQ1ROWHUSDSHUDQGFRQFHQWUDWHGDQGdried to powder.

AnimalsTwenty four months old male Sprague Dawley rats

weighing 300-350 g (Conn et al., 1980; Sanchez, et al., 2008) were obtained from Institute of Medical Research (IMR, Kuala Lumpur, Malaysia), Kuala Lumpur and housed under standard laboratory conditions (25 2C; 12 hr light and dark cycles). The animals were given access to an animal diet and tap water ad libitum. The rats were placed in polypropylene cages with three animals per cage and were allowed to acclimatize one week pri-or to treatment.

Animals were randomly assigned into four groups with

eight rats per group. Control animals received saline alone. Centella asiatica extract (CAE) alone group of rats were administered 300 mg/kg body weight (dissolved in 0.9% saline and administrated orally) of C. asiatica extract for 21 days (Kumar and Gupta, 2002; Gupta and Flora, 2006; Subathra et al., 2005). MPTP alone group received 20 mg/kg body weight of MPTP (Sigma-Aldrich, St. Louis, MO, USA) (HCl salt, dissolved in 0.9% saline, intra-peritoneally), twice at 20 min interval and the rats were maintained for 21 days (Gevaerd et al., 2001; Ramirez et al., 2003; Kwok-Tung et al., 2008). In the fourth group, the rats were challenged with neurotoxin-MPTP and simultaneously administered with CAE for 21 days. All the experimental procedures described were approved by Institutional Research and Ethics committee.

On completion of experimental period, animals were sacrificed by decapitation under sodium pentobarbi-tol anesthesia. Brain tissues were excised immediate-ly. Regions were separated according to the method of Glowinski and Iversen (1966) and immersed in ice cold saline. The tissues were homogenized using 0.01 M Tris-HCl buffer (pH 7.4) using glass homogenizer and super-QDWDQWZDVVWRUHGDW&WLOOIXUWKHUDQDO\VLV)URPWKHhomogenate samples, lipid hydroperoxides (LPO), protein-FDUERQ\OFRQWHQW3&&[DQWKLQHR[LGDVH;2VXSHU-oxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and total antioxidants (TA) were assayed using ELISA kits (Cayman Chemicals and Pierce Bio-technology, Ann Arbor, MI, USA). The levels of enzym-ic and non-enzymic antioxidants, LPO, protein carbonyls and TA were expressed as units per milligrams of proteins of samples. Protein levels of the samples were estimated by protein assay kits obtained from Cayman Chemicals (Cayman Chemicals and Pierce Biotechnology).

Statistical analysisData were presented as Mean standard deviation. Sta-

tistical analysis was done by SPSS v 14.0 software pack-DJH'LIIHUHQFHVDPRQJYDULRXVJURXSVDQGWKHVLJQL-cance were calculated by non-parametric Kruskal Wallis H and Mann- Whitneys U-Tests. P value less than 0.05 ZDVFRQVLGHUHGVWDWLVWLFDOO\VLJQLFDQW

RESULTS

LPOCorpus striatum and hippocampus lipid hydroperoxide

OHYHOVLQFUHDVHGVLJQLFDQWO\LQ0373WUHDWPHQWJURXSVFRPSDUHG WRFRQWUROJURXSV 3$VLJQLILFDQWdecrease in serum LPO was seen with C. asiatica alone WUHDWHGJURXSV3,QJURXSVWUHDWHGZLWK&$(

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N. Haleagrahara and K. Ponnusamy

DORQJZLWK0373WKHUHZDVDVLJQLFDQWGHFUHDVH3LQ/32OHYHOVDQGWKLVZDVVLJQLFDQWO\OHVVWKDQWKHFRQWUROJURXSV37DEOHVDQG

PCCA significant increase in striatum and hippocampus

3&&ZHUHUHFRUGHGDIWHU0373WUHDWPHQW3The level of protein carbonyl was decreased in CAE with 0373JURXSV3(YHQWKRXJKWKHOHYHORI3&&ZHUHVLJQLFDQWO\UHGXFHGZLWK&$(WUHDWPHQWLQ0373treated rats, it did not significantly decrease below the control levels both in striatum and hippocampus samples (Tables 1 and 2).

XO7KHUHZDVDVLJQLILFDQW LQFUHDVHLQ;2OHYHOVERWK

in striatum and hippocampus after 21 days exposure WR037337UHDWPHQWZLWKC. asiatica VLJQL-cantly reduced these changes and the enzyme level was

GHFUHDVHGWRFRQWUROOHYHOV37UHDWPHQWZLWKCAE alone showed significant increase in antioxidant HQ]\PHOHYHOV37DEOHVDQG

SOD6HUXP62'GHFUHDVHGVLJQLFDQWO\LQVWULDWXPDIWHU

H[SHULPHQWDOO\LQGXFHG3DUNLQVRQLVP3EXWWUHDWPHQWZLWK&$(VLJQLFDQWO\LQFUHDVHGWKH62'OHY-HOVDIWHUGD\V37DEOH6LPLODUFKDQJHVwere observed in SOD levels in hippocampus homoge-nates. More significant decrease in hippocampus SOD OHYHOZDVREVHUYHGDIWHU03733DQGFRQFXUUHQWtreatment with C. asiaticaVLJQLFDQWO\HOHYDWHGWKH62'levels (Table 2).

GPxCAE alone when injected at a dose of 300 mg/kg sig-

QLILFDQWO\LQFUHDVHGWKHFRUSXVVWULDWDO*3[OHYHOV30.05). But MPTP treatment decreased the GPx levels sig-

Table 1. Effect of CAE on antioxidant status in the corpus striatum of ratsParameters Control C. asiatica MPTP C. asiatica + MPTPLPO (nmol/mg of protein) 3.17 0.8 2.76 0.09* 4.56 0.03** 2.98 0.05 *

PCC (nmol/mg protein) 1.68 0.09 1.54 0.08 2.48 0.09** 1.65 0.05

;20PJSURWHLQ 3.23 0.03 2.61 0.07 4.89 0.04** 3.81 0.03

SOD (units/mg of protein) 18.20 0.92 22.54 0.86* 14.52 0.97*** 17.17 1.74

&$70PJRISURWHLQV 45.28 1.90 53.71 1.12* 32.20 1.74*** 41.73 1.23

GPx (nmol/min/mg of protein) 21.65 1.17 26.50 0.96* 16.30 1.14** 24.12 1.34

TA (mM/mg of protein) 3.22 0.08 3.45 0.5 1.52 0.09*** 2.97 0.09

Results are expressed as means S.D. of eight rats per group.*P < 0.05; **P < 0.01; ***P < 0.001 - Control with other groups.P < 0.05; P < 0.01; P < 0.001 - MPTP with C. asiatica + MPTP.

Table 2. Effect of CAE on antioxidant status in the hippocampus of ratsParameters Control C. asiatica MPTP C. asiatica + MPTPLPO (nmol/mg of protein) 2.96 0.03 2.42 0.24 3.56 0.08** 2.67 0.27

PCC (nmol/mg protein) 1.76 0.07 1.69 0.18 2.26 0.18** 1.52 0.15

;20PJSURWHLQ 3.82 0.09 2.79 0.18 5.12 0.19** 3.17 0.21

SOD (units/mg of protein) 14.50 0.32 17.57 0.13* 11.75 0.87*** 16.62 1.88

&$70PJRISURWHLQV 47.76 1.74 56.24 1.30** 38.52 1.82*** 46.77 1.92

GPx (nmol/min/mg of protein) 19.35 1.57 24.66 0.98* 14.40 1.08** 21.82 1.73

TA (mM/mg of protein) 3.47 0.12 3.09 0.25 1.74 0.05*** 2.65 0.16

Results are expressed as means S.D. of eight rats per group.*P < 0.05; **P < 0.01; ***P < 0.001 - Control with other groups.P < 0.05; P < 0.01; P < 0.001 - MPTP with C. asiatica + MPTP.

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Effect of Centella asiatica extract on Parkinsonism in rats

QLFDQWO\3DQGWUHDWPHQWZLWK&$(DORQJZLWKMPTP was able to increase the serum GPx levels. Serum GPx level reached near normal in Parkinsons group with C. asiatica37DEOH$VLPLODUWUHQGZDVREVHUYHGLQWKH*3[OHYHOLQKLSSRFDPSXVRIUDWV30.01) (Table 2).

CAT7KHUHZDVVWDWLVWLFDOO\VLJQLFDQWGHFUHDVHLQVWULDWDO

&$7OHYHOVDIWHU0373WUHDWPHQWIRUGD\V3&$(DORQHGLGDOVREULQJDERXWDVLJQLFDQWFKDQJH30.01) in serum CAT levels. But CAE administered for 21 days along with MPTP was able to increase the CAT lev-HOVPRUHWKDQ0373DORQH3DQGWKH&$7OHY-el reached more than control group (Table 1). Hippocam-pus CAT level also significantly decreased after MPTP IRUGD\V3C. asiatica reversed this change FDXVHGE\0373DV&$7OHYHOZDVIRXQGWREHVLJQL-FDQWO\KLJKHULQKLSSRFDPSXV37DEOH

TA7KHUHZDVDVLJQLFDQWGHFUHDVHLQKLSSRFDPSXVDQG

VWULDWXP7$DIWHUH[SRVXUHRIUDWVWR03733&RQFXUUHQWWUHDWPHQWZLWK&$(VLJQLFDQWO\LQFUHDVHGVHUXPWRWDODQWLR[LGDQWOHYHOV30373WUHDWPHQWIRUGD\VVLJQLFDQWO\GHFUHDVHGWKHKLSSRFDPSXV7$3DQG&$(WUHDWPHQWZDVDEOHWRLQFUHDVHWKHDQWLR[LGDQWFRQWHQWVDIWHUGD\V37DEOHVand 2).

DISCUSSION

In the present study, adult Sprague Dawley rats chal-OHQJHGZLWK0373VKRZHGKLJKO\VLJQLFDQWLQFUHDVHLQ/32SURWHLQFDUERQ\OFRQWHQWVDQG;2OHYHOVLQFRUSXVVWULDWXPDQGKLSSRFDPSXV7KHUHZDVDOVRDVLJQLFDQWdecrease in TA levels with MPTP treatment. The differ-ences in the levels of LPO products observed in various brain regions may be attributed to the differences in their iron content and diverse metabolism, which influenced the generation of ROS. Certain brain regions like stria-tum and hippocampus are highly enriched with non-heme iron, which is catalytically involved in the production of ROS (Hill and Switzer, 1984). Exposure to MPTP might have lead to the peroxidation of membrane lipids even-WXDOO\OHDGLQJWRORVVRIPHPEUDQHLQWHJULW\DQGQDOO\to cell death in these brain regions. MPTP is believed to induce selective toxicity at central dopaminergic neurons via the end products of its oxidation, 1-methyl-4-phe-nylpyridinium (MPP+) which inhibits oxidative metabo-lism at complex I of the mitochondrial respiratory chain

E\LWVVSHFLFLQKLELWLRQRI1$'+XELTXLQRQHR[LGRUH-ductase (Ebadi et al., 2001). Although the mechanism by which it induces selective dopaminergic cell death has not been fully elucidated, it was described that MPTP acts as an inhibitor of complex I of mitochondrial respirato-ry pathway and produces a decrease in tissue ATP content and increases ROS formation (Lane et al., 2008). Malo-nised dopaminergic neurones have been reported to be more susceptible to neurodegeneration in PD and MPTP toxicity (Iczkiewicz et al., 2006).

During oxidative stress in the neuronal cells there is an increase in intracellular Ca2+ levels in the brain (Annunziato et al., 2003). This increased intracellular Ca2+can induce the irreversible conversion of xanthine dehy-GURJHQDVH;'+WR;2ZKLFKLQWXUQFDWDO\]HVWKHR[L-dation of xanthine to provide a source of O-2. In addition, auto-oxidation of dopamine in brain could also serve as a source of superoxide anion (Olanow, 1993). These mech-anisms could be the main reasons for the increased lev-HORI;2DQGUHGXFWLRQLQDFWLYLW\RI62'OHDGLQJWRDQoverload of oxygen radicals and repression of antioxidant enzymes with MPTP exposure.

Dysfunction of cholinergic neuronal activities has been considered to be a cause of age-related impairment of brain function among animal species, including human beings. It has been documented that mitochondrial com-plex I activity is decreased in the substantia nigra of PD patients (Lestienne et al., 1990; Gadaleta et al., 1990). Studies on rodent brains have indicated age-related reduc-WLRQVLQFKROLQHUJLFDFWLYLWLHVVXFKDVKLJKDIQLW\FKROLQHuptake, acetylcholine synthesis and acetyl choline release (Curti et al., 1989; Vogelsberg et al., 1997; Rodrigues et al., 2007). The reduced acetylcholine release is thought to be directly related to a decreased synaptic transmis-sion in cholinergic neurons. The acetylcholine release per synapse was later shown to be decreased in aged rats in experiments using synaptosomes (Rylett et al., 1993). Oxidation of L-3,4-dihydroxy phenylalanine (L-DOPA) and dopamine (DA) to generate semiquinones / quinones, oxygen radicals, and other ROS may play a vital role in neuronal cell death in PD. In particular, semiquinones / quinones can form conjugates with thiol compounds such as GSH and cysteine. Exposure of L-DOPA, DA, and oth-er catacholamines to a system generating O2- radical led to O2- -dependent depletion of added GSH (or cysteine), accompanied by the formation of thiol-DA or thiol-DOPA adducts. The substantia nigra of rodents, primates and humans contain lower levels of glutathione than oth-er brain regions (Migdalis et al., 2001). The concentra-tions of GPx, CAT and SOD and the TA were found to EHGHFUHDVHGVLJQLFDQWO\LQWKHVWULDWXPDQGKLSSRFDP-

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N. Haleagrahara and K. Ponnusamy

pus of MPTP treated rats as compared to the control rats.The present study showed that CAE administration

UHGXFHG/32VLJQLFDQWO\LQDJHGUDWVDQGDJHGUDWVFKDO-lenged with MPTP. The reduction in LPO levels may be due to the electron and H+ donating capacity of polyphe-nols present in CAE, which is attributed the termination of LPO chain reaction based on their reducing power. 6HYHUDOVWXGLHVKDYHVKRZQWKDWDYRQRLGVLQWHUDFWZLWKcell membranes, improving their stability, thereby pro-tecting them from LPO (Saija et al., 1995). Antioxidant property of CAE may possibly be attributed to the nitrig-enomic phenolic compounds present which are also effec-tive hydrogen donors, which makes them good antioxi-dants (Rice-Evans et al., 1995). A dose of 300 mg/kg of C. asiatica extract has decreased the LPO and protein car-bonyls, which are the products of lipid peroxidation and a measure of free radical production. There was a signif-icant increase in SOD levels, which is the only enzyme using the super oxide anions as a substrate and produc-ing the hydrogen peroxide as metabolite, with C. asiati-FDWUHDWPHQW7KHUHZDVDOVRDVLJQLFDQWLQFUHDVHLQ*3[levels, which protects the cells and neurons against free radicals induced damage. C. asiatica treatment produced a significant increase in the CAT levels, compared to MPTP alone treated group, demonstrating that the extract of C. asiatica scavenges the hydrogen peroxide generated by SOD (Schulz et al., 2000; Kumar and Gupta, 2002). Thus, our study has shown that the C asiatica extract has mitigated MPTP induced peroxide production in striatum and hippocampus and reversed the oxidative damage, as evidenced by increased TA and antioxidant enzyme lev-els in these regions.

The active constituents present in the C. asiatica extract are triterpens like asiatic acid and asiaticoside. Differ-ent parts of the C. asiatica were also found to have high level of phenolic contents. Mandel et al. (2006) reported that phenolic compounds acts as a potent iron-chelating, nitrigenomic antioxidants. The phenolic compounds such as quercetin and catechins present in the CAE may have different functional property such as scavenging of ROS (Hatano et al., 1989; Zainol et al., 2003), inhibition of the generation of free radicals and chain-breaking activ-ity (Laranjinha et al., 1995). They may act as hydrogen-donating radical scavengers by scavenging lipid alkoxyl and peroxyl radical and spares overall antioxidant status (Bors et al6WXGLHVKDYHGRFXPHQWHGWKDWELR-avanoids and triterpenes exerts free radical scavenging, membrane stabilizing, iron-chelating, lipotrophic, vasoac-WLYHLPPXQRPRGXODWRU\DQWLPXWDJHQLF;2LQKLELWR-ry, anti-apoptotic, anti-ageing and neuroprotective effects and increases cytoprotective heat shock proteins (Lahouel

et al., 2006; Broncel et al., 2007; Greenrod et al., 2005; Kalfon et al., 2007; Mo et al., 2007; Luangaram et al., 2007). Thus the observed free radical scavenging and antioxidant effect of C asiatica in the present study could be attributed to the triterpenes and phenolic content of the whole plant.

In conclusion, the present study implies that Centella asiatica enriched with polyphenols and triterpenes may be particularly useful xenobiotics detoxifying agents as it could decrease lipid peroxidation and enhance brain anti-R[LGDQWVDQGVLJQLFDQWO\SUHYHQWWKHEUDLQIURPQHXUR-toxic effects of MPTP. Therefore, C. asiatica could offer a useful support to the Parkinsonism therapy by acting as a neuroprotective antioxidant and thus prevent the neuro-nal damage in brain regions associated with Parkinson-LVP7KXV&$(H[WUDFWHQULFKHGZLWKELRDYDQRLGVDQGtriterpenes may be considered as a powerful neuropro-tective agent for membrane molecular medicine. Further studies may be necessary to elucidate the exact molecular mechanisms of actions of the various constituents in CAE against MPTP induced neurotoxicity.

ACKNOWLEDGMENT

This work was supported by a grant-in aid for Scientif-ic Research, from International Medical University (IMU Research Grant No: 115/2006), Kuala Lumpur, Malaysia.

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